How microtubules get fluorescent speckles.

The dynamics of microtubules in living cells can be seen by fluorescence microscopy when fluorescently labeled tubulin is microinjected into cells, mixing with the cellular tubulin pool and incorporating into microtubules. The subsequent fluorescence distribution along microtubules can appear "speckled" in high-resolution images obtained with a cooled CCD camera (Waterman-Storer and Salmon, 1997. J. Cell Biol. 139:417-434). In this paper we investigate the origins of these fluorescent speckles. In vivo microtubules exhibited a random pattern of speckles for different microtubules and different regions of an individual microtubule. The speckle pattern changed only after microtubule shortening and regrowth. Microtubules assembled from mixtures of labeled and unlabeled pure tubulin in vitro also exhibited fluorescent speckles, demonstrating that cellular factors or organelles do not contribute to the speckle pattern. Speckle contrast (measured as the standard deviation of fluorescence intensity along the microtubule divided by the mean fluorescence intensity) decreased as the fraction of labeled tubulin increased, and it was not altered by the binding of purified brain microtubule-associated proteins. Computer simulation of microtubule assembly with labeled and unlabeled tubulin showed that the speckle patterns can be explained solely by the stochastic nature of tubulin dimer association with a growing end. Speckle patterns can provide fiduciary marks in the microtubule lattice for motility studies or can be used to determine the fraction of labeled tubulin microinjected into living cells.     

Direct visualization of microtubule flux during metaphase and anaphase in crane-fly spermatocytes

Microtubule flux in spindles of insect spermatocytes, long-used models for studies on chromosome behavior during meiosis, was revealed after iontophoretic microinjection of rhodamine-conjugated (rh)-tubulin and fluorescent speckle microscopy. In time-lapse movies of crane-fly spermtocytes, fluorescent speckles generated when rh-tubulin incorporated at microtubule plus ends moved poleward through each half-spindle and then were lost from microtubule minus ends at the spindle poles. The average poleward velocity of approximately 0.7 microm/min for speckles within kinetochore microtubules at metaphase increased during anaphase to approximately 0.9 microm/min. Segregating half-bivalents had an average poleward velocity of approximately 0.5 microm/min, about half that of speckles within shortening kinetochore fibers. When injected during anaphase, rhtubulin was incorporated at kinetochores, and kinetochore fiber fluorescence spread poleward as anaphase progressed. The results show that tubulin subunits are added to the plus end of kinetochore microtubules and are removed from their minus ends at the poles, all while attached chromosomes move poleward during anaphase A. The results cannot be explained by a Pac-man model, in which 1) kinetochore-based, minus end-directed motors generate poleward forces for anaphase A and 2) kinetochore microtubules shorten at their plus ends. Rather, in these cells, kinetochore fiber shortening during anaphase A occurs exclusively at the minus ends of kinetochore microtubules.     

Tubulin dynamics in cultured mammalian cells

Bovine neurotubulin has been labeled with dichlorotriazinyl- aminofluorescein (DTAF-tubulin) and microinjected into cultured mammalian cells strains PTK1 and BSC. The fibrous, fluorescence patterns that developed in the microinjected cells were almost indistinguishable from the pattern of microtubules seen in the same cells by indirect immunofluorescence. DTAF-tubulin participated in the formation of all visible, microtubule-related structures at all cell cycle stages for at least 48 h after injection. Treatments of injected cells with Nocodazole or Taxol showed that DTAF-tubulin closely mimicked the behavior of endogenous tubulin. The rate at which microtubules incorporated DTAF-tubulin depended on the cell-cycle stage of the injected cell. Mitotic microtubules became fluorescent within seconds while interphase microtubules required minutes. Studies using fluorescence redistribution after photobleaching confirmed this apparent difference in tubulin dynamics between mitotic and interphase cells. The temporal patterns of redistribution included a rapid phase (approximately 3 s) that we attribute to diffusion of free DTAF-tubulin and a second, slower phase that seems to represent the exchange of bleached DTAF-tubulin in microtubules with free, unbleached DTAF- tubulin. Mean half times of redistribution were 18-fold shorter in mitotic cells than they were in interphase cells.     

