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Localization of tubulin in the mitotic apparatus of mammalian cells by immunofluorescence and immunoelectron microscopy (1977)

Pepper, Daniel A. ; Brinkley, B. R.

Springer

Chromosoma 60 (1977), S. 223-235

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ISSN: 1432-0886

Source: Springer Online Journal Archives 1860-2000

Topics: Biology , Medicine

Notes: Abstract Antitubulin antibody was used as an immunofluorescent and immunoelectron microscopic probe to localize tubulin in components of the mitotic apparatus of rat kangaroo (strain PtK1) cells in vitro. In addition to the detection of tubulin in the spindle microtubules and centrioles, other structures were found to display specific staining including kinetochores, amorphous pericentriolar material and small virus-like particles associated with the centrioles. The kinetochores consisted of a densely stained outer layer about 400 Å thick which is separated from an inner layer of the same dimension by a lightly staining middle layer. Microtubules were primarily associated with the outermost plate of the kinetochore but tubulin was uniformly distributed in both outer and inner plates. Colcemid treatment prevented the assembly of spindle microtubules and resulted in specific alterations of the kinetochore but failed to diminish the staining of the kinetochores. These observations suggest that tubulin molecules may comprise an important structural component of the kinetochore.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1007/BF00329772

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Tubulin nucleation and assembly in mitotic cells: Evidence for nucleic acids in kinetochores and centrosomes (1980)

Pepper, Daniel A. ; Brinkley, B. R.

New York, NY : Wiley-Blackwell

Cell Motility and the Cytoskeleton 1 (1980), S. 1-15

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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.

Additional Material: 45 Ill.

Type of Medium: Electronic Resource

URL: http://dx.doi.org/10.1002/cm.970010102

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