Surface functions during mitosis in rat basophilic leukemia cells

At the entry into mitosis, cells abruptly lose membrane activities such as phagocytosis, pinocytosis, and capping. The present studies test if mitotic cells also resist functional responses to cell surface ligand-receptor interactions. The IgE receptors of RBL-2H3 rat basophilic leukemia cells were labeled with anti-dinitrophenol IgE (anti-DNP-IgE) and then cross-linked with multivalent ligands (DNP-bovine serum albumin [BSA]; DNP-B-phycoerythrin; DNP-BSA-gold). IgE-receptor cross-linking modulates cell surface organization and function and releases serotonin and other mediators of allergic and asthmatic reactions from interphase cells (Pfeiffer, J. R., JC. Seagrave, B. H. Davis, G. G. Deanin, and J. M. Oliver, 1985, J. Cell Biol., 101:2145-2155). It was found that anti-DNP-IgE-receptor complexes are preserved on the cell surface throughout mitosis; they continue to bind DNP-proteins, and the resulting antigen-IgE-receptor complexes can redistribute to coated pits on the cell surface. Furthermore, there is no loss of [3H]serotonin through mitosis. Nevertheless, antigen-stimulated [3H]-serotonin release is strongly impaired in mitotic-enriched as compared with mixed interphase or G1-enriched cell populations. In addition, antigen binding transforms the surface of interphase cells from a microvillous to a plicated topography and stimulates the uptake of fluorescein isothiocyanate-conjugated dextran by fluid pinocytosis. Mitotic cells maintain a microvillous surface topography after antigen treatment, and fluid pinocytosis virtually ceases from prometaphase to telophase. Phorbol myristate acetate, a tumor promoter that activates protein kinase C, restores surface ruffling activity to mitotic cells. Thus, the mitosis-specific freezing of membrane and secretory responses is most likely due to the failure of transmembrane signaling.     

The blocked pinocytic activity of mitotic cells is restored in mitotic—Interphase hybrids

During mitosis there is an abrupt inhibition of a wide range of membrane functions, including fluid-phase and adsorptive pinocytosis. We have used cell hybrids formed between mitotic and interphase cells to approach the mechanism of this inhibition. We report that fluid pinocytosis is reactivated in the mitotic partner of hybrids formed between mitotic and interphase Chinese hamster ovary (CHO) cells. It thus appears that the interphase cell provides some necessary element(s) for membrane activity during mitosis. This dominance of interphase membrane properties stands in contrast with earlier evidence that mitotic nuclear properties dominate in similar mitotic-interphase hybrids.     

Delay of HeLa cell cleavage into interphase using dihydrocytochalasin B: retention of a postmitotic spindle and telophase disc correlates with synchronous cleavage recovery

The molecular signals that determine the position and timing of the cleavage furrow during mammalian cell cytokinesis are presently unknown. We have studied in detail the effect of dihydrocytochalasin B (DCB), a drug that interferes with actin assembly, on specific late mitotic events in synchronous HeLa cells. When cleavage furrow formation is blocked at 10 microM DCB, cells return to interphase by the criteria of reformation of nuclei with lamin borders, degradation of the cyclin B component of p34cdc2 kinase, and loss of mitosis specific MPM-2 antigens. However, the machinery for cell cleavage is retained for up to one hour into G1 when cleavage cannot proceed. The components retained consist prominently of a "postmitotic" spindle and a telophase disc, a structure templated by the mitotic spindle in anaphase that may determine the position and timing of the cleavage furrow. Upon release from DCB block, G1 cells proceed through a rapid and synchronous cleavage. We conclude that the mitotic spindle is not inevitably destroyed at the end of mitosis, but persists as an integral structure with the telophase disc in the absence of cleavage. We also conclude that cell cleavage can occur in G1, and is therefore an event metabolically independent of mitosis. The retained telophase disc may indeed signal the position of furrow formation, as G1 cleavage occurs only in the position where the retained disc underlies the cell cortex. The protocol we describe should now enable development of a model system for the study of mammalian cell cleavage as a synchronous event independent of mitosis.     