Dynamics of a fluorescent calmodulin analog in the mammalian mitotic spindle at metaphase

We have compared the exchange kinetics of fluorescein-labeled calmodulin and tubulin in the spindles of living mitotic cells at metaphase. Cultured mammalian cells in early stages of mitosis were microinjected with labeled calmodulin or tubulin and returned to an incubator to allow equilibration of the fluorescent protein with the endogenous protein pools. Calmodulin becomes concentrated in the mitotic spindle, and treatments with inhibitors of tubulin assembly show that this concentration is dependent on the presence of microtubules. The steady-state exchange rates of both tubulin and calmodulin were measured by an analysis of fluorescence redistribution after photobleaching (FRAP), using cells pre-equilibrated to either 26 +/- 2 degrees C or 36 +/- 2 degrees C. A pulse of laser light focused to a 5-microns diameter column was used to destroy the fluorescence at one pole of a metaphase mitotic spindle. Ratios of fluorescence intensity from the two half-spindles and from the two polar regions were calculated for each image in a post-bleach time series to determine the rates and extents of FRAP. For tubulin, we confirm earlier observations concerning the temperature dependence of the extent of FRAP, but our data do not show a significant temperature dependence for the rate of FRAP. We hypothesize that the reduced extent of tubulin FRAP at the lower temperatures is a result of microtubules that are stable to depolymerization at 26 degrees C and are thus less likely to exchange subunits. Calmodulin's FRAP, however, does not exhibit any of the temperature dependence observed with fluorescent tubulin. At 26 +/- 2 degrees C calmodulin exchanges rapidly with the relatively stable population of microtubules, suggesting that calmodulin is bound, either directly or indirectly, to microtubule walls.     

Microtubules of the kinetochore fiber turn over in metaphase but not in anaphase

In previous work we injected mitotic cells with fluorescent tubulin and photobleached them to mark domains on the spindle microtubules. We concluded that chromosomes move poleward along kinetochore fiber microtubules that remain stationary with respect to the pole while depolymerizing at the kinetochore. In those experiments, bleached zones in anaphase spindles showed some recovery of fluorescence with time. We wished to determine the nature of this recovery. Was it due to turnover of kinetochore fiber microtubules or of nonkinetochore microtubules or both? We also wished to investigate the question of turnover of kinetochore microtubules in metaphase. We microinjected cells with x- rhodamine tubulin (x-rh tubulin) and photobleached spindles in anaphase and metaphase. At various times after photobleaching, cells were detergent lysed in a cold buffer containing 80 microM calcium, conditions that led to the disassembly of almost all nonkinetochore microtubules. Quantitative analysis with a charge coupled device image sensor revealed that the bleached zones in anaphase cells showed no fluorescence recovery, suggesting that these kinetochore fiber microtubules do not turn over. Thus, the partial fluorescence recovery seen in our earlier anaphase experiments was likely due to turnover of nonkinetochore microtubules. In contrast fluorescence in metaphase cells recovered to approximately 70% the control level within 7 min suggesting that many, but perhaps not all, kinetochore fiber microtubules of metaphase cells do turn over. Analysis of the movements of metaphase bleached zones suggested that a slow poleward translocation of kinetochore microtubules occurred. However, within the variation of the data (0.12 +/- 0.24 micron/min), it could not be determined whether the apparent movement was real or artifactual.     

