ALBERT

Notes: We describe an instrument designed to monitor molecular motions in multiphasic, weakly fluorescent microscopic systems. It combines synchrotron radiation, a low irradiance polarized microfluorimeter, and an automated, multiframing, single-photon-counting data acquisition system, and is capable of continually accumulating subnanosecond resolved anisotropy decays with a real-time resolution of about 60 s. The instrument has initially been built to monitor ligand–receptor interactions in living cells, but can equally be applied to the continual measurement of any dynamic process involving fluorescent molecules, that occurs over a time scale from a few minutes to several hours. As a particularly demanding demonstration of its capabilities, we have used it to monitor the environmental constraints imposed on the peptide hormone epidermal growth factor during its endocytosis and recycling to the cell surface in live cells. © 1998 American Institute of Physics.

Notes: We describe an instrument based on the novel combination of synchrotron radiation, a high sensitivity time-resolved microfluorimeter, and a multiframe single photon counting data acquisition system. This instrument has been designed specifically to measure kinetic events in live cells using fluorescence resonance energy transfer, and is capable of rapidly collecting multiple consecutive decay profiles from a small number of fluorophores. The low irradiance on the samples (〈10 mW/cm2) greatly reduces probe photobleaching and specimen photodamage during prolonged exposures. The Daresbury Synchrotron Radiation Source provides fully wavelength tunable light pulses that have a full width half-maximum of 160 ps at a repetition rate of 3.125 MHz, with the high temporal stability required for continuous measurements over periods of hours. A very low limit of detection (〈104 molecules/mW/cm2) is accomplished by combining a high-gain single photon counting detection system with a low fluorescence background optical layout. The latter is achieved by the inclusion of collimating optics, a reflecting objective, and a specially designed beam stop situated in the epi-fluorescence light-path. A typical irradiance of 8 mW/cm2 on a sample of ∼105 fluorescein molecules gives, in under 20 s, a fluorescence decay profile with a peak height of 104 counts, over 400 channels, and a signal to background ratio better than 40. The data acquisition system has been developed to have a real-time time-resolved fluorescence collection capability (denoted as TR2) so that fluorescence lifetime data can be continually collected throughout a changing process. To illustrate the potential of this instrument, we present the results of a TR2 experiment in which lifetime measurements of fluorescence resonance energy transfer are used to monitor the degree of clustering of epidermal growth factor receptors during endocytosis, over a period of about 1 h, with a 5 s resolution. © 1996 American Institute of Physics.

Notes: Summary Results were obtained from contracting frog muscles by collecting high quality time-resolved, two-dimensional, X-ray diffraction patterns at the British Synchrotron Radiation Source (SERC, Daresbury, Laboratory). The structural transitions associated with isometric tension generation were recorded under conditions in which the three-dimensional order characteristic of the rest state is either present or absent. In both cases, new layer lines appear during tension generation, subsequent to changes from activation events in the filaments. Compared with the ‘decorated’ actin layer lines of the rigor state, the spacings of the new layer lines are similar whereas their intensities differ substantially. We conclude that in contracting muscle an actomyosin complex is formed whose structure is not like that in rigor, although it is possible that the interacting sites are the same. Transition from rest to plateau of tension is accompanied by approximately 1.6% increase in the axial spacing of the myosin layer lines. This is explained as arising from the axial disposition of the interacting myosin heads in the actomyosin complex. Model calculations are presented which support this view. We argue that in a situation where an actomyosin complex is formed during contraction, one cannot describe the diffraction features as being either thick or thin filament based. Accordingly, the layer lines seen during tension generation are referred to as actomyosin layer lines. It is shown that these layer lines can be indexed as submultiples of a minimum axial repeat of approximately 218.7 nm. After lattice disorder effects are taken into account, the intensity increases on the 15th and 21st AM layer lines at spacings of approximately 14.58 and 10.4 nm respectively, show the same time course as tension rise. However, the time course of the intensity increase of the other actomyosin layer lines and of the spacing change (which is the same for both phenomena) shows a substantial lead over tension rise. These findings suggest that the actomyosin complex formed prior to tension rise is a non-tension-generating state and that this is followed by a transition of the complex to a tension-generating state. The intensity increase in the 15th actomyosin layer line, which parallels tension rise, can be accounted for assuming that in the tension-generating state the attached heads adopt (axially) a more perpendicular orientation with respect to the muscle axis than is seen at rest or in the non-tension-generating state. This suggests the existence of at least two structurally distinct interacting myosin head conformations. The results of comparing the meridional intensities between the myosin layer lines at rest and the actomyosin layer lines at the plateau of tension (measured to a resolution of approximately 2.6 nm) are interpreted to indicate that the majority of the myosin heads in the actomyosin complex do not perform random axial rotations with a mean value greater than approximately 3.0 nm. From this we conclude that the extent of axial order in the interacting heads must be at least as high as is that of resting heads.

Notes: Summary Using the facilities at the Daresbury Synchrotron Radiation Source, meridional diffraction patterns of muscles at ca 8°C were recorded with a time resolution of 2 or 4 ms. In isometric contractions tetanic peak tension (P 0) is reached in ca 400 ms. Under such conditions, following stimulation from rest, the timing of changes in the major reflections (the 38.2 nm troponin reflection, and the 21.5 and 14.34/14.58 nm myosin reflections) can be explained in terms of four types of time courses: K 1, K 2, K 3 and K 4. The onset of K 1 occurs immediately after stimulation, but that of K 2, K 3 and K 4 is delayed by a latent period of ca 16 ms. Relative to the end of their own latent periods the half-times for K 1, K 2, K 3 and K 4 are 14–16, 16, 32 and 52 ms, respectively. In half-times, K 1, K 2, K 3 lead tension rise by 52, 36 and 20 ms, respectively. K 4 parallels the time course of tension rise. From an analysis of the data we conclude that K 1 reflects thin filament activation which involves the troponin system; K 2 arises from an order-disorder transition during which the register between the filaments is lost; K 3 is due to the formation of an acto-myosin complex which (at P 0) causes 70% or more of the heads to diffract with actin-based periodicities; and K 4 is caused by a change in the axial orientation of the myosin heads (relative to thin filament axis) which is estimated to be from 65–70° at rest to ca 90° at P 0. Isotonic contraction experiments showed that during shortening under a load of ca 0.27 P 0, at least 85% of the heads (relative to those forming an acto-myosin complex at P 0) diffract with actin-based periodicities, whilst their axial orientation does not change from that at rest. During shortening under a negligible load, at most 5–10% of the heads (relative to those forming an acto-myosin complex at P 0) diffract with actin-based periodicities, and their axial orientation also remains the same as that at rest. This suggests that in isometric contractions the change in axial orientation is not the cause of active tension production, but rather the result of it. Analysis of the data reveals that independent of load, the extent of asynchronous axial motions executed by most of the cycling heads is no more than 0.5–0.65 nm greater than at rest. To account for the diffraction data in terms of the conventional tilting head model one would have to suppose that a few of the heads, and/or a small part of their mass perform the much larger motions demanded by that model. Therefore we conclude either that the required information is not available in our patterns or that an alternative hypothesis for contraction has to be developed.