Different cyclic changes in the surface membrane of normal and malignant transformed cells

Transformed fibroblasts in interphase and normal fibroblasts in mitosis were agglutinated by Con A and the lectin from wheat germ, whereas normal fibroblasts in interphase and transformed fibroblasts in mitosis were not agglutinated by these lectins. The percentage of fluorescent cells at non-saturation concentrations of fluorescent ConA was also higher with transformed interphase and normal mitotic cells, than with normal interphase and transformed mitotic cells. Under the same conditions, a similar number of radioactively labeled ConA molecules were bound to normal and transformed cells in interphase and mitosis. Our results indicate different cyclic changes in the surface membrane of normal and transformed fibroblasts, so that regarding interaction with these lectins, normal mitotic cells resemble transformed interphase cells and transformed mitotic resemble normal interphase cells. The data suggest that there is a reversed cyclic change in the mobility of specific surface membrane sites in normal and transformed cells.     

Surface functions during mitosis. II. Quantitation of pinocytosis and kinetic characterization of the mitotic cycle with a new fluorescence technique

The profound depression of fluid pinocytosis observed in mitotic cells (Berlin, R. D., et al. 1978. Cell. 15:327--341) is documented by quantitative microspectrofluorimetry of fluorescein-labeled dextran uptake in single cells. In J774.2 macrophages, fluid pinocytosis is reduced 30-fold during mitosis. The depression develops within 30 s of entry into prophase and recovers with equal rapidity upon emergence from telophase into G1. This characteristic pattern of fluid pinocytosis forms the basis of a new method for detailed kinetic analysis of the duration of mitosis and its phases. The analysis is applied to the J774.2 macrophage cell line but should be generally applicable to other lines. Effects of ouabain and colchicine on the length of mitosis and its phases are evaluated, revealing a selective prolongation of metaphase by ouabain and suggesting a role for microtubules in the transition from G2 into mitosis.     

Localization and activation of the ARF6 GTPase during cleavage furrow ingression and cytokinesis

The ARF6 GTPase mediates cell shape changes in interphase cells through its effects on membrane cycling and actin remodeling. In this study, we focus our attention on the dynamics of cell division and present evidence supporting a novel role for ARF6 during cleavage furrow ingression and cytokinesis. We demonstrate that endogenous ARF6 redistributes during mitosis and concentrates near the cleavage furrow during telophase. Constitutively activated ARF6 localizes to the plasma membrane at the site of cleavage furrow ingression and midbody formation, and dominant negative ARF6 remains cytoplasmic. By using a novel pull-down assay for ARF6-GTP, we find an abrupt, but transient, increase in ARF6-GTP levels as cells progress through cytokinesis. Whereas high levels of expression of a GTPase-defective ARF6 mutant induce aberrant phenotypes in cells at cytokinesis, cells expressing low levels of ARF6 mutants do not display a significant mitotic delay or cytokinesis defect, presumably due to compensatory or redundant mechanisms that allow cytokinesis to proceed when the ARF6 GTPase cycle is disrupted. Finally, actin accumulation and phospholipid metabolism at the cleavage furrow are unchanged in cells expressing ARF6 mutants, suggesting that ARF6 may be involved in membrane remodeling during cytokinesis via effector pathways that are distinct from those operative in interphase cells.     

Electron microscopic observations on the maintenance of the tight junction during cell division in the epithelium of the mouse small intestine.

Dividing epithelial cells in the mouse small intestine were examined by thin-section electron microscopy with special attention given to the mode of cytokinesis. As the columnar epithelial cells entered mitosis in the crypt, they became rounded, maintaining their junctional complexes with neighboring cells while detaching themselves from the basal lamina. In such rounded cells the mitotic apparatus was formed with its long axis parallel to the luminal surface. Replicated centrioles moved down from the apical region to locate themselves lateral to the nucleus, where they served as the poles of the mitotic spindle. During mitosis the cell retained microvilli on its luminal surface, though the terminal web became much thinner. At telophase the formation of a cleavage furrow proceeded asymmetrically from the basal side alone, and thus the contractile ring which was prominent at the base of the furrow, merged with the terminal web. Eventually, an intercellular bridge with a midbody was formed on the luminal surface. The space in the furrow was occupied by the flattened cytoplasmic processes of the neighboring cells. The tight junction was also seen on the basolateral surface of the intercellular bridge with the underlying neighboring cells. At very late telophase the intercellular bridge was disconnected from the neighboring cells and protruded into the lumen. These observations have led us to propose a mode by which the simple columnar epithelium maintain the tight junctional seal during cell division in the crypt of the small intestinal epithelium.     