Microinjection of biotin-tubulin into anaphase cells induces transient elongation of kinetochore microtubules and reversal of chromosome-to- pole motion

During prometaphase and metaphase of mitosis, tubulin subunit incorporation into kinetochore microtubules occurs proximal to the kinetochore, at the plus-ends of kinetochore microtubules. During anaphase, subunit loss from kinetochore fiber microtubules is also thought to occur mainly from microtubule plus-ends, proximal to the kinetochore. Thus, the kinetochore can mediate both subunit addition and loss while maintaining an attachment to kinetochore microtubules. To examine the relationship between chromosome motion and tubulin subunit assembly in anaphase, we have injected anaphase cells with biotin-labeled tubulin subunits. The pattern of biotin-tubulin incorporation was revealed using immunoelectron and confocal fluorescence microscopy of cells fixed after injection; chromosome motion was analyzed using video records of living injected cells. When anaphase cells are examined approximately 30 s after injection with biotin-tubulin, bright "tufts" of fluorescence are detected proximal to the kinetochores. Electron microscopic immunocytochemistry further reveals that these tufts of biotin-tubulin-containing microtubules are continuous with unlabeled kinetochore fiber microtubules. Biotin-tubulin incorporation proximal to the kinetochore in anaphase cells is detected after injection of 3-30 mg/ml biotin-tubulin, but not in cells injected with 0.3 mg/ml biotin-tubulin. At intermediate concentrations of biotin-tubulin (3-5 mg/ml), incorporation at the kinetochore can be detected within 15 s after injection; by approximately 1 min after injection discrete tufts of fluorescence are no longer detected, although some incorporation throughout the kinetochore fiber and into nonkinetochore microtubules is observed. At higher concentrations of injected biotin-tubulin (13 mg/ml), incorporation at the kinetochore is more extensive and occurs for longer periods of time than at intermediate concentrations. Incorporation of biotin-tubulin proximal to the kinetochore can be detected in cells injected during anaphase A, but not during anaphase B. Analysis of video records of microinjection experiments reveals that kinetochore proximal incorporation of biotin-tubulin is accompanied by a transient reversal of chromosome-to-pole motion. Chromosome motion is not altered after injection of 0.3 mg/ml biotin-tubulin or 5 mg/ml BSA. These results demonstrate that kinetochore microtubules in anaphase cells can elongate in response to the elevation of the tubulin concentration and that kinetochores retain the ability to mediate plus-end-dependent assembly of KMTs and plus-end-directed chromosome motion after anaphase onset.     

Multiple-color fluorescence imaging of chromosomes and microtubules in living cells

Microscopic observation of fluorescently-stained intracellular molecules within a living cell provides a straightforward approach to understanding their temporal and spatial relationships. However, exposure to the excitation light used to visualize these fluorescently-stained molecules can be toxic to the cells. Here we describe several important considerations in microscope instrumentation and experimental conditions for avoiding the toxicity associated with observing living fluorescently-stained cells. Using a computer-controlled fluorescence microscope system designed for live observation, we recorded time-lapse, multi-color images of chromosomes and microtubules in living human and fission yeast cells. In HeLa cells, a human cell line, microtubules were stained with rhodamine-conjugated tubulin, and chromosomes were stained with a DNA-specific fluorescent dye, Hoechst33342, or with rhodamine-conjugated histone. In fission yeast cells, microtubules were stained with alpha-tubulin fused with the jellyfish green fluorescent protein (GFP), and chromosomes were stained with Hoechst33342.     

Functional states of kinetochores revealed by laser microsurgery and fluorescent speckle microscopy

The impact of mechanical forces on kinetochore motility was investigated using laser microsurgery to detach kinetochores with associated chromatin (K fragment) from meiotic chromosomes in spermatocytes from the crane fly Nephrotoma suturalis. In spermatocytes, elastic tethers connect telomeres of homologues during anaphase A of meiosis I, thus preventing complete disjunction until mid- to late anaphase A. K fragments liberated from tethered arms moved at twice the normal velocity toward their connected poles. To assess functional states of detached and control kinetochores, we loaded cells with fluorescently labeled tubulin for fluorescent speckle microscopy on kinetochore microtubules. Control kinetochores added fluorescent speckles at the kinetochore during anaphase A, whereas kinetochores of K fragments generally did not. In cases in which speckles reappeared in K-fragment K fibers, speckles and K fragments moved poleward at similar velocities. Thus detached kinetochores convert from their normal polymerization (reverse pac-man) state to a different state, in which polymerization is not evident. We suggest that the converted state is "park," in which kinetochores are anchored to plus ends of kinetochore microtubules that shorten exclusively at their polar ends.     

