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  • Analytical Chemistry and Spectroscopy  (9,774)
  • Life and Medical Sciences  (9,191)
  • 1995-1999  (6,118)
  • 1980-1984  (7,734)
  • 1975-1979  (5,113)
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  • 1
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 2
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 229-229 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 4
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 230-231 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 5
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 232-236 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 6
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 343-362 
    ISSN: 0886-9383
    Keywords: straight line calibration ; errors in both axes ; uncertainties ; linear regression ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Calibration is a fundamental step in the calculation of the unknown concentration of analyte in most analytical methods. It is known that for certain methodologies, if only the errors in the independent variable are taken into account, there may be considerable errors in the estimation of the value of the regression coefficients, the derived statistical parameters and in some cases the sought for response and concentration values. This paper reviews the calibration methods including some references to procedures for the detection of outliers and robust regression when there are errors in both axes. The advantages and limitations of the different approaches are discussed and a comparative study is made of the approaches of several techniques for which computer programmes have been developed based on the algorithms put forward by the different authors. Finally, some trends of future development in this field are envisaged.
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  • 7
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    Journal of Chemometrics 9 (1995), S. 363-372 
    ISSN: 0886-9383
    Keywords: PLS regression ; orthogonal expansion ; optimization ; Lagrange multipliers ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A simple iteration algorithm that is faster and less memory-intensive than the NIPALS iteration algorithm for PLS regression is presented. The iteration algorithm is obtained by treating the orthogonal expansion or decomposition of a matrix X as an extremum problem subject to normalization and orthogonality constraint conditions and then solving the problem by use of the method of Lagrange multipliers. The main idea in this method is to find the transformation vector r. The latent variable t is expressed exactly as the linear combination of X-variables with the vector r so that the final regression coefficients can be conveniently provided. In the algorithm the recursion of the orthogonal projection is needed, which is derived by use of a matrix inverse formula. Algorithms are established from the equation for calculating the vector r that are suitable for dealing with three cases of large data sets. The first case is when the number of objects is very large, the number of variables is relatively small and the number of Y-variables is equal to or greater than the number of X-variables. The second case is when the number of objects is very large, the number of variables is relatively small and the number of X-variables is greater than the number of Y-variables. The last case is when the number of variables, either X- or Y-variables, or both, is very large and the number of objects is small.
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  • 8
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 431-432 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 9
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 451-457 
    ISSN: 0886-9383
    Keywords: non-linear regression ; optimization ; robust methods ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Several robust regression methods, including a new proposal, are described and their properties discussed. Resistance to various types of outliers and non-normality is demonstrated. The techniques are applied to non-linear regression models from chemical kinetics and calibration. Optimization of the types of objective functions encountered when applying robust regression is considered.
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  • 10
    ISSN: 0886-9383
    Keywords: PLS ; kernel algorithm ; multivariate calibration ; EM algorithm ; cross-validation ; missing data ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: This is Part II of a series concerning the PLS kernel algorithm for data sets with many variables and few objects. Here the issues of cross-validation and missing data are investigated. Both partial and full crossvalidation are evaluated in terms of predictive residuals and speed and are illustrated on real examples. Two related approaches to the solution of the missing data problem are presented. One is a full EM algorithm and the second a reduced EM algorithm which applies when the number of missing values is small. The two examples are multivariate calibration data sets. The first set consists of UV-visible data measured on mixtures of four metal ions. The second example consists of FT-IR measurements on mixtures consisting of four different organic substances.
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  • 11
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 439-439 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 12
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995) 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 13
    ISSN: 0886-9383
    Keywords: sequential response surface modelling ; Williamson ether synthesis ; SN2 ; optimization ; reaction kinetics ; reaction mechanism ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In this work the utility of a new method for determining kinetic parameters by sequential response surface modelling, previously described (Part 1), is shown by applying it to an experimental study of a reaction with known kinetics. The nucleophilic substitution reaction between ethoxide and benzyl chloride, the Williamson ether synthesis, was selected as a model reaction. This reaction is known to proceed with second-order kinetics. The method gives access to estimates of initial reaction rate which can be further used to obtain estimates of activation energy and reaction order of reactants. The results obtained are in good agreement with the estimated values of these parameters obtained with conventional kinetic experiments.
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  • 14
    Electronic Resource
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    Journal of Chemometrics 9 (1995), S. 483-487 
    ISSN: 0886-9383
    Keywords: fractional factorial design ; multiresponse ; PLS ; PCA ; reduced-rank regression ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: This paper presents an interpretation of PLS applied to orthogonal explanatory variables. In particular, it is shown that in fractional factorial multiresponse experiments PLS2 gives identical results to ordinary least squares applied to principal components of the response variables. The general relationship is that the reduced-rank regression algorithm which first projects Y onto the X-space and then truncates this matrix by principal component analysis before performing ordinary least squares estimation gives the same predictor as PLS2 and SIMPLS if all the non-zero eigenvalues of XTX are identical.
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  • 15
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 471-481 
    ISSN: 0886-9383
    Keywords: batch prediction ; continuum regression ; multivariate calibration ; sequential prediction ; simultaneous prediction ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: In analytical chemistry a single fitted calibration model is used repeatedly to predict the level of the analyte of interest for the specimens comprising the prediction set. Unlike the calibration (or training) set, which is often limited in size, the prediction set can be very large.In the case of multivariate calibration a number of methods such as PLS and PCR are commonly used to construct the calibration model. The set of instrumental measurements and the reference analyte level are available for each specimen in the calibration set. For specimens in the prediction set, only the instrumental measurements are available, since the problem is to predict the analyte level for these specimens. It is not widely recognized that predictions of the analyte levels for individual specimens can be improved by utilizing seemingly unrelated information from the instrumental measurements associated with the other members of the prediction set. In the case of PCR there exists a very straightforward procedure for doing this. A description of the various sources of prediction errors is provided to explain the ability of PCR to utilize this additional information. The use of PCR in this context is illustrated with both a synthetic and a real example.
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  • 16
  • 17
    Electronic Resource
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    New York, NY : Wiley-Blackwell
    Journal of Chemometrics 9 (1995), S. 531-531 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 18
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    Journal of Chemometrics 9 (1995), S. 509-520 
    ISSN: 0886-9383
    Keywords: canonical variates ; discriminant analysis ; partial least squares ; principal components ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A new set of derived variables is proposed for exhibiting group separation in multivariate data on for preprocessing such data prior to discriminant analysis. The technique combines optimal features of canonical variate analysis and principal component analysis: the derived variables are linear combinations of the original variables that optimize the canonical variate criterion (ratio of between-group to within-group variance) but subject to the orthogonality constraints of principal components. In this formulation the canonical variates can be derived even when the within-group matrix is singular (i.e. when there are more variables than objects in the data matrix). A simple computational algorithm for extraction of these variables is proposed. The methods are illustrated on several data sets and compared with alternative techniques such as principal component analysis and partial least squares.
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  • 19
    Electronic Resource
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    Journal of Chemometrics 9 (1995), S. 489-507 
    ISSN: 0886-9383
    Keywords: QSAR ; partial least squares ; robust regression ; CoMFA ; weighted regression ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A robust implementation of partial least squares (PLS) is developed in which the method of iteratively reweighted least squares is adapted for use with PLS. The result is a PLS algorithm which is robust to outliers and is easy to implement. Examples and case studies are presented, followed by two Monte Carlo studies designed to explore the behavior of the method.The paper begins with the motivation and intended applications for the procedure. A discussion is given of the method of interatively reweighted least squares (IRLS) for outlier detection. The procedure, given the name IRPLS, is then presented. Three case studies illustrate how the procedure works on various types of data and how it should be used. The first Monte Carlo study is designed to determine whether the IRPLS procedure correctly identifies multiple outliers in a wide variety of configurations. The second Monte Carlo study is designed to estimate the breakdown bound of the procedure.
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  • 20
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 21
    Electronic Resource
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    Journal of Chemometrics 9 (1995), S. 309-322 
    ISSN: 0886-9383
    Keywords: simulated annealing ; messy genetic algorithms ; optimization of multimodal objective functions ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: The method of simulated annealing is modified so that the concept of messy chromosomes is applied. Constituent genes of messy chromosomes are specified by their respective names (indices) and values (alleles) simultaneously. Unlike simple chromosomes (binary vectors), messy chromosomes may be either under- or overspecified with respect to the problem being solved. The messy simulated annealing algorithm is a very robust and efficient stochastic optimization method which is able to find correct minima of deceptive or highly multimodal objective functions. This is shown by way of a number of simulations.
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  • 22
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    Journal of Chemometrics 9 (1995), S. 323-326 
    ISSN: 0886-9383
    Keywords: partial least squares ; biased regression ; ordinary least squares ; minimum length least squares ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: An algebraic proof is given that in partial least squares (PLS) regression the Euclidean length of the estimator is shrunk in comparison with the ordinary least squares estimator or with PLS estimators based on a larger number of dimensions.
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  • 23
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    Journal of Chemometrics 9 (1995), S. 329-330 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 24
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    Journal of Chemometrics 9 (1995) 
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 25
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    Journal of Chemometrics 9 (1995), S. 389-409 
    ISSN: 0886-9383
    Keywords: multivariate image analysis ; principal component analysis ; exploratory data analysis ; projection in multivariate space ; graphical visualization ; noise ; Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: Multivariate image analysis (MIA) is a powerful tool for many image segmentation and classification problems, but the interpretation and understanding of the original and resulting multidimensional (multivariate) data are not always easy. A strategy for MIA has been proposed which describes its usage on multivariate images for segmentation tasks. MIA starts with principal component analysis (PCA) and then continues with interactive analysis of the output from PCA. In this paper a number of extensions to MIA are proposed. The extensions are the suggestion to incorporate preprocessing of the multivariate image in MIA, the suggestion to use synthetic multivariate image models which create a clear-cut situation, and new visualization tools which improve the interactivity and understanding of the results. Extended MIA is applied on synthetic multivariate image data simulating a possible application with large noise, positron emission tomography (PET). As a result of the interactive analysis, suggestions for preprocessing emerge. The developed methodology for handling the noise is then applied on real PET image data with good results.
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  • 26
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 27
    ISSN: 0886-9383
    Keywords: Chemistry ; Analytical Chemistry and Spectroscopy
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
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  • 28
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    Cell Motility and the Cytoskeleton 1 (1980) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 29
    Electronic Resource
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    Cell Motility and the Cytoskeleton 1 (1980), S. 1-15 
    ISSN: 0886-1544
    Keywords: centrosomes ; kinetochores ; microtubule initiation ; nuclease enzymes ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A lysed cell system was used to study the organelle structure and nucleation of exogenous tubulin at kinetochores and centrosomes in mitotic PtK2 cells. We have used this lysed cell system in conjunction with nuclease digestion experiments to determine which specific nucleic acids (DNA or RNA) are involved in either the structure and/or microtubule-initiating capacity of kinetochores and centrosomes. The results indicate that DNase I specifically decondenses the kinetochore plate structure, with the eventual loss in the ability of the chromosomes to nucleate microtubule assembly. DNase I had no effect on either the structure or nucleating capacity of centrosomes. Both RNase T1 and RNase A specifically attacked the amorphous pericentriolar material of the centrosomes, with a concomitant loss in the ability of this material to nucleate microtubule formation. Neither RNase appeared to affect the structure or nucleating capacity of the kinetochore. Therefore, the two types of nucleases appear to exert preferential effects on the different types of microtubule initiation sites in mitotic mammalian cells. The results suggest that DNA is a major component of the kinetochore, while RNA is a major component of the amorphous pericentriolar material. These findings support the concept that microtubule initiation sites in mitotic cells contain nucleic acids which are essential for the structural and functional integrity of the sites.