An electron microscopic study of mitosis in mouse duodenal crypt cells confirms that the prophasic condensation of chromatin begins during the DNA-synthesizing (S) stage of the cycle

The phases of mitosis were examined in the columnar cells at the base of duodenal crypts in adult male mice given an intravenous injection of 3H-thymidine and sacrificed 20 min later. The duodenum was fixed by immersion into glutaraldehyde-formaldehyde, and the cells were examined in the electron microscope, with or without processing for radioautography. Interphase nuclei are characterized by the distribution of chromatin; aside from the cortical chromatin spread along nuclear envelope and nucleolus, there are chromatin accumulations that belong mainly in two different classes: 1) numerous chromatin "specks" ranging in size from about 5 to 70 nm and averaging 47 nm; 2) a few roughly circular or elongated chromatin "packets" measuring from 70 to 230 nm. Early prophase nuclei differ mainly by a large increase in the number of chromatin packets to 20-30 or more per nuclear profile; their average diameter is 128 nm. During mid-prophase, the chromatin packets enlarge gradually to an average 221 nm diameter. Between mid- and late prophase, there is a further increase in diameter to 679 nm. At metaphase, the packets take on the appearance of mature chromosomes, and their diameter increases to 767 nm. At anaphase, daughter chromosomes migrate to each pole, where they fuse into a compact chromatin mass. At telophase, nucleoplasmic areas progressively enlarge within the chromatin mass and separate strands of chromatin, which gradually become segmented into chromatin clumps. Counts of mitotic cells show a high proportion of prophase and telophase nuclei. Calculation from the counts yields the duration of the phases, that is, 5.6, 0.2, 0.1, and 1.6 hr, respectively, for pro-, meta-, ana-, and telophase. Finally, radioautography 20 min after 3H-thymidine injection shows labeling in 54% of the interphase nuclei, 85% of early prophase nuclei, and 73% of mid-prophase nuclei, while there is no label in late prophase, metaphase, anaphase and telophase nuclei. In confirmation of previous light microscopic work, the S stage of the cycle begins when a cell is in interphase and continues through the early prophase and part of mid-prophase. Moreover, the main sites of DNA synthesis are the chromatin specks during interphase and the cortical chromatin during early and mid-prophase. The chromosome condensation taking place in the meantime may be separated into two main steps: 1) a slow, moderate condensation of the chromatin packets during early and mid-prophase and 2) a rapid, pronounced one during late prophase and prometaphase when the packets become chromosomes.     

Different activity regulation and subcellular localization of LIMK1 and LIMK2 during cell cycle transition

LIM kinases (LIMK1 and LIMK2) regulate actin cytoskeletal reorganization through phosphorylating and inactivating cofilin, an actin-depolymerizing factor of actin filaments. Here, we describe a detailed analysis of the cell-cycle-dependent activity of LIMK2, and a subcellular localization of LIMK1 and LIMK2. The activity of LIMK2, distinct from LIMK1, toward cofilin phosphorylation did not change in the normal cell division cycle. In contrast, LIMK2 was hyperphosphorylated and its activity was markedly increased when HeLa cells were synchronized at mitosis with nocodazole treatment. Immunofluorescence analysis showed that LIMK1 was localized at cell-cell adhesion sites in interphase and prophase, redistributed to the spindle poles during prometaphase to anaphase, and accumulated at the cleavage furrow in telophase. In contrast, LIMK2 was diffusely localized in the cytoplasm during interphase, redistributed to the mitotic spindle, and finally to the spindle midzone during anaphase to telophase. These findings suggest that LIMK2 is activated in response to microtubule disruption, and that LIMK1 and LIMK2 may play different roles in regulating for the mitotic spindle organization, chromosome segregation, and cytokinesis during the cell division cycle.     

