Characterization and dynamics of cytoplasmic F-actin in higher plant endosperm cells during interphase, mitosis, and cytokinesis

We have identified an F-actin cytoskeletal network that remains throughout interphase, mitosis, and cytokinesis of higher plant endosperm cells. Fluorescent labeling was obtained using actin monoclonal antibodies and/or rhodamine-phalloidin. Video-enhanced microscopy and ultrastructural observations of immunogold-labeled preparations illustrated microfilament-microtubule co-distribution and interactions. Actin was also identified in cell crude extract with Western blotting. During interphase, microfilament and microtubule arrays formed two distinct networks that intermingled. At the onset of mitosis, when microtubules rearranged into the mitotic spindle, microfilaments were redistributed to the cell cortex, while few microfilaments remained in the spindle. During mitosis, the cortical actin network remained as an elastic cage around the mitotic apparatus and was stretched parallel to the spindle axis during poleward movement of chromosomes. This suggested the presence of dynamic cross-links that rearrange when they are submitted to slow and regular mitotic forces. At the poles, the regular network is maintained. After midanaphase, new, short microfilaments invaded the equator when interzonal vesicles were transported along the phragmoplast microtubules. Colchicine did not affect actin distribution, and cytochalasin B or D did not inhibit chromosome transport. Our data on endosperm cells suggested that plant cytoplasmic actin has an important role in the cell cortex integrity and in the structural dynamics of the poorly understood cytoplasm-mitotic spindle interface. F-actin may contribute to the regulatory mechanisms of microtubule-dependent or guided transport of vesicles during mitosis and cytokinesis in higher plant cells.     

Localization of myosin II to chromosome arms and spindle fibers in PtK1 cells: a possible role for an actomyosin system in mitosis

Summary. The enzymes of importance in moving chromosomes are called motor proteins and include dynein, kinesin, and possibly myosin II. These three molecules are all included in the category of ATPases, in that they have the ability to convert chemical energy into mechanical energy. Both dynein and kinesin have been documented as molecules that “walk” along microtubules in the mitotic spindle, carrying cargo such as chromosomes. Myosin II, analogous to the muscle contraction system, transiently interacts along actin filaments and associates with kinetochore microtubules. In this paper we present evidence that a third ATPase, myosin II, may act as a “thruster” to propel chromosomes during the mitotic process. Double-label immunocytochemistry to actin and myosin II shows that myosin II is localized on chromosome arms at the beginning of mitosis and remains localized to the chromosomes throughout mitosis. Specific staining of myosin II is relegated to the outside of chromosomes with the highest density of staining occurring between the spindle poles and the chromosomes. This specific localization could account for the movement of chromosomes during mitosis, since they segregate towards the spindle poles, along kinetochore microtubules containing actin filaments, after aligning at the equatorial region of the cell at metaphase. We conclude from this study that there is an actomyosin system present in the mitotic spindle and that myosin is attached to chromosome arms and may act as a thruster in moving chromosomes during the mitotic process. Correspondence and reprints: Department of Biological Sciences, University of Denver, 2190 E Iliff Avenue, Denver, CO 80208, U.S.A.     

Involvement of actin filaments in chloroplast division of the algaClosterium ehrenbergii