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  • 30
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    Cell Motility and the Cytoskeleton 1 (1980), S. 31-40 
    ISSN: 0886-1544
    Keywords: actin ; fascin ; actin cross-linking proteins ; fertilization ; microvilli ; sea urchin eggs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Following fertilization, the sea urchin egg cortex undergoes a structural change involving the assembly and organization of actin filaments into microvilli. Antifascin localizes this actin cross-linking protein in the microvilli of the fertilized egg cortex but no organized staining is present in the unfertilized cortex. Determination of the actin content of eggs using the DNAase I inhibition assay indicates that actin is about 1.4% of the total protein. Approximately 90% of this actin is soluble in low calcium isotonic extracts of unfertilized eggs while only 60-65% can be recovered in identical extracts of fertilized eggs. Similar measurements for fascin using a radioimmunoassay indicate this molecule represents about 0.3% of the total egg protein, essentially all of which is recovered in low calcium isotonic extracts of unfertilized eggs. After fertilization only 65-70% of this actin cross-linking protein is in the soluble phase. These results demonstrate a markedly different solubility for actin and fascin after fertilization, when the indirect immunofluorescence staining localizes fascin in the microvilli, and are consistent with the idea that fascin organizes newly polymerized actin filaments into the microvillar cores. A consideration of the amounts of actin and fascin incorporated into the cortex after fertilization and the number of microvilli on the egg surface indicates that the measured values are sufficient to account for the observed microvillar elongation.
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  • 31
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    Cell Motility and the Cytoskeleton 1 (1980), S. 17-29 
    ISSN: 0886-1544
    Keywords: Ca-ion ; Labyrinthula ; contraction ; glycerination ; Ca-reservoir ; cell movement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Colonies of Labyrinthula, a colonial marine protist, expand by protrusive movements of the specialized slimeways. The movements recorded in time-lapse films are of two types - filopodial and lamellipodial - and occur at rates equivalent to those of cell translocation.Evidence is presented that Ca2+ regulates the contraction of the actomyosin system of filaments present in the slimeways of Labyrinthula. In glycerinated models or in colonies exposed to ionophore A23187 contraction is evidenced by the occurrence of periodic contractions of the slimeways, giving them the appearance of strings of beads. Glycerinated slimeways contract on the addition of Ca2+ and ATP while slimeways provided with ionophore A23187 contract on addition of Ca2+ alone. The concentration required is 1.1 × 10-7 M Ca2+ while concentrations of 6.2 × 10-8 or lower were ineffective. Rates of contraction were measured in time-lapse films which provide evidence that contractions and beading occur everywhere in the slimeway system. When beading occurs, the 6-nm filaments transform from an array of parallel single filaments into an interwoven meshwork.We have identified by pyroantimonate-OsO4 fixation, as possible Ca2+ reservoirs, deposits of Ca2+ in bothrosomes - structures through which cell secretions pass into the slimeways. The electron-dense deposits are located at the base of the bothrosome and disappear after incubation with EGTA. We propose that the translocation of cells as well as the movements of slimeways may be regulated by the cells through the local measured liberation of Ca2+ from the bothrosome where it is sequestered.
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  • 32
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    Cell Motility and the Cytoskeleton 1 (1980), S. 41-61 
    ISSN: 0886-1544
    Keywords: mitosis ; mitotic spindle ; kinetochore ; microtubule ; micronucleus ; Tetrahymena ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Mitotic micronuclei were isolated from Tetrahymena thermophila and data on spindle ultrastructure were obtained from serial, transverse sections. Comparison of data from nuclei at meta- and early anaphase with data from nuclei at late anaphase showed that during anaphase, sister kinetochores move from the equator to the spindle poles, but kinetochore translocation occurs without any apparent change in either the number or length of kinetochore microtubules. This unprecedented result is ascribed significance with regard to the mechanism of kinetochore transport since there are only a limited number of ways that result could be achieved. The organization of the peripheral sheath changes during anaphase as evidenced by gaps in the sheath at late anaphase. Numerous kinetochore and non-kinetochore microtubules are located in polar regions of the spindle at late anaphase, whereas those regions contained only peripherally arranged microtubules at earlier stages. Tracking of individual kinetochore microtubules in late anaphase nuclei showed that some of them appeared to become incorporated into the peripheral sheath near the pole. At early and late anaphase, crossbridges connect adjacent microtubules throughout the spindle poleward to the kinetochores, as well as in the interzone.
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  • 33
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    Cell Motility and the Cytoskeleton 1 (1980), S. 63-71 
    ISSN: 0886-1544
    Keywords: Physarum polycephalum ; myosin light chains ; polyacrylamide gel electrophoresis ; calcium ; cytoplasmic streaming ; actomyosin ATPase regulation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myosin from the slime mold Physarum polycephalum contains three sizes of polypeptides: a heavy chain and two light chains, LC-1 and LC-2. Using a simple qualitative test for calcium binding by comparing electrophoretic migration of the polypeptides in sodium dodecy1 sulfate (SDS) acrylamide gels in the presence and absence of calcium, we have found that Physarum myosin light chain LC-2 migrates with an apparent molecular weight of 16,900 daltons in the presence of the metal ion chelator ethylene glycol bis (B-aminoethyl ether) N,N′-tetraacetic acid (EGTA). However, if calcium chloride is added to the sample prior to electrophoresis, the apparent molecular weight decreases to 16,100. Lanthanide and cadmium ions, but not magnesium, can substitute for calcium. Because the ionic radii of Ca2+, La3+, and Cd2+ are almost identical, we conclude that Physarum myosin LC-2 possesses a very size-specific binding site for calcium. Physarum myosin LC-1 and the heavy chain give no evidence for binding calcium by this test. Since cytoplasmic streaming in the plasmodium of Physarum requires calcium, our evidence indicates that the calcium-binding property of Physarum myosin LC-2 may be important in regulating the production of force by actomyosin in the ectoplasm. Unexpectedly, the myosin light chain in Physarum capable of binding calcium, LC-2, is the essential light chain, while LC-1 is a member of the regulatory class of myosin light chains [V. T. Nachmias, personal communication]. Until now, essential myosin light chains have not been shown to have high affinity divalent cation binding sites. This means a new version of the myosin-based model for actomyosin regulation by calcium may be required to explain cytoplasmic movement in Physarum, and perhaps in other motile systems involving cytoplasmic myosins as well.
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  • 34
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    Cell Motility and the Cytoskeleton 2 (1982), S. 273-285 
    ISSN: 0886-1544
    Keywords: actin gelation ; F-actin nucleation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A new actin binding protein has been purified to homogeneity from amoebae of Dictyostelium discoideum. This protein is a single polypeptide with a molecular weight of 120,000 upon sodium dodecyl sulfate gel electrophoresis. It is soluble and trypsin-sensitive, contains no carbohydrate, increases the viscosity and sedimentation rate of F actin, and inhibits the actin-stimulated Mg ATPase of rabbit muscle heavy meromyosin. The interaction of 120,000-dalton protein with F actin is not inhibited by millimolar ATP, pyrophosphate, or micromolar calcium. The 120,000-dalton actin binding protein increases the initial rate of actin polymerization and decreases the critical concentration of actin at steady state.These properties demonstrate that 120,000-dalton protein from Dictyostelium discoidum is not a myosinlike protein. Rather, this protein is probably involved in regulating the assembly of the actin cytoskeletion.
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  • 35
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    Cell Motility and the Cytoskeleton 2 (1982), S. 287-308 
    ISSN: 0886-1544
    Keywords: actin-binding protein ; Dictyostelium ; cytoskeleton ; amoeboid movement ; calcium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A protein from Dictyostelium discoideum with an apparent subunit molecular weight of 95,000 daltons (95K protein) was previously identified as an actin-binding protein ‘Hellewell and Taylor, 1979’. In this paper, we present a method for purifying the protein, and characterize some important aspects of its structure and function. Purification of the 95K protein is achieved by fractionation with ammonium sulfate followed by chromatography on DEAE-cellulose, gel filtration on 6% agarose, and final purification on hydroxyapatite. The 95K protein is a dimer, composed of apparently identical subunits. It is a rod-shaped molecule, 38 nm in length, with a Stokes radius of 74 Å. In these structural properties, the 95K protein is similar to muscle and nonmuscle α-actinins. The 95K protein and filamin are equally competent, when compared on a weight basis, to enhance the apparent viscosity of actin as determined by falling ball viscometry. The apparent viscosity of mixtures of the 95K protein and actin is dramatically reduced at pH greater than 7.0 or free ‘Ca2+’ greater than 10-7 M. We also examine the mechanism by which calcium regulates the interaction of the 95K protein and actin. A change in free ‘Ca2+’ induces no detectable change in the quaternary structure of the 95K protein. Our experiments indicate that the 95K protein does not dramatically alter the length distribution of actin filaments in the presence of micromolar free ‘Ca2+’. A large fraction of the 95K protein cosediments with actin in the presence of low free ‘Ca2+’ (ca. 3 × 10-8M), but not in the presence of high free ‘Ca2+’ (ca. 4 × 10-6M). We conclude that increased free ‘Ca2+’ inhibits gelation of actin by the 95K protein by reducing the affinity of the 95K protein for actin. We propose that 95K protein is an important component of the cytoskeletal/contractile system in D. discoideum amoebae.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 317-332 
    ISSN: 0886-1544
    Keywords: cytoskeleton ; platelets ; actin-binding protein ; actin ; myosin ; thrombin activation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When human blood platelets were immersed in an ice-cold solution containing 1% Triton ×-1200, 40 mM KCl, 10 mM EGTA, 10 mM imidazole-HCl, and 2 mM NaN3 pH 7.0, a flocculent precipitate appeared immediately in the tube. This precipitate was collected at 3,000g and SDS-polyacrylamide gel analysis showed it to consist mainly of actin, α-actinin, actin-binding protein (ABP), and varying amounts of myosin.Any modifications of this solution used to isolate the platelets' Triton-insoluble cytoskeleton caused profound changes in the nature of the cytoskeleton isolated. Increasing the KCl concentration resulted in a lower yield of cytoskeletal actin and ABP. Inclusion of EDTA in the solution resulted in an increased amount of myosin associated with the cytoskeleton, whereas including MgATP decreased the myosin yield.Experiments with the purified proteins showed that ABP and myosin can each protect the actin from depolymerizing when dialyzed into the Triton solubilization solution. In addition, it was found that when platelets were stimulated with thrombin for 2 min prior to the addition of the Triton solution, 3-4 times more myosin was associated with the cytoskeletal precipitate.The results suggest, therefore, that any variations in solution conditions used for isolating the cytoskeleton from resting platelets, which results in alterations in the amount of ABP, may have profound effects on the state of actin polymerization. Likewise, in thrombin-activated platelets, it is suggested that the increased association of myosin with the cytoskeleton results in a greater stabilization of the F-actin associated with the cytoskeleton. These factors must be considered when interpreting the results regarding the nature of actin transformations in the resting and activated platelet.