Rab24 is Required for Normal Cell Division

Rab24 is an atypical member of the Rab GTPase family whose distribution in interphase cells has been characterized; however, its function remains largely unknown. In this study, we have analyzed the distribution of Rab24 throughout cell division. We have observed that Rab24 was located at the mitotic spindle in metaphase, at the midbody during telophase and in the furrow during cytokinesis. We have also observed partial co‐localization of Rab24 and tubulin and demonstrated its association to microtubules. Interestingly, more than 90% of transiently transfected HeLa cells with Rab24 presented abnormal nuclear connections (i. e. chromatin bridges). Furthermore, in CHO cells stably transfected with GFP‐Rab24wt, we observed a large percentage of binucleated and multinucleated cells. In addition, these cells presented an extremely large size and multiple failures in mitosis, as aberrant spindle formation (metaphase), delayed chromosomes (telophase) and multiple cytokinesis. A marked increase in binucleated, multinucleated and multilobulated nucleus formation was observed in HeLa cells depleted of Rab24. We also present evidence that a fraction of Rab24 associates with microtubules. In addition, Rab24 knock down resulted in misalignment of chromosomes and abnormal spindle formation in metaphase leading to the appearance of delayed chromosomes during late telophase and failures in cytokinesis. Our findings suggest that an adequate level of Rab24 is necessary for normal cell division. In summary, Rab24 modulates several mitotic events, including chromosome segregation and cytokinesis, perhaps through the interaction with microtubules.     

Ultrastructural labeling of cell surface lectin receptors during the cell cycle.

The distribution of specific surface receptors in the course of the cell cycle has been studied on two transformed cell lines by means of ultrastructural labelling techniques employing Concanavalin A (ConA) and wheat germ agglutinin (WGA). Synchronized cultures of Cl₂TSV₅, an SV40-transformed hamster cell line and of CHO cells were labelled as monolayers or in suspension in the different phases of the cell cycle. In cells labelled in monolayers, a moderately discontinuous pattern of surface labelling was present during G 1, S, and G 2. On cells in mitosis, however, this pattern changes strikingly and becomes very discontinuous. These results indicate that the degree of receptor clustering is greater in mitosis than in interphase. In cells labelled in suspension, the differences in pattern between mitosis and interphase were absent. Colcemid treatment did not modify the distribution of the label, either in interphase or in mitosis. Moreover, cells in mitosis collected by Colcemid treatment and labelled at a moment in which parallel unblocked cultures had completed mitosis and passed into G 1 showed an interphase-type labelling pattern; this indicates that a certain dissociation exists between surface events and nuclear events during mitosis. These results are discussed in terms of several factors that may contribute to the production of receptor clustering, namely, direct lectin action, surface movement and membrane flow, participation of cytoplasmic structures and, finally, attachment of cells to a substratum.     

Spectrin redistributes to the cytosol and is phosphorylated during mitosis in cultured cells

