Behaviour of microtubules and actin filaments in living Drosophila embryos

We describe the preparation of novel fluorescent derivatives of rabbit muscle actin and bovine tubulin, and the use of these derivatives to study the behaviour of actin filaments and microtubules in living Drosophila embryos, in which the nuclei divide at intervals of 8 to 21 min. The fluorescently labelled proteins appear to function normally in vitro and in vivo, and they allow continuous observation of the cytoskeleton in living embryos without perturbing development. By coinjecting labelled actin and tubulin into the early syncytial embryo, the spatial relationships between the distinct filament networks that they form can be followed second by second. The dynamic rearrangements of actin filaments and microtubules observed confirms and extends results obtained from previous studies, in which fixation techniques and specific staining were used to visualize the cytoskeleton in the Drosophila embryo. However, no tested fixation method produces an exact representation of the in vivo microtubule distribution.     

Actin based motility on retraction fibers in mitotic PtK2 cells.

When PtK2 cells round up in mitosis they leave retraction fibers attached between the substrate and the cell body. Retraction fibers and the region where they meet the cell body are rich in actin filaments as judged by phalloidin staining and electron microscopy. Video microscopy was used to study actin dependent motile processes on retraction fibers. Small, phase-dense nodules form spontaneously on the fibers, and move in to the cell body at a rate of 3 microns/minute. As they move in they increase progressively in phase-density. This movement appears to be related to actin dependent centripetal movement which has been previously studied in lamellipodia. Despite its generality, the mechanism of such movement is unknown, and retraction fibers present some special advantages for its study. Cytochalasin treatment causes nodules to stop moving and dissolve. Withdrawal of the drug causes them to reform and start moving. Surprisingly, movement after cytochalasin withdrawal was often outward, indicating a local reversal of cortical polarity. After a few minutes correct polarity is reestablished by a global control mechanism. The implications of these observations for the mechanism and polarity of actin dependent motility is discussed.     

Direct observation of steady-state microtubule dynamics

Different types of unusual dynamic behavior have been reported for steady-state microtubules. While almost all earlier reports relied on kinetic measurements of bulk polymerization, we have directly visualized the steady-state addition of subunits to individual microtubules through the use of tubulin derivitized with biotin. Biotinylated tubulin was used both as an internal "seed" for polymerization and as a marker for assembly onto the ends of microtubules composed of purified tubulin. Biotinylated segments were distinguished from unmodified tubulin by double-label immunofluorescence. Microtubule lengths, number concentrations, and segment lengths have been monitored with time at steady state under two buffer conditions. The results indicate that the microtubule steady state under these conditions is a balance between a majority of slowly growing microtubules and a minority of rapidly depolymerizing ones as described by the "dynamic instability" model (Mitchison T., and M. Kirschner, 1984, Nature (Lond.)., 312:232-242). Microtubules show no evidence of treadmilling; instead most show progressive growth off both ends at steady state. Although solvent conditions markedly influence the growth rates, qualitatively the behavior is unchanged.     

Assembly properties of fluorescein-labeled tubulin in vitro before and after fluorescence bleaching

Brain tubulin has been conjugated with dichlorotriazinyl- aminofluorescein (DTAF) to form a visualizable complex for the study of tubulin dynamics in living cells. By using several assays we confirm the finding of Keith et al. (Keith, C. H., J. R. Feramisco, and M. Shelanski, 1981, J. Cell Biol., 88:234-240) that DTAF-tubulin polymerizes like control tubulin in vitro. The fluorescein moiety of the complex is readily bleached by the 488-nm line from an argon ion laser. When irradiations are performed over short times (less than 1 s) and in the presence of 2 mM glutathione, a mixture of DTAF-tubulin and control protein (as occurs after microinjection of the fluorescent conjugate into living cells) will retain full polymerization activity. Slow bleaching (approximately 5 min) or bleaching without glutathione promotes formation of covalent cross-links between neighboring polypeptides and kills the polymerization activity of DTAF-tubulin, including some molecules that are neither cross-linked nor bleached. Even under conditions that damage DTAF-tubulin, however, DTAF- microtubules are not destroyed by bleaching. They will continue to elongate by addition of DTAF-tubulin subunits to their free ends, and they neither bind nor exchange subunits along their lateral surfaces. These results suggest that DTAF-tubulin is a suitable analog for tubulin, both in studies of protein incorporation and for investigations of fluorescence redistribution after photobleaching.     

Microtubule flux in mitosis is independent of chromosomes, centrosomes, and antiparallel microtubules.

We investigated the mechanism of poleward microtubule flux in the mitotic spindle by generating spindle subassemblies in Xenopus egg extracts in vitro and assaying their ability to flux by photoactivation of fluorescence and low-light multichannel fluorescence video-microscopy. We find that monopolar intermediates of in vitro spindle assembly (half-spindles) exhibit normal poleward flux, as do astral microtubule arrays induced by the addition of dimethyl sulfoxide to egg extracts in the absence of both chromosomes and conventional centrosomes. Immunodepletion of the kinesin-related microtubule motor protein Eg5, a candidate flux motor, suggests that Eg5 is not required for flux. These results suggest that poleward flux is a basic element of microtubule behavior exhibited by even simple self-organized microtubule arrays and presumably underlies the most elementary levels of spindle morphogenesis.     

THE DIRECT ISOLATION OF THE MITOTIC APPARATUS

A method for isolating the mitotic apparatus from dividing sea urchin eggs without the use of ethyl alcohol or of detergents is described. In the present method, the eggs are dispersed directly in a medium containing 1 M (to 1.15 M) sucrose, 0.15 M dithiodiglycol, and 0.001 M Versene at pH 6, releasing the visibly intact mitotic apparatus. The method is designed for studies of enzyme activities, lipid components, and the variables affecting the stability of the apparatus.