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    Cell Motility and the Cytoskeleton 2 (1982) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 2 (1982), S. 369-383 
    ISSN: 0886-1544
    Keywords: motility ; flagella ; cilia ; microtubules ; Gregarines ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The male gametes of the parasitic protozoan, Lecudina tuzetae, have a motile flagellum with a “6 + O” ultrastructure ‘Schrével and Besse, 1975’. These gametes were isolated from the cysts in which they develop and were observed and photographed under a variety of conditions. The flagella beat continuously, without stopping and starting, with a beat period of about 2 sec. They can beat in solutions whose viscosities are greater than 0.5 Nsm-2 (l Nsm-2 = 103 cP). The waveform can be approximated by a series of helical arcs and interconnecting straight regions that travel from the base to the tip. The helical regions have a radius of curvature of 3.2 μm and subtend a final angle of 1.7 radians. The straight portions are 2.0 μm in length. There are two sets of opposing bends, but they do not originate in the same plane. The resulting waveform is an approximately helical coil, with a pitch of 9.8 μm, a pitch angle of 0.6 radian and a peak-to-peak amplitude of 2.3 μm. The sense of the coil is left handed. The axoneme twists during beating. The main differences between the movement of this flagellum and that of typical 9 + 2 flagella are a low beat frequency and three-dimensional bends that produce relatively little forward movement of the cell. Twisting is discussed as a means of discriminating between some types of models of flagellar motility.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 393-403 
    ISSN: 0886-1544
    Keywords: motility ; Ca2+ ; ionophores ; spirulina subsalsa ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Motility of the marine filamentous cyanobacterium Spirulina subsalsa is both Ca2+ and Na+ dependent, and replacement of Na+ by mannitol arrests it. The data presented suggest that Ca2+ interacts with sites on the surface of the cell membrane. The inhibitory effect of dicyclohexylcarbodiimide (DCCD) hints at the possibility that the role of Ca2+ may be associated with a membrane bound Ca-ATPase. Motility is pH dependent, being nil at pH 〈 6.5 and 〉 10.0, with an optimum at 8.5. Norepinephrine abolishes most of the inhibitory effect of low pH on motility. Ca2+ has an “all-or-none” effect on motility that is triggered at 5 mM. Acetylcholine lowers the threshold of Ca2+ necessary for triggering motility.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 445-455 
    ISSN: 0886-1544
    Keywords: clot structure ; platelet contractility ; protein networks ; rheological techniques ; viscoelasticity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When citrated plasma is recalcified, it forms a viscoelastic gel-a clot. The relationship between platelet contractility and clot rigidity was studied by using a rheological technique which simultaneously measured both the dynamic rigidity modulus and the contractile force during gel formation with platelet rich plasma (PRP). Protein network formation in a clot was accompanied by a contractile force throughout the clotting process. PRP demonstrated a maximum elastic modulus of 6,000 dynes/cm2 and a maximum contractile force/area of 1,500 dynes/cm2. The values of these parameters for a platelet-free clot (PFP) were 700 dynes/cm2 and less than 100 dynes/cm2 respectively. Sonicated control PRP and PRP from a Glanzmann thrombasthenia patient both clotted in a manner similar to PFP. Metabolic inhibitors, 2-deoxy-D-glucose and KCN (5 mM each), retarded the clotting curves of PRP. Cytochalasin B and E suppressed both structural rigidity and force generation in a concentration-dependent manner similar to their inhibitory effect on actin polymerization in platelets. Colchicine (2.5 mM) or vinblastine (0.11 mM) did not affect these clotting curves. Thrombi-activated, fixed platelets did not generate any force, nor did they significantly increase clot rigidity. Streptokinase induced a concurrent decrease of both rigidity and force in PRP clots. The elastic modulus of a PFP clot could be increased to 2,500 dynes/cm2 by externally straining the network with an axial force/area of 1,500 dynes/cm2. Our results indicate that clot structure formation in PRP is strongly coupled to the contractile force generated by the platelet microfilament system and that this force modulates clot rigidity.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 457-470 
    ISSN: 0886-1544
    Keywords: fragmin ; critical actin concentration ; nucleation ; filament growth ; pointed end ; barbed end ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: As reported previously, fragmin isolated form Physarum plasmodia restricts the polymerization of actin to produce short F-actin filaments in the presence of Ca2+ ions. Here it is shown that when actin is polymerized at low concentrations of salts, fragmin increases the critical concentration of actin for polymerization. This effect of fragmin on the critical concentration is independent of the molar ratio of fragmin to actin. The addition of actin monomers onto heavy meromyosin-decorated F-actin fragments treated with fragmin occurs unidirectionally at the pointed end of each fragment. These results suggest that fragmin binds to the barbed ends of F-actin filaments and inhibits association and dissociation of actin monomers at this end. Fragmin accelerates the initial stage of polymerization of actin. When a constant amount of G-actin is polymerized in the presence of small amounts of fragmin, the inverse of the half-polymerization time increases in proportion to the square root of the amount of fragmin added. This means that fragmin acts as a potent promoter of the nucleation step in actin polymerization. Both functions of fragmin-promotion of nucleation and capping at the barbed end of F-actin-require micromolar concentrations of Ca2+.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 471-482 
    ISSN: 0886-1544
    Keywords: calmodulin ; myosin ; antibody ; immunofluorescence ; amoeba ; Dictyostelium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A rabbit antiserum was raised against calmodulin from the eukaryotic microorganism Dictyostelium discoideum. In double immunodiffusion experiments, the antiserum formed an immunoprecipitation line with Dictyostelium calmodulin but not bovine brain calmodulin; competition radioimmunoassays showed no cross reactivity between the antiserum and calmodulins from bovine brain and spinach. The calmodulin content of vegetative Dictyostelium amoebae, determined by competition radioimmunoassay, was 0.5 μg/mg protein; similar levels were found in developing cells. The antiserum was used to visualize the distribution of calmodulin in Dictyostelium amoebae by indirect immunofluorescence. Cells were examined under various conditions: in suspension, attached to a substrate, and while phagocytosing yeast cells. In all cases, anticalmodulin staining was concentrated in the cell cortex. Parallel experiments using a monoclonal antibody against Dictyostelium myosin showed that this protein is also enriched in the cortical region of the cell.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 355-367 
    ISSN: 0886-1544
    Keywords: proacrosome migration ; nuclear pore redistribution ; nuclear membrane fluidity ; electron microscopy ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Electron microscopic examination of thin sections and freeze fractures of Locusta spermatids revealed that the proacrosome docks to the nuclear membrane and glides around the nucleus during sperm development. Whereas nuclear pore complexes occur in groups distributed at random over the entire nucleus of the early spermatid, they are found only in a narrow ring closely surrounding the centriolar adjunct in later spermatids. The pores appear to be swept caudally in the nuclear envelope, perhaps by a process like capping; they are not merely excluded by structures adhering to the nucleus. The observations suggest that both proacrosome migration and the deployment of pores are facilitated by the inherent fluidity of the nuclear membranes.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 343-354 
    ISSN: 0886-1544
    Keywords: NBD-phallacidin ; actin ; ocular tissues ; wound repair ; stress fibers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The fluorescent derivative of the actin-binding toxin phallacidin, 7-nitrobenz-2-oxa-1,3 diazole phallacidin, has been used to cytologically demonstrate the presence of actin in lens epithelium, corneal endothelium, and retinal pigment epithelium. In these noninjured tissues, no stress fibers are observed and fluorescence is confined mainly to an area at or near the cell membrane, although some diffuse cytoplasmic staining can also be seen. However, following injury to either the lens epithelium or corneal endothelium of rats and frogs, stress fibers are detected, but only in those cells that migrate into the wound area. Cells on the periphery of each tissue do not partake in would repair and thus maintain their normal appearance. After the tissue has regenerated, stress fibers disappear, and those cells involved in the injury response return to their normal morphology.When rabbit corneal endothelium is placed in tissue culture, stress fibers are observed as the cells migrate away from the initial explant. Upon reaching confluency, these cells spread out and each is surrounded by thick actin-containing bands. Furthermore, they exhibit some stress cables within their cytoplasm. This is in contrast to their appearance in vivo where stress fibers are absent and fluorescence is limited to a region near the cell membrane.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 385-391 
    ISSN: 0886-1544
    Keywords: ciliated cell ; basal body apparatus ; microtubules ; microfilaments ; respiratory epithelium ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: This is a descriptive study showing the three-dimensional interrelationship of cytoskeletal elements at the apex of ciliated cells of rat respiratory epithelium. Tissue specimens were serially thin sectioned in various planes and examined by transmission electron microscopy. Thicker sections were also cut at various angles and analyzed stereoscopically. Other specimens were cleared of soluble molecules by glycerination or Triton-X 100 treatment and sectioned as described above. It was found that C microtubules from the triplets of each basal body diverge from the A and B microtubules, run a short distance, and converge at the basal foot. These microtubules or other microtubules arising anew then dispersed deeper into the cytoplasm. The C fibers also interdigitated with other microtubules running perpendicular to them and parallel to the ciliated surface. Ten-nanometer intermediate filaments were organized in parallel sheets between adjacent basal bodies. Sixnanometer actin filaments were distributed throughout the apical cytoplasm. Neighboring basal bodies were linked to one another by microtubules and microfilaments. Basal bodies from each cell appear to be structured for stability, flexibility, and arranged to operate as a single unit.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 429-443 
    ISSN: 0886-1544
    Keywords: 21S dynein ; tubulin ; binding stoichiometry ; ATP sensitivity ; binding cooperativity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The binding properties of Tetrahymena 21S dynein to doublet A and B subfiber microtubules were analyzed by both a turbidimetric assay (Δ A350 nm) and electron microscopy. KCl-extracted, sucrose-gradient, purified 21S dynein binds to each of the two kinds of axonemal microtubules in both ATP-insensitive and ATP-sensitive modes, even though only a single type of binding occurs to each of the subfibers in situ. Total dynein bound to axonemal microtubules is a composite of binding that is sensitive to dissociation by ATP and binding that is insensitive to ATP. Each exhibits a different binding profile. Total binding exhibits a sigmoid profile (h = 1.93) and saturates at 1.49 mg D/mg T. ATP-sensitive binding likewise exhibits a sigmoid profile (h = 2.66) but saturates at 1.06 mg D/mg T. Binding occurs with a similar affinity for both A and B subfibers. The Hill coefficient (h) for ATP-sensitive binding implies positive cooperativity between binding events. ATP-insensitive binding was studied independently in 20 μM ATP, 10 μM vanadate, which blocks ATP-sensitive binding. ATP-insensitive binding exhibits a hyperbolic profile (h = 1.0) and likewise occurs along each of the two kinds of axonemal tubules. Binding saturates at 0.87 mg D/mg T. The binding data suggest that the tubulin dimer has conserved both ATP-sensitive and ATP-insensitive binding sites for 21S dynein, even though the sites may not be expressed in vivo.