Summary Studies have been made of whether actin filaments and microtubules are involved in the chloroplast division ofClosterium ehrenbergii (Conjugatae). Fluorostaining with rhodamine-phalloidin showed 5 types of localization of F-actin: (1) cables of actin filaments running in the cortical cytoplasm along the cell's long axis, (2) condensed actin filaments at the septum, (3) perinuclear distribution of actin filaments, (4) F-actins in a marking pin-like configuration adjacent to the nucleus of semicells just before completion of chloroplast kinesis, and (5) actin filaments girdling the isthmus of the constricted and dividing chloroplasts. Cytochalasin D (CD) at a concentration of 6 to 25 M caused significant disruption of actin filaments and the arrest of chloroplast kinesis, nuclear division, septum formation and cytoplasmic streaming within 3 to 6h. Chloroplast kinesis and cytoplasmic streaming recovered when cells were transferred to the medium without CD after CD treatment, or were subjected to prolonged contact with CD for more than 9h. In these cells there was a coincidental reappearance of actin filaments. A tubulin inhibitor, amiprophos-methyl at 330 M, did not inhibit chloroplast kinesis but did inhibit division and positioning of the nucleus. These results suggest that actin filaments do play a role in the mechanism of chloroplast kinesis but that microtubules do not appear to be involved in the process.Abbreviations APM amiprophos-methyl - CD cytochalasin D - DAPI 4,6-diamidino-2-phenylindole - DIC Nomarski differential interference contrast - DMSO dimethyl sulfoxide - Rh-Ph rhodamine-phalloidin     

Regulated assembly of the mitotic spindle: a perspective from two ends

Chromosome segregration and cell division requires the regulated assembly of the mitotic spindle apparatus. This mitotic spindle is composed of condensed chromosomes attached to a dynamic array of microtubules. The microtubule array is nucleated by centrosomes and organized by associated structural and motor proteins. Mechanical linkages between sister chromatids and microtubules are critical for spindle assembly and chromosome segregation. Defects in either chromosome or centrosome segregation can lead to aneuploidy and are correlated with cancer progression. In this review, we discuss current models of how centrosomes and chromosomes organize the spindle for their equal distribution to each daughter cell.     

Phallacidin stains the kinetochore region in the mitotic spindle of the green algaeOedogonium spp.

Summary We found previously that in living cells ofOedogonium cardiacum andO. donnellii, mitosis is blocked by the drug cytochalasin D (CD). We now report on the staining observed in these spindles with fluorescently actin-labeling reagents, particularly Bodipy FL phallacidin. Normal mitotic cells exhibited spots of staining associated with chromosomes; frequently the spots appeared in pairs during prometaphase-metaphase. During later anaphase and telophase, the staining was confined to the region between chromosomes and poles. The texture of the staining appeared to be somewhat dispersed by CD treatment but it was still present, particularly after shorter (<2 h) exposure. Electron microscopy of CD-treated cells revealed numerous spindle microtubules (MTs); many kinetochores had MTs associated with them, often laterally and some even terminating in the kinetochore as normal, but the usual bundle of kinetochore MTs was never present. As treatment with CD became prolonged, the kinetochores became shrunken and sunk into the chromosomes. These results support the possibility that actin is present in the kinetochore ofOedogonium spp. The previous observations on living cells suggest that it is a functional component of the kinetochore-MT complex involved in the correct attachment of chromosomes to the spindle.Abbreviations CD cytochalasin D - EM electron microscopy - MBS m-maleimidobenzoyl N-hydroxysuccinimide ester - MTs microtubules     

Chromosome attachment to the spindle in crane-fly spermatocytes requires actin and is necessary to initiate the anaphase-onset checkpoint

Summary We investigated the possible involvement of actin in the attachment of chromosomes to spindles in crane-fly primary spermatocytes. In a previous study, cytochalasin D, an inhibitor of actin polymerisation, prevented bivalent attachment to microtubules when applied at prophase, but did not cause the detachment of already attached bivalents. We were able to detach the already attached bivalents by first treating prometaphase cells with an antitubulin drug, nocodazole, to disrupt spindle microtubules. 2 min after nocodazole addition, we added cytochalasin D, to disrupt actin filaments; then 2 min later nocodazole was removed, and the cells were kept in cytochalasin D until the time of normal anaphase. Double treatment with nocodazole and cytochalasin D blocked reattachment of bivalents to the spindle. Single treatment with nocodazole alone caused chromosome detachment but did not prevent reattachment when nocodazole was washed out. Extended treatment with cytochalasin D alone starting in prometaphase did not cause bivalents to detach from the spindle. These data suggest that actin is needed for attachment of bivalents to spindle microtubules. This protocol is relevant to the anaphase-onset checkpoint. From previous experiments it was argued that the anaphase-onset checkpoint recognises unattached chromosomes only after those chromosomes first interact with (become attached to) the spindle. Our experiments showed that anaphase disjunction occurred at normal times when bivalents were prevented from attaching to the spindle (by adding cytochalasin D in prophase), while anaphase disjunction was greatly delayed when previously attached bivalents were detached (with nocodazole) and then prevented from re-attaching (with cytochalasin D) in the double treated cells. Thus the anaphaseonset checkpoint recognises only those unattached bivalents that previously were attached to the spindle. Other results provided further indication that actin-microtubule interactions are important in spindle organisation. Nocodazole treatment for 4 min caused most microtubules to disappear: bivalents aggregated around remnant microtubules. When cytochalasin D treatment followed nocodazole treatment, remnant spindle microtubules were not seen, suggesting that actin interactions help stabilise those microtubules.Abbreviations CD cytochalasin D - NMBD nuclear-membrane breakdown - NOC nocodazole     