Dramatic changes in morphology and extensive reorganization of membrane-associated actin filaments take place during mitosis in cultured cells, including rounding up; appearance of numerous actin filament-containing microvilli and filopodia on the cell surface; and disassembly of intercellular and cell-substratum adhesions. We have examined the distribution and solubility of the membrane-associated actin-binding protein, spectrin, during interphase and mitosis in cultured CHO and HeLa cells. Immunofluorescence staining of substrate-attached, well-spread interphase CHO cells reveals that spectrin is predominantly associated with both the dorsal and ventral plasma membranes and is also concentrated at the lateral margins of cells at regions of cell-cell contacts. In mitotic cells, staining for spectrin is predominantly in the cytoplasm with only faint staining at the plasma membrane on the cell body, and no discernible staining on the membranes of the microvilli and filopodia (retraction fibers) which protrude from the cell body. Biochemical analysis of spectrin solubility in Triton X-100 extracts indicates that only 10-15% of the spectrin is soluble in interphase CHO or HeLa cells growing attached to tissue culture plastic. In contrast, 60% of the spectrin is soluble in mitotic CHO and HeLa cells isolated by mechanical "shake-off" from nocodazole-arrested synchronized cultures, which represents a four- to sixfold increase in the proportion of soluble spectrin. This increase in soluble spectrin may be partly due to cell rounding and detachment during mitosis, since the amount of soluble spectrin in CHO or HeLa interphase cells detached from the culture dish by trypsin-EDTA or by growth in spinner culture is 30-38%. Furthermore, mitotic cells isolated from synchronized spinner cultures of HeLa S3 cells have only 2.5 times as much soluble spectrin (60%) as do synchronous interphase cells from these spinner cultures (25%). The beta subunit of spectrin is phosphorylated exclusively on serine residues both in interphase and mitosis. Comparison of steady-state phosphorylation levels of spectrin in mitotic and interphase cells demonstrates that solubilization of spectrin in mitosis is correlated with a modest increase in the level of phosphorylation of the spectrin beta subunit in CHO and HeLa cells (a 40% and 70% increase, respectively). Two-dimensional phosphopeptide mapping of CHO cell spectrin indicates that this is due to mitosis-specific phosphorylation of beta-spectrin at several new sites. This is independent of cell rounding and dissociation from other cells and the substratum, since no changes in spectrin phosphorylation take place when cells are detached from culture dishes with trypsin-EDTA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Specific visualization of the distribution of the calcium dependent regulatory protein of cyclic nucleotide phosphodiesterase (modulator protein) in tissue culture cells by immunofluorescence microscopy: mitosis and intercellular bridge.

Monospecific antibodies against the homogeneous Ca++ dependent regulatory protein of cyclic nucleotide phosphodiesterase (CDR protein) from bovine brain were used in indirect immunofluorescence microscopy to visualize the cytoplasmic organization of this key regulatory protein in growing tissue culture cells. Although cells during interphase reveal only a weak general cytoplasmic fluorescence, a dramatic reorganization of CDR protein occurs with the onset of mitosis. Throughout the different mitotic stages CDR protein is strongly concentrated in the two polar parts of each half spindle. After completion of telophase (CDR protein appears at both cytoplasmic ends of the intercellular bridge which still connects the two daughter cells. Parallel use of monospecific antibodies against CDR protein and tubulin emphasizes the spatial restriction in the localization of CDR protein during mitosis and early G1 phase of the cell cycle.     

The degradation of two mitotic cyclins contributes to the timing of cytokinesis

BACKGROUND: Cytokinesis occurs just as chromosomes complete segregation and reform nuclei. It has been proposed that cyclin/Cdk kinase inhibits cytokinesis until exit from mitosis; however, the timer of cytokinesis has not been experimentally defined. Whereas expression of a stable version of Drosophila cyclin B blocks cytokinesis along with numerous events of mitotic exit, stable cyclin B3 allows cytokinesis even though it blocks late events of mitotic exit. We examined the interface between mitotic cyclin destruction and the timing of cytokinesis. RESULTS: In embryonic mitosis 14, the cytokinesis furrow appeared 60 s after the metaphase/anaphase transition and closed 90 s later during telophase. In cyclin B or cyclin B3 mutant cells, the cytokinesis furrow appeared at an earlier stage of mitosis. Expression of stable cyclin B3 delayed and prolonged furrow invagination; nonetheless, cytokinesis completed during the extended mitosis. Reduced function of Pebble, a Rho GEF required for cytokinesis, also delayed and slowed furrow invagination, but incomplete furrows were aborted at the time of mitotic exit. In functional and genetic tests, cyclin B and cyclin B3 inhibited Pebble contributions to cytokinesis. CONCLUSIONS: Temporal coordination of mitotic events involves inhibition of cytokinesis by cyclin B and cyclin B3 and punctual relief of the inhibition by destruction of these cyclins. Both cyclins inhibit Pebble-dependent activation of cytokinesis, whereas cyclin B can inhibit cytokinesis by additional modes. Stable cyclin B3 also blocks the later return to interphase that otherwise appears to impose a deadline for the completion of cytokinesis.     