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    Cell Motility and the Cytoskeleton 2 (1982) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 2 (1982), S. i 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 2 (1982), S. 573-582 
    ISSN: 0886-1544
    Keywords: Euglena flagella ; laser microsurgery ; stigma ; Mg2+ ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: When the area of the stigma of Euglena was irradiated with an infrared laser beam at a dose too low to cause permanent loss of motility, a reduction in flagellar motility was observed only when the external medium contained less than 1 mM Mg2+. At these low Mg2+ concentrations, the laser caused a decrease in flagellar frequency and a tendency for the flagellar waveform to shift towards that taken during reversed swimming. This suggests that the effect of the laser irrdiation was to deplete the cells of Mg2+. After the laser pulse the reversal response remained sensitive to the wavelength of the illuminating light. In white light (420-700 nm) 60% of the Euglena showed a reversed waveform; in orange light (530-700 nm) this increased to 90%. This shows that the photoreceptor was not destroyed by the laser irradiation.These experiments were performed on cells that had been impaled on a microelectrode. If direct electric current was passed into the laser-irradiated cells, the current necessary to cause flagellar arrest was 2 to 4 times less than that for cells not laser irradiated.It is concluded that an internal Mg2+ store is present in the Euglena, localized in the area of the paraflagellar swelling; and that the laser irradiation eliminates this Mg2+ store, but at the power used it does not destroy the ability of the stigmaparaflagella to control the flagellar activity.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 599-614 
    ISSN: 0886-1544
    Keywords: monoclonal antibodies to tubulin ; radioimmune assay ; immunoautoradiography ; Western blots ; immunofluorescence ; tubulin heterogeneity ; eukaryotic flagellar motility ; immunomotility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two monoclonal antibodies reactive for α-tubulin but not for β-tubulin have been prepared, characterized in terms of their relative binding to tubulins from differnt sources by a solid-phase binding assay, immunoautoradiography, and indirect immunofluorescence, and utilized to study flagellar motility. Our results demonstrate that α-tubulins from different species, and even from different tissues of the same species, are nonidentical. Especially interesting was the observation that one of the antibodies, Ab2, immunofluorescently stained microtubules of chick embryo fibroblast cells, but was completely unreactive for microtubules of rat kangaroo (PtK2) fibroblasts; a different antibody, Ab1, stained both cell types. Results of these and additional experiments clearly show that Ab1 and Ab2 recognize discrete and different epitopes on α-tubulin.Monoclonal antitubulins Ab1 and Ab2 each inhibited the bend amplitude of reactivated sea urchin spermatozoa without affecting beat frequencies or the ability of the outer doublet microtubules to slide past each other in elastase-digested models. These results, together with those obtained previously using rabbit polyclonal antitubulin antibodies [Asai and Brokaw, 1980], demonstrate that inhibition of bend amplitude is a common property of antitubulin antibodies and is not due to the binding of antibodies to one specific site on the axoneme. Our results suggest that tubulin subunit conformational changes may occur on the outer doublet lattice and may be integrally involved in the mechanism and control of flagellar bending.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 19-24 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 52
    ISSN: 0886-1544
    Keywords: videomicroscopy ; differential interference microscopy ; streaming ; reticulopodial motility ; Allogromia ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A new method called Allen Video-enhanced Contrast, Differential Interference Contrast (AVEC-DIC) microscopy is shown to be sufficiently sensitive to detect several new features of microtubule-related motility in the reticulopodial network of the foraminifer, Allogromia. The method takes advantage of the variable gain and offset features of a binary video camera to operate the DIC microscope under conditions highly favorable for video imaging, but in which the optical image is virtually invisible to the eye yet retains its full information when viewed by a suitable video camera. The improvements are made possible by setting a dé Senarmont compensator to λ/9-λ/4 at maximal working aperture of internally corrected planapochromatic objectives. Under these conditions, the offset feature of the video camera can reject so much stray light from the instrument and specimen that contrast compares favorably with that observed in high-extinction images, and polarizing rectifiers offer scarcely any advantage. Freed from the constraints of the light-limited conditions of DIC microscopy, video images can be recorded 60 times per second, or over 1,000 times the rate of photomicrographs at comparable magnifications under high-extinction conditions.Application of this method to the reticulopodial network of Allogromia has shown that cytoplasmic organelles are translocated only in contact with single microtubules or bundles of microtubules, and that these organelles fail to move when separated from microtubules. Microtubules themselves undergo both axial translatory (“sliding”) and lateral “zipping and unzipping” movements that have been suggested to occur during mitosis and other biological processes.
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    Cell Motility and the Cytoskeleton 1 (1981), S. 329-347 
    ISSN: 0886-1544
    Keywords: actin ; microfilaments ; heavy meromyosin ; mammary gland ; secretion ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Cytochalasin B, a microfilament-altering drug, inhibits lactose synthesis in lactating guinea pig mammary gland [Biochim. Biophys. Acta 392:20, 1975] but not primarily by inhibiting glucose transport [Eur. J. Cell Biol. 20:150, 1979]. In order to study the possible role of microfilaments in lactose synthesis and secretion, we isolated both the alveolar (milk-secreting) and myoepithelial (contractile) cells from lactating mammary gland. Light microscopy shows that the alveolar cell fraction (viability approximately 71%) is homogenous and that the cells retain strong polarity of secretory structures in the apical region. Two proteins were extracted from the alveolar cell fraction. One (mol wt 42,000) comigrates with skeletal muscle actin on SDS-PAGE gels. The other, a high-molecular-weight (180,000) protein (HMWP) may be analogous to actin-binding protein or clathrin. An extract from the myoepithelial cell fraction also contains a protein that comigrates with actin but no HMWP. Whole tissue extract contains the 42K protein, and a 185K HMWP. Examination of the alveolar cell extract by electron microscopic (EM) negative staining revealed meshworks of multistranded, interconnecting filaments, with attached globular structures (100-200 A) (possibly the HMWP) and single filaments (40-60 A diameter) branching off. To localize these filamentous structures in situ, whole tissue was glycerinated and incubated with rabbit skeletal muscle heavy meromyosin (HMM). Masses of filaments in myoepithelial cells served as convenient standards for HMM decoration. Decorated filaments have cross-arms or projections, unlike the narrow, smooth filaments of control tissue. Decorated filaments in alveolar cells are located beneath the plasma membrane, in close association with secretory vacuoles, and near the Golgi apparatus; filaments near the latter two are often oriented perpendicular to the plasma membrane. Microvesicles are embedded in meshworks under the plasmalemma and near the Golgi apparatus. Intermediate-sized (85-115 A diameter), non-decorated filaments diverge from the meshworks of decorated filaments. Microvesicles are associated with intermediate-sized filaments as well. The association of actin-like filaments with secretory vacuoles and microvesicles and their location in areas of the cell concerned with biosynthetic activities suggest a possible function in the intracellular transport of secretory products.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 131-150 
    ISSN: 0886-1544
    Keywords: flagella ; Chlamydomonas ; motility ; flagellar reversal ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using a uniflagellate mutant of Chlamydomonas and flash photomicrography at 300 Hz, we have obtained detailed information on the forward and reverse beating modes of Chlamydomonas flagella and on the relationship between rotation of the uniflagellate cell and the bending cycle of the forward mode. Flagella ranging in length from 5 to 15.5 μm were photographed. There is a decrease in wavelength and an increase in curvature in the principal bends when the length of the flagellum is less than the normal length of 12-13 μm, but these changes are not sufficient to maintain similarity of the bending pattern. In the reverse mode, the flagellum propagates symmetrical, planar, undulatory waves with a shear amplitude which is the same as in the forward mode: there is a 19% increase in beat frequency and a similar decrease in wave length. The reorientation of the flagellar beat direction towards the axis of the cell in the reverse mode is caused both by the decrease in asymmetry of beat and by activation of sliding in the principal bends at an earlier time in the beat cycle, relative to the time of activation of sliding in reverse bends. There are additional rare modes of beating which may be related to intermediate stages in the transition between forward and reverse beating modes.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 123-130 
    ISSN: 0886-1544
    Keywords: taxol ; microtubules ; flagellar outer doublets ; tubulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Taxol induces the in vitro assembly of calcium stable microtubules from flagellar tubulin solubilized from sea urchin (Strongylocentrotus purpuratus) sperm tail outer doublets by sonication. Assembly occurs in the presence or absence of exogenous GTP. The drug (10 μM) reduces the critical concentration of protein required for assembly to ≤0.04 mg/ml. 3H-Taxol binds specifically to both isolated flagellar outer doublets and to reassembled microtubules with calculated maximal binding ratios of 0.25 and 1.32 moles taxol/mole polymerized flagellar tubulin dimer, respectively. We suggest that the discrepancy in maximal binding ratios may result from the presence of an endogenous molecule(s) along the surface of outer doublet microtubules that restricts taxol binding to that structure.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 185-197 
    ISSN: 0886-1544
    Keywords: dynein ; microtubules ; cell motility ; fibroblasts ; in vitro ; phagokinetic tracks ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Patients with Kartagener syndrome (KS) show defects in ciliary and flagellar movement that are usually associated with the partial or total absence of dynein side arms from axonemal microtubules. Dynein is essential for such movements, but its involvement in other cellular (particularly microtubule-related) processes is unknown. It has recently been reported that neutrophils from KS patients show impaired motility including responses to chemotactic stimuli, suggesting that dynein-like proteins may be generally involved in motile processes. In support of this, we have now found that spontaneous motility of cultured skin fibroblasts from KS patients is also markedly impaired. Three cell lines derived from skin explants of KS patients with deficient dynein side arms in nasal cilia and eight cell lines derived from normal volunteers were studied. Fibroblasts were seeded into dishes containing colloidal gold-coated cover glasses [Albrecht-Buehler, 1977], incubated for 24 h at 37°C, and the area of cell “phagokinetic” tracks determined.Each cell line studied in this manner reproducibly displayed an amount of spontaneous motility characteristic for that cell line. The mean track area (± SE) for all control cells studied was 14.6 ± 0.5 × 103μm2 whereas for KS fibroblasts was 8.7 ± 0.4 × 103μm2 (P 〈 0.001). Immunofluorescence microscopy using antitubulin and antihuman 210 K MAP antibodies revealed no differences in the staining patterns between control and KS fibroblasts. Pinocytic rates were identical, and the complement of tubulin and major microtubule associated proteins as seen on one-dimensional SDS polyacrylamide gel autoradio-graphs appeared similar for control and KS cells. Thus, the observed motility defect is probably not the result of alterations in the occurrence or distribution of microtubules or in the occurrence or binding of the major microtubule-associated proteins. This defect in cellular motility may be related to the absence of dynein or may reflect another independent cellular defect.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 321-332 
    ISSN: 0886-1544
    Keywords: microtubule sliding ; interdoublet links ; radial spokes ; bend formation ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ciliary axonemes from Tetrahymena extracted by KCl to remove the dynein arms reveal an orderly array of interdoublet links connecting adjacent A-B or A-A subfibers. The links repeat every 96 nm at a stable site on the A subfiber positioned near the bases of radial spokes 2 and 3. Both links and radial spokes are in lateral register across the nine successive doublets of unbent axonemes. In contrast, bent axonemes or those reactivated by ATP to undergo partial sliding disintegration exhibit systematic displacement of the interdoublet links. The links show no evidence of having elastic or other extendable properties and, therefore, must have undergone intermittent attachment with nonstructural binding sites on the adjacent subfiber. These observations suggest a more dynamic role for the interdoublet links in ciliary motion than previously has been envisioned.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 213-226 
    ISSN: 0886-1544
    Keywords: microtubules ; fertilization ; cell division ; sea urchin ; cytoskeleton ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The microtubule-containing structures that appear in eggs during fertilization and cell division in the sea urchins Lytechinus variegatus and Arbacia punctulata were detected by antitubulin immunofluorescence microscopy of detergent extracted cytoskeletal preparations. The extraction buffer, which is composed of 0.55 mM MgCl2, 10 mM EGTA, 25 mM MES, 25% glycerol, 1% Nonidet P-40, and 25 μM PMSF, pH 6.7, allows for dramatically improved fluorescent images compared to those obtained using conventional staining procedures, with residual background staining being reduced to near zero.The immunofluorescent images obtained using this technique provide information on several motile events that occur during the first cell cycle. This technique demonstrates that all of the cytoplasmic microtubules are associated with the incorporated sperm's centrioles during female pronuclear migration. This changes during the centration of the male and female pronuclei at which time a monastral array of microtubules forms in the egg's cytoplasm. A large proportion of the monastral microtubules do not appear to be associated with the centrioles. At prophase and early metaphase, the centrioles are the dominant microtubule organizing centers (MTOCs) consistent with mitotic theories that the kinetochore catches, but does not initiate, microtubules. Observations of intercentriolar distances show that there are three stages of pole separation during the first cell cycle. The initial separation occurs during pronuclear centration, the second during the streak stage, and the final one during the late stages of mitosis. At telophase, polar microtubules appear to extend into the cortex supporting the cell surface at all regions except the presumptive cleavage site.