Interzone microtubule behavior in late anaphase and telophase spindles

We have studied microtubule behavior in late anaphase and telophase spindles of PtK1 cells, using fluoresceinated tubulin (DTAF-tubulin), microinjection, and laser microbeam photobleaching. We present the results of two novel tests which add to the evidence that DTAF-tubulin closely mimics the behavior of native tubulin in vivo. (a) Microinjected DTAF-tubulin was as effective as injected native tubulin in promoting division of taxol-dependent mitotic mutant cells that had been deprived of taxol. (b) Microinjected colchicine-DTAF-tubulin complex was similar to injected colchicine-native tubulin complex in causing depolymerization of spindles. Immediately after microinjection of DTAF-tubulin into wild-type cells during late anaphase or telophase, fluorescence incorporation by microtubules was seen in chromosomal half-spindles and just behind the chromosomes, but there was no fluorescence incorporation near the middle of the interzone. Over the next few minutes, tubulin fluorescence accumulated at the center of the interzone (the equator), becoming progressively more intense. In other experiments, cells were microinjected with DTAF-tubulin at prophase and allowed to equilibrate for 30 min. Cells that had progressed to late anaphase were then photobleached to reduce the fluorescence in the central portion of the interzone. Over a period of several minutes, the only substantial redistribution of fluorescence was the appearance of a bright area at the equator of the interzone. Both the site of fluorescence incorporation and the photobleaching data suggest that tubulin adds to the elongating spindle interzone near the equator where the plus ends of the interdigitated microtubules are located. In further experiments, several dark lines were photobleached perpendicular to the pole-to-pole axis of fluorescent anaphase-telophase spindles. Time-dependent changes in the spacings between the lines indicated that the two halves of the interzone lying on opposite sides of the spindle equator moved away from one another. This shows that the interdigitated microtubules, which make up most of the interzone, can undergo antiparallel sliding. Our data support a model for anaphase B in which plus end elongation of interdigitated microtubules and antiparallel sliding contribute to chromosome separation.     

Sites of Tubulin Polymerization in PC 12 Cells

The site at which tubulin enters into polymer in the neuritic process is a very important datum in terms of our understanding of the mechanism of transport of the microtubular cytoskeleton out the axon. If the form of tubulin being transported out the axon is the microtubule, then assembly of tubulin into microtubules should occur at or near the cell body; if, however, the form of tubulin transported is free tubulin dimer, then assembly can occur at any free microtubule end out the neurite. We have injected a fluorescent analog of tubulin into differentiated PC 12 cells and used differential extraction protocols to extract free dimer but not microtubules. We have imaged these cells before and after extraction by low-light-level video fluorescence microscopy and have used image analysis to examine the sites of tubulin incorporation into polymer or other unextracted components as a function of time. We find that tubulin in the distal reaches of the neurite is found initially as monomer and that its appearance in the unextracted component occurs later. This pattern of appearance of fluorescent tubulin initially in the soluble fraction and later in the unextractable component is qualitatively similar to that reported by other workers for biotinylated tubulin, but we see a larger gap between the rates of appearance in soluble fraction and in polymer. Quantitative analysis of fluorescence intensities in the two compartments with distance out the neurite reveals substantial variation between different neurites: In some neurites, the pattern of variation of unextracted/total tubulin suggests that tubulin enters into the unextracted component primarily near the cell body and that this unextracted component moves out the neurite with time, and in other neurites it suggest that monomer adds into microtubule ends staggered out the neurite. In no case do we see a pattern suggesting that distal addition predominates. These analyses of fluorescence intensities in extracted and unextracted neurites suggest that both transport of polymerized microtubules and monomer addition onto staggered microtubule ends occur in PC12 neurites and that in individual neurites one or the other of these two behaviors may predominate.     