Phosphorylation-induced rearrangement of the histone H3 NH2-terminal domain during mitotic chromosome condensation

The NH2-terminal domain (N-tail) of histone H3 has been implicated in chromatin compaction and its phosphorylation at Ser10 is tightly correlated with mitotic chromosome condensation. We have developed one mAb that specifically recognizes histone H3 N-tails phosphorylated at Ser10 (H3P Ab) and another that recognizes phosphorylated and unphosphorylated H3 N-tails equally well (H3 Ab). Immunocytochemistry with the H3P Ab shows that Ser10 phosphorylation begins in early prophase, peaks before metaphase, and decreases during anaphase and telophase. Unexpectedly, the H3 Ab shows stronger immunofluorescence in mitosis than interphase, indicating that the H3 N-tail is more accessible in condensed mitotic chromatin than in decondensed interphase chromatin. In vivo ultraviolet laser cross-linking indicates that the H3 N-tail is bound to DNA in interphase cells and that binding is reduced in mitotic cells. Treatment of mitotic cells with the protein kinase inhibitor staurosporine causes histone H3 dephosphorylation and chromosome decondensation. It also decreases the accessibility of the H3 N-tail to H3 Ab and increases the binding of the N-tail to DNA. These results indicate that a phosphorylation-dependent weakening of the association between the H3 N-tail and DNA plays a role in mitotic chromosome condensation.     

Effect of inhibition of the catalytic activity of cyclic AMP-dependent protein kinase on mitosis in PtK1 cells

Evidence has suggested that cyclic AMP, acting through activation of the type II cyclic AMP-dependent protein kinase, may play a role in the regulation of interphase and mitotic microtubules. In order to examine the potential role of the type II cAMP-dependent kinase during mitosis, dividing PtK1 cells were microinjected with two specific inhibitors of the catalytic activity of the type II kinase. These inhibitors were a specific protein inhibitor of cAMP-dependent protein kinase (PKI) and an affinity-purified polyclonal antiserum (anti-C) directed against the catalytic subunit of the kinase. Both have been shown previously to inhibit kinase activity in vitro. Microinjection of PKI during early- to mid-prophase significantly delayed the progression of the cells through mitosis, with the greatest delay occurring in metaphase. PKI injected during prometaphase also delayed progression through mitosis but to a lesser extent. Microinjection of anti-C during early- to mid-prophase also caused a significant delay in the completion of mitosis, with many cells becoming "hung up" in prometaphase. Anti-C injected during prometaphase had little effect on subsequent progression through mitosis. Microinjection of either anti-C or PKI during metaphase had no discernible effect. No effect on anaphase movement of chromosomes was observed with any treatment. These results provide further evidence that cAMP-dependent phosphorylation may be involved in the regulation of mitosis, although whether it acts directly through regulation of mitotic spindle microtubules is unclear.     

Identification and partial characterization of mitotic centromere- associated kinesin, a kinesin-related protein that associates with centromeres during mitosis

Using antipeptide antibodies to conserved regions of the kinesin motor domain, we cloned a kinesin-related protein that associates with the centromere region of mitotic chromosomes. We call the protein MCAK, for mitotic centromere-associated kinesin. MCAK appears concentrated on centromeres at prophase and persists until telophase, after which time the localization disperses. It is found throughout the centromere region and between the kinetochore plates of isolated mitotic CHO chromosomes, in contrast to two other kinetochore-associated microtubule motors: cytoplasmic dynein and CENP-E (Yen et al., 1992), which are closer to the outer surface of the kinetochore plates. Sequence analysis shows MCAK to be a kinesin-related protein with the motor domain located in the center of the protein. It is 60-70% similar to kif2, a kinesin-related protein originally cloned from mouse brain with a centrally located motor domain (Aizawa et al., 1992). MCAK protein is present in interphase and mitotic CHO cells and is transcribed as a single 3.4-kb message.     