What moves chromosomes, microtubules or microfilaments?

An investigation of the spindle apparatus of crane-fly (Nephrotoma suturalis) spermatocytes has been undertaken using methods that permit combined light and electron microscopy of selected cells. At the ultrastructural level, spindles contain microtubules in a granular matrix. Microtubules have been classified as kinetochore microtubules (which connect to kinetochores of chromosomes) and non-kinetochore microtubules (not attached to kinetochores). Kinetochore microtubules are distributed in densely packed bundles, which are the birefringent chromosomal fibers seen in living cells. Actin filaments were not observed in spindles of unglycerinated cells or in cells fixed in glutaraldehyde containing tannic acid, which negatively stains F-actin in situ and thus can be used to aid the localization of actin filaments in non-muscle cells. The absence of actin filaments in the spindle coupled with their presence in the "contractile ring" of spermatocytes fixed during cytokinesis is evidence against the hypothesis that chromosome movements are microfilament-based. The results are compatible with the hypothesis that microtubules are involved in the mechanism of chromosome transport. The details of that mechanism remain to be clarified.     

Spoke: a 120-kD protein associated with a novel filamentous structure on or near kinetochore microtubules in the mitotic spindle

We have characterized an antiserum that recognizes a single 120-kD protein in CHO cells which is soluble and cytoplasmically localized in interphase, but which is associated with a novel filamentous structure localized on or near kinetochore microtubules in mid-mitosis. These filaments, one per sister chromatid, run from near the mitotic spindle pole to within approximately 0.3 microns of each kinetochore. In metaphase, the staining pattern shows considerable substructure at light microscopy resolution, appearing as bright nodes or striations, often with a kinked or helical appearance. This overall localization pattern is retained throughout anaphase, with the filaments shortening as the chromosomes move toward the mitotic spindle poles. Also in anaphase, a separate ring-like structure lacking a tubulin-staining component appears near the spindle poles. As cells exit mitosis, the amount of this antigen in the cell decreases seven- to tenfold. The unusual staining pattern and the specific localization of this antigen on or near kinetochore microtubules in mid-mitosis indicate that the 120-kD protein defines or is associated with an important and previously unrecognized structural element of the mitotic spindle.     

Chromatin diminution and early cleavage in Parascaris univalens (Nematoda)