Polewards microtubule flux in the mitotic spindle: evidence from photoactivation of fluorescence

I have synthesized a novel derivative of carboxyfluorescein that is nonfluorescent, but can be converted to a fluorescent form by exposure to 365-nm light. This photoactivable, fluorescent probe was covalently attached to tubulin and microinjected into mitotic tissue culture cells, where it incorporated into functional spindles. To generate a fluorescent bar across the mitotic spindle, metaphase cells were irradiated with a slit microbeam. This bar decreased in intensity over the first minute, presumably due to turnover of nonkinetochore microtubules. The remaining fluorescent zones, now presumably restricted to kinetochore microtubules, moved polewards at 0.3-0.7 microns/min. This result provides strong evidence for polewards flux in kinetochore microtubules. In conjunction with earlier biotin-tubulin incorporation experiments (Mitchison, T. J., L. Evans, E. Schulze, and M. Kirschner. 1986. Cell. 45:515-527), I conclude that microtubules polymerize at kinetochores and depolymerize near the poles throughout metaphase. The significance of this observation for spindle structure and function is discussed. Local photoactivation of fluorescence should be a generally useful method for following molecular dynamics inside living cells.     

Spindle microtubule dynamics in sea urchin embryos: analysis using a fluorescein-labeled tubulin and measurements of fluorescence redistribution after laser photobleaching

The rate of exchange of tubulin that is incorporated into spindle microtubules with dimeric tubulin in the cytoplasm has been measured in sea urchin eggs by studying fluorescence redistribution after photobleaching (FRAP). Dichlorotriazinyl amino fluorescein (DTAF) has been used to label bovine brain tubulin. DTAF-tubulin has been injected into fertilized eggs of Lytechinus variegatus and allowed to equilibrate with the endogenous tubulin pool. Fluorescent spindles formed at the same time that spindles were seen in control eggs, and the injected embryos proceeded through many cycles of division on schedule, suggesting that DTAF-tubulin is a good analogue of tubulin in vivo. A microbeam of argon laser light has been used to bleach parts of the fluorescent spindles, and FRAP has been recorded with a sensitive video camera. Laser bleaching did not affect spindle structure, as seen with polarization optics, nor spindle function, as seen by rate of progress through mitosis, even when one spindle was bleached several times in a single cell cycle. Video image analysis has been used to measure the rate of FRAP and to obtain a low resolution view of the fluorescence redistribution process. The half-time for spindle FRAP is approximately 19 s, even when an entire half-spindle is bleached. Complete exchange of tubulin in nonkinetochore spindle and astral microtubules appeared to occur within 60-80 s at steady state. This rate is too fast to be explained by a simple microtubule end-dependent exchange of tubulin. Efficient microtubule treadmilling would be fast enough, but with current techniques we saw no evidence for movement of the bleached spot during recovery, which we would expect on the basis of Margolis and Wilson's model (Nature (Lond.)., 1981, 293:705)-- fluorescence recovers uniformly. Microtubules may be depolymerizing and repolymerizing rapidly and asynchronously throughout the spindle and asters, but the FRAP data are most compatible with a rapid exchange of tubulin subunits all along the entire lengths of nonkinetochore spindle and astral microtubules.     