Effect of phagocytosis on receptor distribution and endocytic activity in macrophages

Phagocytosis requires the internalization of a significant fraction of the plasma membrane and results in the intracellular deposition of large particles. We evaluated the effect of phagocytosis on the cellular distribution of recycling receptors and uptake of ligand to determine whether phagocytosis affects receptor behavior. Phagocytosis of zymosan, latex particles, or IgG-coated red blood cells by rabbit alveolar macrophages did not decrease the number of cell surface receptors for transferrin, alpha 2-macroglobulin X protease complexes, maleylated proteins, or mannosylated proteins. The number of surface receptors for transferrin was also unaltered in J774 cells, a macrophage-like cell line. In both cell types extensive phagocytosis did not affect the rate of receptor-mediated endocytosis or the distribution of receptors between the endosome and the cell surface. However, fluid phase pinocytosis was reduced by phagocytosis. The major reduction appeared to be not in the rate of internalization but rather in the delivery of fluid to the lysosome. These results demonstrate that internalization of a significant amount of the plasma membrane during phagocytosis does not diminish the number of receptors on the cell surface and has no effect on receptor-mediated ligand uptake.     

Actin dynamics during the cell cycle in Chlamydomonas reinhardtii.

We have used two monoclonal antibodies to demonstrate the presence and localization of actin in interphase and mitotic vegetative cells of the green alga Chlamydomonas reinhardtii. Commercially available monoclonal antibodies raised against smooth muscle actin (Lessard: Cell Motil. Cytoskeleton 10:349-362, 1988; Lin: Proc. Natl. Acad. Sci. USA 78:2335-2339, 1981) identify Chlamydomonas actin as a approximately 43,000-M(r) protein by Western immunoblot procedures. In an earlier study, Detmers and coworkers (Cell Motil. 5:415-430, 1985) first identified Chlamydomonas actin using NBD-phallacidin and an antibody raised against Dictyostelium actin; they demonstrated that F-actin is localized in the fertilization tubule of mating gametes. Here, we show by immunofluorescence that vegetative Chlamydomonas cells have an array of actin that surrounds the nucleus in interphase cells and undergoes dramatic reorganization during mitosis and cytokinesis. This includes the following: reorganization of actin to the anterior of the cell during preprophase; the formation of a cruciate actin band in prophase; reorganization to a single anterior actin band in metaphase; rearrangement forming a focus of actin anterior to the metaphase plate; reextension of the actin band in anaphase; presence of actin in the forming cleavage furrow during telophase and cytokinesis; and finally reestablishment of the interphase actin array. The studies presented here do not allow us to discriminate between G and F-actin. None the less, our observations, demonstrating dynamic reorganization of actin during the cell cycle, suggest a role for actin that may include the movement of basal bodies toward the spindle poles in mitosis and the formation of the cleavage furrow during cytokinesis.     

Differential dynamics of splicing factor SC35 during the cell cycle

Pre-mRNA splicing factors are enriched in nuclear domains termed interchromatin granule clusters or nuclear speckles. During mitosis, nuclear speckles are disassembled by metaphase and reassembled in telophase in structures termed mitotic interchromatin granules (MIGs). We analysed the dynamics of the splicing factor SC35 in interphase and mitotic cells. In HeLa cells expressing green fluorescent protein (GFP)-SC35, this was localized in speckles during interphase and dispersed in metaphase. In telophase, GFP-SC35 was highly enriched within telophase nuclei and also detected in MIGs. Fluorescence recovery after photobleaching (FRAP) experiments revealed that the mobility of GFP-SC35 was distinct in different mitotic compartments. Interestingly, the mobility of GFP-SC35 was 3-fold higher in the cytoplasm of metaphase cells compared with interphase speckles, the nucleoplasm or MIGs. Treatment of cells with inhibitors of cyclin-dependent kinases (cdks) caused changes in the organization of nuclear compartments such as nuclear speckles and nucleoli, with corresponding changes in the mobility of GFP-SC35 and GFP-fibrillarin. Our results suggest that the dynamics of SC35 are significantly influenced by the organization of the compartment in which it is localized during the cell cycle.