Summary In Parascaris developmental commitment to the germ line and somatic lineages is indicated by the orientation of the mitotic spindle in blastomeres, the topology of cells in the embryo, and chromatin diminution in presomatic blastomeres. Using three different experimental techniques: transient pressure treatment, application of cytochalasin B, and isolation of blastomeres, we have succeeded in uncoupling several developmental processes during cleavage of P. univalens. The following results were obtained: (1) Following mitotic nondisjunction we observed identical behavior of all chromatids in each blastomere. Thus chromosome differentiation by differential replication does not occur. (2) Chromosome fragments obtained by pressure treatment of egg cells underwent chromatin diminution. Thus this process does not require an intact germ-line chromosome. However, chromosomes immobilized on a monopolar spindle did not undergo chromatin diminution. Thus diminution appears to require segregation of chromatids. (3) Blastomeres that completely lacked chromosomes as a result of mitotic nondisjunction underwent normal early cleavage divisions. (4) Pressure treatment or prolonged treatment with cytochalasin B caused egg cells or germ line blastomeres to lose their germ line quality, as deduced from the coincident occurrence of symmetrical (presomatic-like) cleavage and chromatin diminution. (5) Isolated blastomeres from 2-cell embryos, i.e. 1/2 blastomeres, usually cleaved according to their prospective fates in the whole embryo. However, in some partial embryos derived from such blastomeres, chromatin diminution was delayed for either one or two cleavage mitoses. An activation model as an alternative to a prelocalization model is presented, which can account for early blastomere topogenesis and chromatin diminution.     

Fission yeast cells undergo nuclear division in the absence of spindle microtubules

Mitosis in eukaryotic cells employs spindle microtubules to drive accurate chromosome segregation at cell division. Cells lacking spindle microtubules arrest in mitosis due to a spindle checkpoint that delays mitotic progression until all chromosomes have achieved stable bipolar attachment to spindle microtubules. In fission yeast, mitosis occurs within an intact nuclear membrane with the mitotic spindle elongating between the spindle pole bodies. We show here that in fission yeast interference with mitotic spindle formation delays mitosis only briefly and cells proceed to an unusual nuclear division process we term nuclear fission, during which cells perform some chromosome segregation and efficiently enter S-phase of the next cell cycle. Nuclear fission is blocked if spindle pole body maturation or sister chromatid separation cannot take place or if actin polymerization is inhibited. We suggest that this process exhibits vestiges of a primitive nuclear division process independent of spindle microtubules, possibly reflecting an evolutionary intermediate state between bacterial and Archeal chromosome segregation where the nucleoid divides without a spindle and a microtubule spindle-based eukaryotic mitosis.     

PDIP38 is a novel mitotic spindle-associated protein that affects spindle organization and chromosome segregation

In order to maintain genomic integrity during mitosis, cells assemble the mitotic spindle to separate sister chromosomes to the two daughter cells. A variety of motor- and non motor-proteins are involved in the organization and regulation of this complex apparatus. DNA polymerase δ-interacting protein 38 (PDIP38) is a highly conserved protein and has so far been shown to be a cytoplasmic and nuclear protein. Cell cycle dependent nuclear localization and the interaction with DNA polymerase δ and proliferating cell nuclear antigen (PCNA) indicate a role for PDIP38 in DNA modification and/or proliferation. Here, we show for the first time that PDIP38 localizes to the mitotic spindle throughout mitosis. Using anti-PDIP38 antibody injections and siRNA silencing, we demonstrate that PDIP38 loss-of-function causes problems with spindle organization, aberrant chromosome segregation, and multinucleated cells. Taken together, the data indicate different roles for PDIP38 in safeguarding a proper cell division at various stages of the cell cycle, including DNA synthesis and repair, organization of the mitotic spindle and chromosome segregation.     

Septin 7 interacts with centromere-associated protein E and is required for its kinetochore localization