Microinjection of fluorescent tubulin into dividing sea urchin cells

To follow the dynamics of microtubule (MT) assembly and disassembly during mitosis in living cells, tubulin has been covalently modified with the fluorochrome 5-(4,6-dichlorotriazin-2-yl)aminofluorescein and microinjected into fertilized eggs of the sea urchin Lytechinus variegatus. The changing distribution of the fluorescent protein probe is visualized in a fluorescence microscope coupled to an image intensification video system. Cells that have been injected with fluorescent tubulin show fluorescent linear polymers that assemble very rapidly and radiate from the spindle poles, coincident with the position of the astral fibers. No fluorescent polymer is apparent in other areas of the cytoplasm. When fluorescent tubulin is injected near the completion of anaphase, little incorporation of fluorescent tubulin into polymer is apparent, suggesting that new polymerization does not occur past a critical point in anaphase. These results demonstrate that MT polymerization is very rapid in vivo and that the assembly is both temporally and spatially regulated within the injected cells. Furthermore, the microinjected tubulin is stable within the sea urchin cytoplasm for at least 1 h since it can be reutilized in successive daughter cell spindles. Control experiments indicate that the observed fluorescence is dependent on MT assembly. The fluorescence is greatly diminished upon treatment of the cells with cold or colchicine agents known to cause the depolymerization of assembled MT. In addition, cells injected with fluorescent bovine serum albumin or assembly-incompetent fluorescent tubulin do not exhibit fluorescence localized in the spindle but rather appear diffusely fluorescent throughout the cytoplasm.     

Spindle microtubule dynamics: modulation by metabolic inhibitors

Recent experiments have shown that spindle microtubules are exceedingly dynamic. Measurements of fluorescence recovery after photobleaching (FRAP), in cells previously microinjected with fluorescent tubulin, provide quantitative information concerning the rate of turnover, or exchange, of tubulin subunits with the population of microtubules in living cells at steady state. In an effort to elucidate the pathways and factors that regulate tubulin exchange with microtubules in living cells, we have investigated the energy requirements for tubulin turnover as measured by FRAP. Spindle morphology was not detectably altered in cells incubated with 5 mM sodium azide and 1 mM 2-deoxyglucose (Az/DOG) for 5 minutes, as assayed by polarized light microscopy and antitubulin immunofluorescence. In FRAP experiments on these ATP-depleted cells, the average rate of recovery and the average percent of bleached fluorescence recovered were reduced to 37% and 30% of controls, respectively. When the inhibitors were removed, cells continued through mitosis, and rapid FRAP was restored. In the presence of azide and glucose, the rate of recovery and percent of fluorescence recovered were only slightly reduced, demonstrating that energy production via glycolysis can support microtubule turnover. Longer incubations with Az/DOG altered the microtubule organization in mitotic cells: astral microtubules lengthened and spindle fibers shortened. Furthermore, both astral and spindle microtubules became resistant to nocodazole-induced disassembly under these conditions. Together these observations indicate that microtubule dynamics require ATP and suggest a relationship between microtubule organization and turnover.     

A fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid, as a probe for the colcemid-binding sites of tubulin and microtubules

The synthesis and biological testing of the fluorescent analog of colcemid, N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-colcemid (NBD-colcemid), are here described. NBD-colcemid exhibited a visible absorption maximum at 465 nm and fluoresced in the range of 520-540 nm, highly in environments of low polarity, whereas only slightly in aqueous solution. The addition of NBD-colcemid to bovine brain tubulin was accompanied by a striking enhancement of fluorescence. The fluorescent titration study suggested a stoichiometric binding of NBD-colcemid to tubulin. Assembled microtubules were directly visualized after mixing with NBD-colcemid using a fluorescence microscope. NBD-colcemid reversibly disrupted the metaphase spindles of sea urchin eggs as well as unlabeled colcemid. However, even when the birefringence of spindles was mostly lost, self-quenching properties of the NBD fluorescence allowed tubulin and its oligomers aggregated in higher concentrations in eggs to be visualized under a fluorescence microscope. The results suggest a wide applicability of NBD-colcemid as a fluorescent probe for studying the interactions of colcemid with tubulin and microtubules, as well as for localizing other colcemid-binding structures within cells.     