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    Cell Motility and the Cytoskeleton 3 (1983), S. 281-282 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 3 (1983), S. 307-320 
    ISSN: 0886-1544
    Keywords: contact inhibition ; contact guidance ; growth cones ; cell-cell interactions ; neuronal contact behavior ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The outcome of contact interactions involving neurons and nonneurons varies depending on the cell types involved. When neuronal growth cones from either ciliary (motor) or dorsal root (sensory) ganglia directly contact the lamellipodium of an embryonic heart fibroblast, both neurite elongation and fibroblast locomotion are inhibited. This occurs in spite of the fact that cell-surface activity in both cells continues unabated. Such contact inhibition is not observed when homologous ganglionic nonneurons are involved in the interaction. In fact, these cells become intimately associated with growth cones and/or neuritic shafts as a result of the contact. The detailed nature of the respose to contact exhibited by nerves and nonnerves varies not only with cell type but also with the portion of the cell involved in the contact. Growth cone filopodia tend to actively palpate the fibroblast surface, whereas spread regions, termed “veils,” form areas of apposition with fibroblast lamellipodia. This latter situation resembles the “typical” contact inhibition of locomotion that occurs following embryonic heart fibroblast-fibroblast interactions. Growth cones also frequently exhibit contact guidance when interacting with nonruffling lateral surfaces of heart fibroblasts.
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    Cell Motility and the Cytoskeleton 3 (1983), S. ix 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 1 (1981), S. 455-468 
    ISSN: 0886-1544
    Keywords: intercellular bridge ; intercellular communication ; cytokinesis ; squid ; ultrastructure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Incomplete cytokinesis followed by the disappearance of the midbody and spindle remnant results in intercellular bridges between the cells of the blastoderm of the squid embryo. An electron microscope study of the morphology of the stages of development of the intercellular bridge is presented. Cytokinesis ceased as the furrow base reached a diameter slightly larger than the midbody. As furrowing stopped, a dense material accumulated to form a cylindrical sheath 50 nm thick, lining the inner surface of the furrow base. Proteolytic enzymes showed this material to have a significant protein component. As the midbody broke down, vesicles lined the inner surface of the bridge sheath. In this configuration, there was cyto-plasmic continuity between the cells, and organelles appeared to pass through the bridge.The intercellular bridge could become temporarily closed. Vesicles entered the channel and fused with the vesicles lining the inner surface of the sheath. The vesicles enlarged until the channel became occluded with a series of transverse cisternae, the edges of which were embedded in the material of the sheath. When the bridge reopened, the transverse cisterna appeared to dissociate from the sheath, move out of the channel, and break down. Occasionally bridges were seen in which the bridge wall appeared distorted into lobes. It is suggested that such bridges might be in the porcess of breaking down, resulting in the final separation of the cells.
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    Cell Motility and the Cytoskeleton 1 (1981), S. 469-483 
    ISSN: 0886-1544
    Keywords: microtubules ; nucleation ; mitosis ; nocodazole ; immunocytochemistry ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The reassembly of microtubules is described in mitotic cells after release from nocodazole-induced block. The formation of microtubules was followed by light microscopic immunocytochemical staining using the PAP method, combined with to-luidine blue staining of the chromatin. The light microscopic observations on whole cells were compared with ultrastructural observations on thin sections. This step is essential to ascertain complete destruction of microtubules during the nocodazole treatment and to correlate immunocytochemical staining with the presence of microtubules.Removal of nocodazole (10 or 1 μg/ml) after a sufficiently long incubation to induce a complete disappearance of microtubules resulted in the appearance of tubulin staining specifically associated with the centromeres and with one or two isolated points in the cytoplasm. Electron microscopy confirmed that the staining was due to the massive accumulation of small microtubules at the kinetochores and centrosomes. Kinetochore nucleation was seen only in association with condensed metaphase-stage chromosomes and not with the less-condensed prophase chromosomes.In a second type of experiment cells were allowed to enter mitosis in the presence of an incompletely active concentration of nocodazole (0.1 μg/ml). The construction of the mitotic spindle was arrested; however, short microtubules were assembled at the kinetochores and centrosomes.These experiments demonstrate that in living mitotic PTK2 cells the kinetochores, as well as the centrosomes, exert a nucleating action on tubulin assembly.The further elongation of microtubules after removal of nocodazole was seen to occur preferentially along axes between the centrosomes and the kinetochores. This resulted in the construction of normal metaphases that evolved through anaphase and telophase. We have attempted to formulate a hypothesis that may explain the oriented assembly that seems to be essential in the construction of the spindle.
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    Cell Motility and the Cytoskeleton 1 (1981), S. 485-497 
    ISSN: 0886-1544
    Keywords: actin ; tubulin ; nucleotides ; polymerization ; microfilaments ; microtubules ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Both actin and tubulin, the major proteins of the cytoskeleton, bind nucleotide triphosphate (NTP) and exhibit the phenomenon of “polymerization-coupled” NTP hydrolysis. In this report I review the nature of polymerization-coupled NTP hydrolysis, and its possible role in the cellular function of actin and tubulin. Polymerization-coupled hydrolysis may be viewed as simply reflecting differences in the NTPase activity of free subunit as compared to polymer. Making assumptions concerning the values of various rate constants, it is possible to write expressions for the effects of NTP hydrolysis on the kinetics of polymerization. The role of NTP hydrolysis may be viewed in at least three different ways: (1) Hydrolysis alters the kinetics of assembly and disassembly. This leads to a consideration of the role of subunit flow in microtubule and microfilament function. (2) Hydrolysis is an essentially irreversible step that separates the assembly and disassembly reactions. This suggests a role of NTP in the regulation of polymer content during cellular cycles of assembly and disassembly. (3) NTP may allow transient stabilization of intersubunit bonds. This suggests a role of NTP in nucleation and possible regulation of nonequilibrium states of assembly.
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    Cell Motility and the Cytoskeleton 1 (1981), S. 499-515 
    ISSN: 0886-1544
    Keywords: dynein ; tubulin ; axonemes ; microtubules ; microtubule-associated proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Microtubule-associated proteins (MAPs), isolated from brain tubulin, bound to and saturated outer fibers of Chlamydomonas flagella. MAPs present on these microtubules prevented the subsequent recombination of dynein. MAPs also bound to intact axonemes and thus did not specifically bind to the dynein binding sites on the A subfiber. A molar ratio of 1 mole MAP2 per 27 moles tubulin dimers at saturation of the outer fibers with MAP2 suggested that MAPs could effectively interfere with dynein recombination only if the MAPs were near the dynein binding sites to sterically prevent binding. However, electron microscopic observations indicated that MAPs were not localized but, instead, were dispersed around the outer fibers. In addition, MAP2 present at saturating amounts on in vitro assembled brain microtubules had no significant effect on dynein binding. Dynein-decorated microtubules contained clusters of arms suggesting that there may be cooperative interaction between the arms during dynein binding. Because the A subfiber of axonemes contains sites to which dynein preferentially attaches, MAPs may prevent recombination by interfering with cooperative binding to these specific sites. Dynein presumably binds with equal affinity to any protofilament on in vitro assembled microtubules, and, therefore, the MAPs may not be capable of effectively interfering with cooperative binding of dynein to these microtubules.
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    Cell Motility and the Cytoskeleton 2 (1982) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 2 (1982), S. 1-8 
    ISSN: 0886-1544
    Keywords: microfilaments ; F-actin ; brain actin-depolymerizing factor ; tropomyosin stabilization of microfilaments ; DNase I inhibition assay ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Brain or muscle F-actin is rapidly depolymerized to monomeric actin in vitro by actin-depolymerizing factor, a protein isolated from chick embryo brain. Binding of muscle tropomyosin to muscle F-actin protects the F-actin from depolymerization by this factor. A 8.4/1.0 molar ratio of actin subunits to tropomyosin, achieved by incubation of the F-actin with excess tropomyosin, protects 58% of the F-actin from depolymerization by excess actin-depolymerizing factor for at least 3 hr at 25°C. Thus, actin-depolymerizing factor seems to be specifically directed toward actin filaments lacking tropomyosin.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 9-24 
    ISSN: 0886-1544
    Keywords: amoebae ; actinopoda ; heliozoa ; Actinophrys ; phagocytosis ; pseudopodia ; membrane ; extrusomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Two phases of prey capture by the heliozoon Actinophrys sol are documented by electron microscopy. The phases are those of prey adhesion to the predator and enclosure of the prey by the predator. Adherence is brought about by numerous small pieces of adhesive membrane produced by the predator at the site of prey contact. Some of the heliozoan extrusomes expel their contents at this time, but the significance of this event is unclear. Enclosure of the prey is effected by a funnelshaped pseudopodium. This is drawn over the prey by the action of the leading margin. The ultrastructural appearance of the cytoplasm of the leading margin differs from the rest of the cell, being homogeneous and finely filamentous. Both force and traction for the progression of the pseudopod are generated primarily at the tip. During the development of the funnel-pseudopod, extrusomes expand and fuse with each other and with the plasma membrane. Their investing membrane is thereby made available as food vacuole membrane.