Chromosome segregation in mitosis is orchestrated by dynamic interaction between spindle microtubules and the kinetochore. Septin (SEPT) belongs to a conserved family of polymerizing GTPases localized to the metaphase spindle during mitosis. Previous study showed that SEPT2 depletion results in chromosome mis-segregation correlated with a loss of centromere-associated protein E (CENP-E) from the kinetochores of congressing chromosomes (1). However, it has remained elusive as to whether CENP-E physically interacts with SEPT and how this interaction orchestrates chromosome segregation in mitosis. Here we show that SEPT7 is required for a stable kinetochore localization of CENP-E in HeLa and MDCK cells. SEPT7 stabilizes the kinetochore association of CENP-E by directly interacting with its C-terminal domain. The region of SEPT7 binding to CENP-E was mapped to its C-terminal domain by glutathione S-transferase pull-down and yeast two-hybrid assays. Immunofluorescence study shows that SEPT7 filaments distribute along the mitotic spindle and terminate at the kinetochore marked by CENP-E. Remarkably, suppression of synthesis of SEPT7 by small interfering RNA abrogated the localization of CENP-E to the kinetochore and caused aberrant chromosome segregation. These mitotic defects and kinetochore localization of CENP-E can be successfully rescued by introducing exogenous GFP-SEPT7 into the SEPT7-depleted cells. These SEPT7-suppressed cells display reduced tension at kinetochores of bi-orientated chromosomes and activated mitotic spindle checkpoint marked by Mad2 and BubR1 labelings on these misaligned chromosomes. These findings reveal a key role for the SEPT7-CENP-E interaction in the distribution of CENP-E to the kinetochore and achieving chromosome alignment. We propose that SEPT7 forms a link between kinetochore distribution of CENP-E and the mitotic spindle checkpoint.     

Regulation of APC-Cdc20 by the spindle checkpoint

The spindle checkpoint ensures the fidelity of chromosome segregation in mitosis and meiosis. In response to defects in the mitotic apparatus, it blocks the activity of the anaphase-promoting complex, a large ubiquitin ligase required for chromosome segregation. Recent studies indicate that the spindle checkpoint monitors both the attachment of chromosomes to the mitotic spindle and the tension across the sister chromatid generated by microtubules. Upon checkpoint activation, checkpoint protein complexes containing BubR1(Mad3), Bub3, Mad2 and Cdc20 directly bind to the anaphase-promoting complex and inhibit its ligase activity. Therefore, the checkpoint proteins form a complex intracellular signalling network to inhibit the anaphase-promoting complex.     

Interrelation between the spatial disposition of actin filaments and microtubules during the differentiation of tracheary elements in culturedZinnia cells

Summary Changes in the spatial relationship between actin filaments and microtubules during the differentiation of tracheary elements (TEs) was investigated by a double staining technique in isolatedZinnia mesophyll cells. Before thickening of the secondary wall began to occur, the actin filaments and microtubules were oriented parallel to the long axis of the cell. Reticulate bundles of microtubules and aggregates of actin filaments emerged beneath the plasma membrane almost simultaneously, immediately before the start of the deposition of the secondary wall. The aggregates of actin filaments were observed exclusively between the microtubule bundles. Subsequently, the aggregates of actin filaments extended preferentially in the direction transverse to the long axis of the cell, and the arrays of bundles of microtubules which were still present between the aggregates of actin filaments became transversely aligned. The deposition of the secondary walls then took place along the transversely aligned bundles of microtubules.Disruption of actin filaments by cytochalasin B produced TEs with longitudinal bands of secondary wall, along which bundles of microtubules were seen, while TEs produced in the absence of cytochalasin B had transverse bands of secondary wall. These results indicate that actin filaments play an important role in the change in the orientation of arrays of microtubules from longitudinal to transverse. Disruption of microtubules by colchicine resulted in dispersal of the regularly arranged aggregates of actin filaments, but did not inhibit the formation of the aggregates itself, suggesting that microtubules are involved in maintaining the arrangement of actin filaments but are not involved in inducing the formation of the regularly arranged aggregates of actin filaments.These findings demonstrate that actin filaments cooperate with microtubules in controlling the site of deposition of the secondary wall in developing TEs.Abbreviations DMSO dimethylsulfoxide - EGTA ethyleneglycolbis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate - MSB microtubule-stabilizing buffer - PBS phosphate buffered saline - PIPES piperazine-N,N-bis(2-ethanesulfonic acid) - TE tracheary element     

Actin filaments and microtubules in the preprophase band and phragmoplast of tobacco cells