Analysis of the treadmilling model during metaphase of mitosis using fluorescence redistribution after photobleaching

One recent hypothesis for the mechanism of chromosome movement during mitosis predicts that a continual, uniform, poleward flow or "treadmilling" of microtubules occurs within the half-spindle between the chromosomes and the poles during mitosis (Margolis, R. L., and L. Wilson, 1981, Nature (Lond.), 293:705-711). We have tested this treadmilling hypothesis using fluorescent analog cytochemistry and measurements of fluorescence redistribution after photobleaching to examine microtubule behavior during metaphase of mitosis. Mitotic BSC 1 mammalian tissue culture cells or newt lung epithelial cells were microinjected with brain tubulin labeled with 5-(4,6-dichlorotriazin-2-yl) amino fluorescein (DTAF) to provide a fluorescent tracer of the endogenous tubulin pool. Using a laser microbeam, fluorescence in the half-spindle was photobleached in either a narrow 1.6 micron wide bar pattern across the half-spingle or in a circular area of 2.8 or 4.5 micron diameter. Fluorescence recovery in the spindle fibers, measured using video microscopy or photometric techniques, occurs as bleached DTAF-tubulin subunits within the microtubules are exchanged for unbleached DTAF-tubulin in the cytosol by steady-state microtubule assembly-disassembly pathways. Recovery of 75% of the bleached fluorescence follows first-order kinetics and has an average half-time of 37 sec, at 31-33 degrees C. No translocation of the bleached bar region could be detected during fluorescence recovery, and the rate of recovery was independent of the size of the bleached spot. These results reveal that, for 75% of the half-spindle microtubules, FRAP does not occur by a synchronous treadmilling mechanism.     

Recovery,visualization, and analysis of actin and tubulin polymer flow in live cells: a fluorescent speckle microscopy study

Fluorescent speckle microscopy (FSM) is becoming the technique of choice for analyzing in vivo the dynamics of polymer assemblies, such as the cytoskeleton. The massive amount of data produced by this method calls for computational approaches to recover the quantities of interest; namely, the polymerization and depolymerization activities and the motions undergone by the cytoskeleton over time. Attempts toward this goal have been hampered by the limited signal-to-noise ratio of typical FSM data, by the constant appearance and disappearance of speckles due to polymer turnover, and by the presence of flow singularities characteristic of many cytoskeletal polymer assemblies. To deal with these problems, we present a particle-based method for tracking fluorescent speckles in time-lapse FSM image series, based on ideas from operational research and graph theory. Our software delivers the displacements of thousands of speckles between consecutive frames, taking into account that speckles may appear and disappear. In this article we exploit this information to recover the speckle flow field. First, the software is tested on synthetic data to validate our methods. We then apply it to mapping filamentous actin retrograde flow at the front edge of migrating newt lung epithelial cells. Our results confirm findings from previously published kymograph analyses and manual tracking of such FSM data and illustrate the power of automated tracking for generating complete and quantitative flow measurements. Third, we analyze microtubule poleward flux in mitotic metaphase spindles assembled in Xenopus egg extracts, bringing new insight into the dynamics of microtubule assemblies in this system.     

Cell cycle-dependent changes in the dynamics of MAP 2 and MAP 4 in cultured cells