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  • 69
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    Keywords: cell migration ; epithelial cell culture ; 2-deoxyglucose ; glycolysis ; microtrabecular lattice ; ATP ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using a line of epithelial cells (SCCA5) derived from a spontaneous rat carcinoma, the glucose analogue 2-deoxyglucose (2DG) has been shown by time-lapse cinemicrography to produce a cessation of motility by 1 hour that can be reversed by replacement of the 2DG, and does not occur in equivalent media with or without glucose or in 2DG-containing media with added pyruvate and citrate. The effect on the cells at the edge of an epithelial island is to prevent the formation of new lamellipodia and produce a progressive retraction and condensation of lamellipodia already present. This effect of 2DG on motility corresponds with a significant reduction in the level of ATP that is partially restored after 30 minutes in the recovery incubation. Only a slight reduction in protein synthesis occurs in the presence of 2DG. The external morphology and the cytoplasmic ground substance of the cells were studied by scanning electron microscopy and high voltage electron microscopy respectively. It was found that after incubation in 2DG for 1 hour the outline of the free edges of the cells was distorted resulting in redistribution of microvilli, condensation of cytoplasm into strands, and irregular projections from the edges of residual lamellipodia. The structure of the cytoplasmic ground substance in lamellipodia from cells incubated in 2DG for 3 hours was distinctly different from that in cells incubated for 3 hours in 2DG then recovered for 25 minutes, or in cells incubated in glucose-containing medium for 3 hours. In the 2DG-treated cells the lattice-like structure evident in critical-point-dried cells was condensed into short thick strands that terminated in bulbous ends, whereas in cells recovered for 25 minutes the lattice material was elongated and tapering and the interlattice space relatively expanded. The results obtained support the concept of modulation occuring in the structure of the microtrabecular lattice component of the cytoplasmic ground substance coincident with alterations in cell function and metabolic state.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 497-508 
    ISSN: 0886-1544
    Keywords: cilia ; electric motor control ; ciliates ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have studied the motor responses to membrane hyperpolarization of the marginal cirri in Stylonychia using voltage-clamp, high-speed cinematography, and computer-processing techniques. The cirri started beating when voltage step amplitudes rose beyond 5 mV. The power stroke was oriented toward the posterior cell and (hyperpolarizing motor activation). The frequency rose slightly during a voltage step, and decreased with similar rates for 100 ms following the step end. Amplitude and duration of the step tended to increase the motor response of the cirri. The late response declined exponentially. The time constant of the decay rose with the step amplitude. Among three response parameters tested (frequency, duration, number of cycles), the number of evoked ciliary cycles was best correlated with the amplitude of the hyperpolarization. Comparisons with the responses to depolarizing voltage steps reveal similarities in the relaxation of ciliary activity which appears to be uncoupled, in part, from the electric membrane events during the voltage stimulus.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 583-597 
    ISSN: 0886-1544
    Keywords: endocytic vesicles ; microtubules ; 10-nm filaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Ligand binding to cell surface receptors induces rapid internalization of ligandreceptor complexes by receptor mediated endocytosis. We have examined the intracellular movement of endocytic vesicles, induced by the lectin concanavalin A (Con A), in cultured rat ovarian granulosa cells using fluorescence and electron microscopy. Within 20 minutes of ligand treatment at 37°C, numerous Con A-containing endocytic vesicles form, which migrate to the cell center by 60 minutes. Double label fluorescence microscopy, using fluorescien-Con-A and rhodamine immunofluorescent staining of tubulin or vimentin, indicates that during vesicle migration microtubules and 10-nm filaments are altered in their organization. By 30 minutes, vesicles are associated with microtubule bundles, which subsequently collapse around the nucleus. Similarly, 10-nm filaments accumulate around the nucleus in conjunction with the perinuclear aggregation of endocytic vesicles. Electron microscopy of Con A-horseradish peroxidase-labeled cells demonstrates that endocytic vesicles fuse to form large receptosome-like structures during intracellular migration and these structures are associated with cytoplasmic microtubules and 10-nm filaments. Taxol, a drug that stabilizes microtubules, prevents endocytic vesicle translocation to the Golgi region. Nocodazole, which causes microtubule disassembly, results in the collapse of 10-nm filaments and the central aggregation of endocytic vesicles. The data indicate that the cytoskeleton participates in the directed intracellular movement of endocytic vesicles; the possible subcellular basis for this movement is discussed.
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    Cell Motility and the Cytoskeleton 2 (1982), S. 13-18 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 4 (1984) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 3 (1983), S. 699-719 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Topics: Biology , Medicine
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    Cell Motility and the Cytoskeleton 2 (1982), S. 59-65 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 4 (1984), S. 371-385 
    ISSN: 0886-1544
    Keywords: microtubules ; dynein ; tubulin ; cilia and flagella ; microtubule associated proteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Dynein, obtained from axonemes of Chlamydomonas, binds by both its A and B ends to microtubules assembled from twice cycled (2 ×) and purified (6S) brain tubulin as well as to microtubules in native spindles, thereby inducing microtubule crossbridging. The two ends of the dynein arm exhibit distinct binding characteristics for the different microtubule preparations. Greater than 99% of the dynein arms are bound exclusively by their B ends to microtubules assembled from 6S tubulin in the presence of dynein and decorated to saturation. In contrast, greater than 80% of the dynein arms are bound by both their A and B ends to and, therefore, crossbridge 6S microtubules that are only partially dynein decorated. Binding of the A end of the dynein arm to saturated 6S microtubules can be enhanced by destabilizing the binding of the B end upon addition of ATP and vanadate. These observations suggest that Chlamydomonas dynein arms can bind by their A ends to microtubules assembled from 6S tubulin only when the B ends of the arms either are not bound or are bound but do not occupy all available dynein binding sites. Dynein exhibits a slight preference for binding by its A end to microtubules assembled from 2 × tubulin and containing microtubule associated proteins (MAPs). Approximately 90% of the dynein arms crossbridge adjacent 2 × microtubles that are only partially decorated. But as saturation of these microtubules with dynein is approached, the majority of the arms are bound solely by their A ends, while a smaller percentage are bound by their B ends or by both their A and B ends. These studies indicate that the type of microtubule as well as the degree of saturation of the microtubule with dynein can determine whether microtubule crossbridging occurs.
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    Cell Motility and the Cytoskeleton 4 (1984), S. 431-441 
    ISSN: 0886-1544
    Keywords: dynein ; chromatophores ; permeabilization ; melanosomes ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Teleost chromatophores are filled with individual pigment granules that rapidly aggregate to the cell center or become dispersed throughout the cytoplasm in response to environmental stimuli. Microtubules appear to be required for pigment aggregation (movement toward the cell center), and recent findings have suggested that a dynein-like ATPase may participate in force production. Based on previous studies, however, it has been argued that pigment aggregation does not require energy directly, a view that supports the involvement of an elastic component in granule movement. To examine this point further, we have reinvestigated the energy requirements for pigment aggregation using both intact cells and detergent-permeabilized cell models of Fundulus melanophores. Poisons of oxidative phosphorylation, namely, 2,4 dinitrophenol and NaCN, reversibly inhibit melanosome aggregation in response to adrenaline. Inhibition of movement results directly from depletion of intracellular ATP, since pigment translocation can be reactivated in permeabilized cells by the addition of exogenous ATP to the lysis buffer. Non-hydrolyzable analogues, including β,γ-imidoadenosine-5′-triphosphate (AMPPNP), β,γ-methylene adenosine-5′-triphosphate (AMPPCP), and ATPγS, will not substitute for ATP in reactivation of movement. Similarly, other nucleotides such as ADP, AMP, GTP, CTP, and ITP, have limited ability to support melanosome aggregation in metabolically poisoned cells subjected to detergent lysis. ATP itself has no effect on intact cells. These results indicate that melanosome aggregation is ATP-dependent and energy-driven, and are consistent with a role for a force-transducing ATPase in particle movement.
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    Cell Motility and the Cytoskeleton 4 (1984) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 3 (1983) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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    Cell Motility and the Cytoskeleton 3 (1983), S. 431-438 
    ISSN: 0886-1544
    Keywords: myotendinous junction ; laminin ; type IV collagen ; heparan sulfate proteoglycan ; alpha actinin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The muscle-tendon junction of murine skeletal muscles has been analyzed by a variety of extraction techniques, by myosin subfragment-1 binding experiments, and by ultrastructural immunocytochemistry. The results indicate that the muscle-tendon junction is composed of four distinct domains: an intracellular domain, the internal lamina; a domain connecting the internal lamina with the lamina densa of the external lamina, the connecting domain; the lamina densa; and a domain which attaches the lamina densa to the collagen fibers, the matrix. Each of these domains is distinct with respect to position, three-dimensional organization, and molecular composition, and is therefore considered to have a unique role in the transmission of contractile force.
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  • 81
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    Cell Motility and the Cytoskeleton 3 (1983), S. 579-588 
    ISSN: 0886-1544
    Keywords: calcium-dependent protease ; contractile proteins ; platelets ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
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  • 82
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    Cell Motility and the Cytoskeleton 3 (1983), S. 609-622 
    ISSN: 0886-1544
    Keywords: erythrocyte membrane ; surface elastic shear modulus ; membrane viscosity ; hereditary disorders of blood ; membrane yield ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Measurements of the mechanical properties of the erythrocyte membrane provide a direct assessment of the proper function of its structural components. To assess the effects of alterations in molecular structure on membrane mechanical properties, measurements have been performed on cells from six individuals whose membranes contain inherited, biochemically characterized structural defects. Because the contribution of the memmbrane skeleton to the mechanical behavior of the membrane is most evident in shear deformation, mechanical experiments were performed to measure the material constants which characterize the response of the membrane to shear force resultants. The surface elastic shear modulus characterizes the elastic response of the membrane; the yield shear resultant is the maximum shear force resultant which the membrane can support elastically; and the plastic viscosity coefficient characterizes the rate of membrane deformation when the elastic limit has been exceeded.Generally, it was found that when the molecular defect is found to occur in a region of the skeleton which is stress-supporting, the maximum elastic strength of the membrane is reduced. However, the magnitude of the reduction can be quite different for membranes having similar or even identical defects. In some cases the differences can be attributed to the removal of the most fragile cells of the population by the spleen, but other results indicate that the biochemical description of the defects may be incomplete. These results emphasize the need for further refinements both in the biochemical characterization of membrane skeleton structure and in the description and measurement of membrane mechanical properties.