Summary Treatment with lysine prior to fixation of tobacco BY-2 cells with formaldehyde improved the preservation of actin filaments in the cells and enabled us to observe both networks of actin filaments and microtubules in the same cells. By using this method, we observed that (1) actin filaments were present in the preprophase band; (2) the actin filaments in the preprophase band and phragmoplast were runnig in the same direction as the microtubules in their respective structures; (3) a cortical network of actin filaments was present throughout all stages of cell cycle.The present method did not preserve the cortical actin filaments in interphase cells. The procedure for staining microtubules destroyed them.Abbreviations EGTA Ethyleneglycol-bis(-aminoethyl ether)N,N,N,N-tetraacetic acid - PIPES Piperazine-N,N-bis(2-ethanesulfonic acid) - PMSF Phenylmethylsulfonyl fluoride - TLCK Na-p-tosyl-L-lysine chloromethyl ketone     

Cytoskeletal changes during generative cell division and sperm formation inTradescantia virginiana

Summary Cytoskeletal organization and chromosome behavior were studied inTradescantia generative cells prior to and during sperm formation using in vitro grown pollen tubes and fluorescence staining methods. Before pollen germination, the crescent-shaped generative cell contains a reticulate microtubule (Mt) system. The cell elongates dramatically after germination, and its Mts assume a helical to longitudinal arrangement. Chromosome condensation is evident approximately 3hr after germination. Kinetochores appear as dark interruptions in the Mt array, and thus seem to attach directly to interphase fibers. No metaphase plate typical of other cells is observed with either DAPI or anti-tubulin staining. Instead, the chromosomes adopt a twisted or braided arrangement, with kinetochores distributed along the length of the cell and kinetochore fibers linked to each other and to surrounding fibers. Anaphase is characterized by a staggered, overlapping separation of chromosomes and by elongation of Mt branches connecting opposing kinetochore fibers. Cytokinesis appears to utilize a furrowing process; a phragmoplast or cell plate was never seen. As a result of these events, the sperm directly inherit their cytoskeleton from generative cell Mts involved in division. No actin fibers are observed at any stage using rhodamine-phalloidin staining. The results are discussed in terms of other reports on sperm formation, possible mitotic and cytokinetic mechanisms, and past distinctions between Mt arrays in higher plant somatic cells.Abbreviations CD cytochalasin D - DAPI 46-diamidino-2-phenyl-indole - DMSO dimethylsulfoxide - K-fiber kinetochore fiber - Mf microfilament - Mt microtubule - PPB preprophase Mt band - RP rhodamine phalloidin     

Intracellular distribution of subcellular organelles revealed by antibody against xyloglucan during cell cycle in tobacco BY-2 cells

Summary Immunofluorescence microscopy using an antibody against xyloglucan (XG) revealed its dynamics during the cell cycle. In interphase tobacco BY-2 cells, punctate and scattered fluorescence was observed throughout the cytoplasm. Colocalization of such signals with cortical microtubules (MTs) was clearly observed on the membrane ghosts. They were also associated and accumulated on MT bundles of the preprophase band. Treatment of protoplasts with cytochalasin B prior to the preparation of the ghosts had no effect on the pattern of anti-XG staining, while treatment with propyzamide caused the disappearance of the staining. These results suggest an association of Golgi apparatus and/or Golgi-derived vesicles with MTs. In metaphase cells, the staining was dispersed in the cytoplasm, except in the area occupied by the metaphase spindle. During anaphase, a broad fluorescence band appeared between daughter chromosomes and gradually concentrated at the equatorial plane before formation of the phragmoplast. At telophase, a bright line of fluorescence appeared at the equatorial plane corresponding to the position of the cell plate. The length of the line increased as cytokinesis proceeded. Thus, we showed that immunofluorescence microscopy using anti-XG antibody can be considered as a powerful tool for the analysis of Golgi apparatus and Golgi-derived vesicles containing XG.     