To examine the behavior of microtubule-associated proteins (MAPs) in living cells, MAP 4 and MAP 2 have been derivatized with 6-iodoacetamido-fluorescein, and the distribution of microinjected MAP has been analyzed using a low light level video system and fluorescence redistribution after photobleaching. Within 1 min following microinjection of fluoresceinated MAP 4 or MAP 2, fluorescent microtubule arrays were visible in interphase or mitotic PtK1 cells. After cold treatment of fluorescent MAP 2-containing cells (3 h, 4 degrees C), microtubule fluorescence disappeared, and the only fluorescence above background was located at the centrosomes; microtubule patterns returned upon warming. Loss of microtubule immunofluorescence after nocodozole treatment was similar in MAP-injected and control cells, suggesting that injected fluorescein-labeled MAP 2 did not stabilize microtubules. The dynamics of the MAPs were examined further by FRAP. FRAP analysis of interphase cells demonstrated that MAP 2 redistributed with half-times slightly longer (60 +/- 25 s) than those for MAP 4 (44 +/- 20 s), but both types of MAPs bound to microtubules in vivo exchanged with soluble MAPs at rates exceeding the rate of tubulin turnover. These data imply that microtubules in interphase cells are assembled with constantly exchanging populations of MAP. Metaphase cells at 37 degrees C or 26 degrees C showed similar mean redistribution half-times for both MAP 2 and MAP 4; these were 3-4 fold faster than the interphase rates (MAP 2, t1/2 = 14 +/- 6 s; MAP 4, t1/2 = 17 +/- 5 s). The extent of recovery of spindle fluorescence in MAP-injected cells was to 84-94% at either 26 or 37 degrees C. Although most metaphase tubulin, like the MAPs, turns over rapidly and completely under physiologic conditions, published work shows either reduced rates or extents of turnover at 26 degrees C, suggesting that the fast mitotic MAP exchange is not simply because of fast tubulin turnover. Exchange of MAP 4 bound to telophase midbodies occurred with dynamics comparable to those seen in metaphase spindles (t1/2 = approximately 27 s) whereas midbody tubulin exchange was slow (greater than 300 s). These data demonstrate that the rate of MAP exchange on microtubules is a function of time in the cell cycle.     

Cytoplasmic dynein-mediated assembly of pericentrin and gamma tubulin onto centrosomes

Centrosome assembly is important for mitotic spindle formation and if defective may contribute to genomic instability in cancer. Here we show that in somatic cells centrosome assembly of two proteins involved in microtubule nucleation, pericentrin and gamma tubulin, is inhibited in the absence of microtubules. A more potent inhibitory effect on centrosome assembly of these proteins is observed after specific disruption of the microtubule motor cytoplasmic dynein by microinjection of dynein antibodies or by overexpression of the dynamitin subunit of the dynein binding complex dynactin. Consistent with these observations is the ability of pericentrin to cosediment with taxol-stabilized microtubules in a dynein- and dynactin-dependent manner. Centrosomes in cells with reduced levels of pericentrin and gamma tubulin have a diminished capacity to nucleate microtubules. In living cells expressing a green fluorescent protein-pericentrin fusion protein, green fluorescent protein particles containing endogenous pericentrin and gamma tubulin move along microtubules at speeds of dynein and dock at centrosomes. In Xenopus extracts where gamma tubulin assembly onto centrioles can occur without microtubules, we find that assembly is enhanced in the presence of microtubules and inhibited by dynein antibodies. From these studies we conclude that pericentrin and gamma tubulin are novel dynein cargoes that can be transported to centrosomes on microtubules and whose assembly contributes to microtubule nucleation.     

Tubulin Carboxypeptidase/Microtubules Association can be Detected in the Distal Region of Neural Processes

The association of tubulin carboxypeptidase with microtubules has been demonstrated in crude brain extracts and in living non-nervous cells. Here, we studied this phenomenon in cultured brain cells. To determine the association of the enzyme with neural microtubules we isolated the cytoskeletons (detergent-extraction under microtubule-stabilizing conditions) and measured the content of Tyr, Glu, and 2 tubulin as a function of the in vitro incubation time of the cytoskeletons. The carboxypeptidase was found associated with microtubules in 2 days-cultured cells but not in 7 days-cultured cells. Quantitative analysis of digitized images after immunofluorescent staining revealed that detyrosination during the incubation of the cytoskeletons occurred preferentially in the distal regions of the neural processes. Prolonged taxol-treatment of the cells promoted higher detyrosination but Tyr tubulin was not depleted suggesting the existence of a subset of microtubules that has not associated carboxypeptidase and therefore cannot be detyrosinated even after prolonged taxol-treatment. This hypothesis was supported, although not conclusively, by additional experiments.