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  • 83
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    Cell Motility and the Cytoskeleton 3 (1983), S. 671-682 
    ISSN: 0886-1544
    Keywords: actin ; cytoskeleton ; membrane connections ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Recently, molecules highly related to erythrocyte spectrin have been identified in nonerythroid cells. Here we summarize our current understanding of these molecules and suggest a model for their organization. Significant differences exist between this family of proteins isolated from mammalian cells and avian cells, and this may explain the variability in antibody preparations as well as differences in peptide maps of these subunits which have been reported. We have prepared antibodies specific for the variant subunits of the spectrinlike proteins fodrin, spectrin, and TW260/240 and analyzed the distribution of these variant subunits in different chicken cell types as well as their developmental distribution in the intestine. The results suggest that fodrin is the general member of this family of proteins and can even coexist with other spectrinlike proteins in the same cells.
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  • 84
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    Cell Motility and the Cytoskeleton 30 (1995), S. 17-25 
    ISSN: 0886-1544
    Keywords: rotation ; twisting ; microtubule-dynein complex ; 22S dynein ; dynein-track ; ATP ; sliding ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Applying a new in vitro motility assay system for microtubules and 22S dynein, we recently reported on an ATP-induced extrusion of microtubules from microtubule-dynein α- and β-complexes [Mimori and Miki-Noumura, 1994:Cell Motil. Cytoskeleton 27:180-191]. In the present study, we prepared a γ-complex by copolymerizing porcine brain tubulin and Tetrahymena ciliary 22S dynein, and examined the ATP-induced microtubule movement from the γ-complex. The extrusion process appeared quite similar to that of the β-complex. The sliding velocity was 18.39 ± 2.20 m̈m/sec, which was a value comparable to that of trypsin-digested flagellar axonemes [Yano and Miki-Noumura, 1980:J. Cell Sci. 44:169-186]. Higher velocity may be due to a densely arranged dynein-track with the same polarity, which was detached from the γ-complex and absorbed in rows on a glass surface of the slide. Sometimes a free-floating microtubule in the perfusion chamber was observed riding and sliding on the dynein-track remaining on the slide after extrusion.Unexpectedly, we found that when the front part of the microtubule was fixed to a glass surface, a continuous sliding microtubule at the rear part on the dyneintrack often transformed into a left-handed helix, and subsequently a twisted helix with several turns. The helix formation may be due to some rigidity in the microtubule and a right-handed torque component in the sliding force of 22S dynein. The addition of ATP may release some distortion accumulated in the complex structure during copolymerization of tubulin and 22S dynein, inducing reverse rotation of the microtubule. © 1995 Wiley-Liss, Inc.
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  • 85
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    Keywords: trout ; spermatozoa ; ATP ; cAMP ; axoneme ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Live trout spermatozoa initiate flagellar motility for only a short period (30 sec at 18°C) during which their mean beat frequency decreases steadily from 60 to 20 Hz. Motility then stops abruptly. Investigations of the activation of movement in demembranated sperm points to cyclic-AMP being necessary for reactivation (half effect at 0.5μm) in some conditions. cAMP acts mainly by increasing the percentage of motile cells and not the beat frequency (BF) of the flagellar axoneme. Dibutyryl cAMP does not initiate movement or prolong motility of live sperm.The initiation of movement of demembranted trout sperm was investigated in various incubation conditions relative to previous phases of in vivo movement and to ATP concentration. In the absence of cAMP and in the presence of ATP lower than 25 μM, all sperm celi models were active with BF up to 15-20 Hz whatever their previous physiological condition. In contrast, at ATP concentrations above 100 μM, the fraction of active spermatozoa decreased proportionally but the BF of the active ones increased so that, at 1 mM ATP up to 20 μM restored activity to 100% sperm models with a similar BF of 65 Hz.At ATP concentrations higher than 25 μM, cAMP was necessary in a concentration dependent manner in the reactivation, but not in the demembranation meduim. This dependence was found to be unrelated to a previous in vivo phase of movement. The antagonistic effects of ATP vs. cAMP were tested at various concentrations of both nucleotides: the apparent affinity for cAMP, measured as the concentration restoring movement of 50% cell models, was decreased from 15 nM at 0.1 mM ATP to 0.5 μM at 1 mM ATP; conversely, the affinity for ATP, measured as the concentration giving rise to the half maximal beat frequency, was not significautly affected when the concentration of cAMP was raised to 0.5 mM. Preincubation with phosphodiesterase (PDE) resulted in motility of 100% of sperm models even at low ATP concentration. This tends to show that cAMP must be constantly present to sustain motility.
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  • 86
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    Cell Motility and the Cytoskeleton 31 (1995), S. 147-158 
    ISSN: 0886-1544
    Keywords: actin ; contact guidance ; microfilaments ; microtubules ; orientation ; cytochalasin ; colcemid ; taxol ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The role of the cytoskeleton and cell attachments in the alignment of baby hamster kidney fibroblasts to ridge and groove substratum topography was investigated using confocal scanning microscopy. This was carried out with normal cells and cells treated with the cytoskeleton modifiers cytochalasin D, colcemid, and taxol. Actin was localised with fluorescent phalloidin. Tubulin, Vinculin, and intracellular adhesion molecule-1 were visualised by indirect immunofluoresence. The spreading, elongation, and orientation of the cells after 24 h of culture in these conditions were measured on grooves of 5, 10, and 25 μm width and 0.5, 1, 2, and 5 μm depth. We have also observed events over the first 30 min of cell attachment. Five minutes after cell attachment, F-actin condensations were seen close to the intersection of groove wall and ridge top, that is, at a topographic discontinuity. The condensations were often at right angles to the groove edge and showed a periodicity of 0.6 μm. Vinculin arrangement at the early stages of cell spreading was similar to that of actin. Organisation of the microtubule system followed later, becoming obvious at about 30 min after cell plating. The Curtis and Clark theory (that cell react to topography primarily at lines of discontinuity in the substratum by actin nucleation) is supported by these results. The use of cytoskeletal poisons did not entirely abolish cell reaction to grooves. Colocemid increased cell spreading and reduced cell orientation and elongation. Cytochalasin D reduced cell spreading, orientation, and elongation. Taxol reduced cell elongation but did not affect cell spreading and orientation. We conclude that the aggregation of actin along groove/ridge boundaries is a primary driving event in determining fibroblast orientation on microgrooved substrata.
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  • 87
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    Cell Motility and the Cytoskeleton 30 (1995) 
    ISSN: 0886-1544
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 88
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    Cell Motility and the Cytoskeleton 30 (1995), S. 108-121 
    ISSN: 0886-1544
    Keywords: intercellular junctions ; desmosome ; assembly ; kinase ; phosphatase ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Desmosomes are one component of the intercellular junctional complex in epithelia. In cultures of epithelial cells, desmosome assembly can be regulated by modulating the calcium concentrations of the growth media. At present, very little is known about the intracellular signal transduction mechanisms that regulate desmosome assembly and disassembly in response to changing extracellular calcium concentrations. We have used inhibitors of protein kinases and phosphatases in a combined biochemical and morphological approach to analyze the role of protein phosphorylation in the assembly and disassembly of desmosomes in Madin-Darby canine kidney epithelial cells. Our results suggest that desmosomal proteins (desmoplakins I/II and desmoglein 1) are primarily phosphorylared on serine residues. Electron microscopic analyses of desmosome assembly upon induction of cell-cell contact, in the presence of protein kinase inhibitor, H-7, revealed an apparently normal assembly of desmosomes. However, complete disassembly of desmosomes was inhibited by H-7 upon removal of extracellular calcium. Under these conditions, although desmosomes split, desmosomal plaques and their associated cytokeratin filaments can not be internalized. In contrast, treatment of the cultures with okadaic acid (OA), an inhibitor of protein phosphatases, inhibited desmosome assembly but had no effect on disassembly. In addition, the inhibitory effect of okadaic acid on desmosome assembly was specific to this junction since we observed apparently normal tight junction and adherens junction in okadaic acid-treated cultures. These results suggest that via reversible protein phosphorylation involving both protein kinase and protein phosphatases. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 31 (1995), S. 196-206 
    ISSN: 0886-1544
    Keywords: gelsolin ; actin ; myofibrils ; immunofluorescence ; nebulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: We have investigated the binding of gelsolin to thin myofilaments in situ and their stability against severing. Differentiated myotubes from chicken skeletal muscle containing cross-striated myofibrils were permeabilized with Triton X-100 and incubated with gelsolin. Immunoflurorescence microcopy localized both endogenous and exogenous gelsolin in the I-Z-I-regions of the sarcomers. The staining pattern suggested a binding of the exogenous gelsolin along the entire length of the thin filaments. This binding was Ca2+ dependent, but gelsolin was not removed after subsequent addition of EGTA. The fluorescence staining for actin remained unchanged after gelsolin incubation, indicating that thin filaments in cross-striated myofibrils were resistant to the severing action of gelsolin, in contrast to the microfilaments in stress fibers. After extraction of the permeabilized cells with high ionic strength to remove tropomyosin and myosin, gelsolin stell bound along the entire thin filament and the actin pattern also remained unchanged. After Triton X-100 permeabilization and high ionic strength extraction, the giant protein nebulin was found to be still present as a myofibrillar component. Gelsolin treatment after high salt extraction affected neither actin nor nebulin in the thin filaments. We therefore conclude that nebulin confers the gelsolin resistance to the sarcomeric actin filaments.
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  • 90
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    Cell Motility and the Cytoskeleton 31 (1995), S. 207-214 
    ISSN: 0886-1544
    Keywords: flagella ; Chlamydomonas ; mutant ; high-frequency vibration ; nanometer-scale measurement ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The flageliar axoneme of the mutant pf18 lacking the central pair does not beat, but undergoes a nanometer-scale, high-frequency oscillation (hyper-oscillation) in the presence of ATP [Yagi et al., 1994: Cell Motil, Cytoskeleton 29:177-185]. The present study demonstrates that the amplitude of the hyper-oscillation increases significantly in the simultaneous presence of ATP and ADP. In addition, the hyper-oscillation under these conditions sometimes takes on an exceptionally simple asymmetric pattern, in which the maximal shearing velocity exceeds 50 μm/sec, much higher than the maximal velocity of ordinary dynein-microtubule sliding. The asymmetric oscillation thus appears to be at least partly driven by an internal elastic force. Its amplitude suggests that the axoneme has an elastic component that can be stretched by as long as 0.1 μm. Analyses of the asymmetric pattern further suggests that the axonemal dyneins have a tendency to attach to and detach from the doublets cooperatively and that the mechanochemical cycle of dynein has an inherent refractory period of about 2 msec, during which dynein cannot interact with microtubules.
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  • 91
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    Cell Motility and the Cytoskeleton 31 (1995), S. 225-240 
    ISSN: 0886-1544
    Keywords: cell-substratum adhesion ; lamellar contractility ; locomotion ; silicone rubber ; traction forces ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A means of determining quantitative maps of the tractions exerted by locomoting cells on a substratum has been developed. This method is similar to the Harris silicone substratum assay [Harris et al., 1980: Science 208:177-179], but uses an improved non-wrinkling film that deforms more predictably in response to traction forces. The method also utilizes a mathematical analysis of rubber deformation to produce the final map of the distribution of tractions. The resulting maps consistently showed that fish keratocytes exert a steady-state “pinching” on the substratum, perpendicular to the cell's direction of locomotion. No significant rearward tractions were detected at or near the front edge of the cell. Likewise, no significant forward tractions associated with peeling of adhesions were found at the back of the cell. A second assay uses deflection of a lightly attached glass microneedle to measure the total force exerted by locomoting cells. Forces of approximately 4.5 × 10-3 dyn were required to “stall” locomoting keratocytes. The implications of these findings for cell movement are discussed.