A spindle checkpoint functions during mitosis in the early Caenorhabditis elegans embryo

During mitosis, chromosome segregation is regulated by a spindle checkpoint mechanism. This checkpoint delays anaphase until all kinetochores are captured by microtubules from both spindle poles, chromosomes congress to the metaphase plate, and the tension between kinetochores and their attached microtubules is properly sensed. Although the spindle checkpoint can be activated in many different cell types, the role of this regulatory mechanism in rapidly dividing embryonic animal cells has remained controversial. Here, using time-lapse imaging of live embryonic cells, we show that chemical or mutational disruption of the mitotic spindle in early Caenorhabditis elegans embryos delays progression through mitosis. By reducing the function of conserved checkpoint genes in mutant embryos with defective mitotic spindles, we show that these delays require the spindle checkpoint. In the absence of a functional checkpoint, more severe defects in chromosome segregation are observed in mutants with abnormal mitotic spindles. We also show that the conserved kinesin CeMCAK, the CENP-F-related proteins HCP-1 and HCP-2, and the core kinetochore protein CeCENP-C all are required for this checkpoint. Our analysis indicates that spindle checkpoint mechanisms are functional in the rapidly dividing cells of an early animal embryo and that this checkpoint can prevent chromosome segregation defects during mitosis.     

Colocalization studies of Arp1 and p150Glued to spindle microtubules during mitosis: the effect of cytochalasin on the organization of microtubules and motor proteins in PtK1 cells

Motor proteins play a fundamental role in the congression and segregation of chromosomes in mitosis as well as the formation of the mitotic spindle. In particular, the dynein/dynactin complex is involved in the maintenance of the spindle, formation of astral microtubules, chromosome motion, and chromosome segregation. Dynactin is a multisubunit, high molecular weight protein that is responsible for the attachment of cargo to dynein. There are a number of major subunits in dynactin that are presumed to be important during mitosis. Arp1 is thought to be the attachment site for cargo to the complex while p150(Glued), a side arm of this complex regulates binding to MTs and the binding of dynactin to dynein. We performed colocalization studies of Arp1 and p150(Glued) to spindle microtubules. Both Arp1 and p150(Glued) colocalize with spindle MTs as well as cytoplasmic components. When treated with cytochalasin J, Arp1 concentrates at the centrosomes and is less co-localized with spindle MTs. Cytochalasin J has less of an effect on the colocalization of p150(Glued) with spindle MTs, suggesting that Arp1 may have a cytochalasin J sensitive site.     

Mitotic inhibition and chromosome displacement induced by estradiol in Chinese hamster cells

We tested diethylstilbestrol (DES) and 17 beta-estradiol as mitotic arrestants to determine their effects on chromosome distribution, spindle microtubules, and the cytoplasmic microtubule complex (CMTC) in the Chinese hamster strain Don. Cytological experiments assessed micronuclei induction, chromosome displacement, and anaphase recovery. Indirect immunofluorescence microscopy with antibody to tubulin and electron microscopy were used to illustrate effects on microtubules. Both DES and estradiol were potent inhibitors of mitosis when applied to cells in vitro. Estradiol induced micronuclei at a greater frequency than did DES. Estradiol-arrested metaphases often contained misaligned chromosomes despite the presence of a bipolar spindle and an equatorial plate. Equatorial plates were not observed in DES-arrested cells. Cells recovered quickly from estradiol exposure upon removal of the steroid. The frequency of abnormal metaphases and abnormal anaphases declined as the recovery period increased. Microtubule experiments showed that DES inhibited spindle assembly and disassembled the CMTC, whereas estradiol, at similar concentrations, arrested mitosis in a manner that allowed spindle assembly. A definite effect on the CMTC by estradiol could not be determined. However, changes in cell morphology were observed. In the presence of estradiol, centrosomes organized microtubules that joined with kinetochores of chromosomes at the equatorial plate as well as with those of misaligned chromosomes. Misaligned chromosomes appeared predominantly at polar regions of mitotic cells. Following drug removal, the pole-oriented chromosomes reoriented at the equatorial plate. The unique arresting properties of estradiol may prove useful in studies of chromosome migration and segregation during mitosis.