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  • 92
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    Keywords: microfilamentous cytoskeleton ; actin binding proteins ; actin polymerization ; annealing ; non-muscle cells ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Gelsolin, a Ca++ activated, 90 kd actin binding protein, can regulate actin polymerization in polymorphonuclear leukocytes (PMNs) via severing of filaments to dissolve gels or by capping of filament ends to limit polymerization. In Triton-lysed PMNs, 30% of gelsolin is bound to the Triton-soluble F-actin (TSF) pool and none is bound to the Triton-insoluble F-actin (TIF) pool. Calcium-activated PMNs exhibit concurrent temporal and quantitative TIF growth and TSF and total F-actin loss. To determine if gelsolin plays a role in regulating TSF pool size, we monitored gelsolin-actin interactions and TIF, TSF and G-actin content at 5 second intervals in PMNs activated with the calcium ionophore, ionomycin. Actin pools were measured by NBDphallacidin binding and by gel scans and expressed relative to basal; gelsolin-actin interactions were measured as change in the amount of EGTA-resistant gelsolin:actin (G:A) complexes and by immunoblot quantification of gelsolin in actin pools. In basal PMNs, 33% of PMN gelsolin is bound in 1:1 EGTA-resistant G:A complexes and TSF and TIF retain 30% and 0% of PMN gelsolin, respectively. By 20 seconds after ionomycin addition, TSF decreases, TIF increases and a fraction of gelsolin repartitions from the TSF to the TIF pool. At maximum change (60 seconds), total F-actin (TIF + TSF) and TSF decrease and TIF increases by 25%; gelsolin is bound to both TSF and TIF (35% of total gelsolin in each pool), and 1:1 EGTA-resistant G:A complexes increase from 33% to 70%. No changes occur in cells activated by ionomycin in the absence of Ca++. The data show Ca++ activated TIF growth and TSF loss are temporally and quantitatively associated with an increase in the percent of gelsolin bound to actin and the translocation of gelsolin from TSF to TIF. This is unique, since no other PMN activator is known to repartition gelsolin into TIF actin. Further, the Ca++ activated initial increase in TIF concurrent with a fall in TSF without a change in total F-actin or G-actin content suggest that TIF grows initially only by TSF annealing/cross-linking to TIF. Gelsolin may regulate these events. © 1995 Wiley-Liss, Inc.
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  • 93
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    Cell Motility and the Cytoskeleton 30 (1995), S. 146-152 
    ISSN: 0886-1544
    Keywords: zinc-sheets ; macrotubes ; kinesin ; electron microscopy ; microtubules ; tubulin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Moving along a microtubule, kinesin follows a course parallel to the protofilaments; but it is not known whether kinesin binds exclusively on a single protofilament. The presence of zinc during tubulin polymerization induces sheets where neighboring protofilaments are antiparallel. If kinesin could support the motility of these zinc-sheets, then the binding site for a kinesin molecule would be limited to a single protofilament.Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] reported that kinesin moves along zinc-sheets. We found that zinc-sheets grown under their conditions often had a microtubule-like structure along one edge. We confirmed the possibility that the motility observed by Kamimura and Mandelkow [1992: J. Cell Biol. 118:865-75] is attributed to the microtubule-like structure rather than the zinc-sheet.To resolve the question of whether kinesin can recognize an antiparallel protofilament lattice, we investigated the kinesin-mediated motility of zinc-macrotubes. At higher free zinc concentrations, zinc-sheets roll up as macrotubes, free of edges. In the presence of 10 m̈M taxol and 100 nM free Zn2+ at pH 6.8, the samples were shown by electron microscopy to contain only macrotubes. Under these buffer conditions, kinesin could bind strongly to axonemal doublets in the presence of AMP-PNP, and generate motility in the presence of ATP, but kinesin did not bind to nor move the macrotubes. This shows that kinesin cannot bind efficiently to nor move on the anti-parallel lattice; it is possible (though not necessary) that the groove between two parallel protofilaments is required for kinesin's motility. © 1995 Wiley-Liss, Inc.
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  • 94
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    Cell Motility and the Cytoskeleton 30 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 95
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    Cell Motility and the Cytoskeleton 30 (1995), S. 153-163 
    ISSN: 0886-1544
    Keywords: colchicine binding site ; MTC ; cod microtubules ; bovine microtubules ; MAPs ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Isolated microtubules from cod (Gadus morhua) are apparently more stable to colchicine than bovine microtubules. In order to further characterize this difference, the effect of the colchicine analogue 2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cyclo heptatrien-1-one (MTC) was studied on assembly, as measured by turbidity and sedimentation analysis, and on polymer morphology. MTC has the advantage to bind fast and reversible to the colchicine binding site of tubulin even at low temperatures. It was found to bind to one site in cod brain tubulin, with affinity (6.5 ± 1.5) × 105M 1at both low or high temperature, similarly to bovine brain tubulin. However, the effect of the binding differed. At substoichiometric concentrations of MTC bovine brain microtubule assembly was almost completely inhibited, while less effect was seen on the mass of polymerized cod microtubule proteins. A preformed bovine tubulin-colchicine complex inhibited the assembly of both cod and bovine microtubules at substoichiometric concentrations, but the effect on the assembly of cod microtubules was less. At higher concentrations (5 × 10-5 to 1 × 10-3M), MTC induced a large amount of cold-stable spirals of cod proteins, whereas abnormal polymers without any defined structure were formed from bovine proteins. Spirals of cod microtubule proteins were only formed in the presence of microtubule associated proteins (MAPs), indicating that the morphological effect of MTC can be modulated by MAPs. The effects of colchicine and MTC differed. At 10-5M colchicine no spirals were formed, while at 10-4M and 10-3M, a mixture of spirals and aggregates was found. The morphology of the spirals differed both from vinblastine spirals and from the spirals previously found when cod microtubule proteins polymerize in the presence of high Ca2concentrations. The present data show that even if the colchicine binding site is conserved between many different species, the bindings have different effects which seem to depend on intrinsic properties of the different tubulins. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 31 (1995), S. 298-306 
    ISSN: 0886-1544
    Keywords: Drosophila ; nurse cells ; oocyte ; microfilaments ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The distribution of microfilaments in Drosophila egg chambers stained with rhodamine (Rh)-conjugated phallcidin was studied by laser scanning confocal microscopy and transmission electron microscopy. These techniques revealed new details in the pattern of microfilament localization. We observed in stage 1-3 egg chambers accumulation of filamentous actin in the oocyte cytoplasm between the ring canals connecting the oocyte with adjacent nurse cells. Starting from stages 6-7 short microfilament bundles arranged in basket-like structures were associated with the side of the ring canals facing the nurse cell cytoplasm. We also observed a dramatic decrease in the actin network associated with the cortex of the oocyte in stage 10. During stage 10B the nurse cell cytoplasm was crossed by radial actin bundles that showed a sarcomeric-like cross striation after Rh-phalloidin staining. The ring canals also did not uniformly stain but showed a punctate labeling. The implications of the actin cytoskeleton during oocyte growth are discussed. © 1995 Wiley-Liss, Inc.
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  • 97
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    Keywords: neurofilament ; phosphorylation ; cdk5 ; cdc2 ; cyclin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Using dephosphorylated neurofilament (NF) proteins as substrates, the kinase with a higher activity for in the dephosphorylated NF-H than the phosphorylated form of NF-H was searched for in the porcine brain extract. Most NF-H kinase activity in the brain extract pelleted with microtubules. The NF-H kinase purified from a high salt extract of the microtubule pellets was composed of cdk5 and a 26 kDa protein, a fragment of the 35 kDa regulatory subunit of cdk5. In contrast to the association of the active kinase with microtubules, each of uncomplexed cdk5 and the 35 kDa regulatory subunit was differently distributed in the supernatant fraction and the pellet, respectively, by ultracentrifugation of the brain extract. Dephosphorylated forms of NF-H and NF-M became reactive to antibodies recoginizing in vivo phosphorylation sites (SM131, 34, and 36, JJ31 and 51) by phosphorylation with cdk5/p26. cdk5/p26 showed similar enzymatic properties to p34cdc2/cyclin B kinase; the substrate specificity and inhibition by a p34cdc2 kinase specific inhibitor, butyrolactone I. However, p34cdc2/cyclin B kinase was distinguished from cdk5/p26 by its binding to p13suc1 protein and by its reactivity to anti-p34cdc2 antibodies. In spite of similar enzymatic properties of cdk5/p26 and p34cdc2/cyclin B kinase, cdk5/26 did not display M-phase promoting activity when assayed with a cell-free system of Xenopus egg extract. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995), S. 133-135 
    ISSN: 0886-1544
    Keywords: cytoplasmic dynein ; motor domain ; mutational analysis ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The highly conserved lysine residue in the putative hydrolytic ATP-binding motif of the yeast cytoplasmic dynein heavy chain was replaced with leucine. The mutation was generated by a two-stage transformation method designed for genomic site-directed mutagenesis. Preliminary observations show that the effects of this alteration on the cellular roles of dynein are indistinguishable from those of a disruption mutation in which the entire motor domain is not expressed. © 1995 Wiley-Liss, Inc.
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    Cell Motility and the Cytoskeleton 32 (1995) 
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    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
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  • 100
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    Cell Motility and the Cytoskeleton 32 (1995), S. 233-243 
    ISSN: 0886-1544
    Keywords: caldesmon ; over-expression ; cell cycle ; motility ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Caldesmon is an actin, calmodulin, tropomyosin, and myosin binding protein implicated in the regulation of actomyosin interactions. We have invesigated the effect of overexpression of the higher molecular weight smooth muscle isoform of caldesmon on mouse L cell physiology. Mouse L(TK-) cell were transfected stably with plasmids carrying the TK+ gene and a full length human smooth muscle caldesmon cDNA under control of the adenovirus major late promoter. Two clones displaying four and eight times the level of the endogenous mouse high molecular weight caldesmon were isolated. These cells acquire a distinct phenotype characterized by an altered morphology, including an increased number of processes and larger area due to enhanced cell spreading, and a significantly slower growth rate than that of untransfected control cells, or cells transfected with the TK+ gene alone. The majority of the overexpressed caldesmon appears to be active and localized on cytoskeleton structures as determined by detergent lysis. Immuno-fluorescence analysis of the clones revealed that the caldesmon is localized as punctate staining on stress-fibers and in membrane ruffles. The immunofluores-cence images suggest that caldesmon overexpressing cells have more total filaments than control cells. The effects of excess caldesmon on cell mobility are ambiguous: one clone displayed increased motility compared to the control, while the motility of the second clone was decreased relative to the control. © 1995 Wiley-Liss, Inc.
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