Reactivation of spindle elongation in vitro is correlated with the phosphorylation of a 205 kd spindle-associated protein

Mitotic spindles isolated from the diatom Stephanopyxis turris consist of two half-spindles of closely interdigitating microtubules that slide relative to one another in the presence of ATP, reinitiating spindle elongation (anaphase B) in vitro. Purified spindles that have been exposed to ATP-gamma-S undergo ATP-dependent reactivation more readily than do control spindles. Thiophosphorylated proteins in such spindles are located in the spindle midzone, kinetochores, and a portion of the pole complex. One major thiophosphorylated peptide of 205 kd is detected in extracts prepared from spindles labeled with [35S]ATP-gamma-S, and is also localized in the spindle midzone by using an antibody that recognizes thiophosphorylated proteins. It is likely that this 205 kd peptide is either a positive regulator or mechanochemical transducer of microtubule sliding when it is in a phosphorylated state.     

The mechanism of anaphase spindle elongation

At anaphase chromosomes move to the spindle poles (anaphase A) and the spindle poles move apart (anaphase B). In vitro studies using isolated diatom spindles demonstrate that the primary mechanochemical event responsible for spindle elongation is the sliding apart of half-spindle microtubules. Further, these forces are generated within the zone of microtubule overlap in the spindle mid-zone.     

Myosin-10 and actin filaments are essential for mitotic spindle function

Mitotic spindles are microtubule-based structures responsible for chromosome partitioning during cell division. Although the roles of microtubules and microtubule-based motors in mitotic spindles are well established, whether or not actin filaments (F-actin) and F-actin-based motors (myosins) are required components of mitotic spindles has long been controversial. Based on the demonstration that myosin-10 (Myo10) is important for assembly of meiotic spindles, we assessed the role of this unconventional myosin, as well as F-actin, in mitotic spindles. We find that Myo10 localizes to mitotic spindle poles and is essential for proper spindle anchoring, normal spindle length, spindle pole integrity, and progression through metaphase. Furthermore, we show that F-actin localizes to mitotic spindles in dynamic cables that surround the spindle and extend between the spindle and the cortex. Remarkably, although proper anchoring depends on both F-actin and Myo10, the requirement for Myo10 in spindle pole integrity is F-actin independent, whereas F-actin and Myo10 actually play antagonistic roles in maintenance of spindle length.     

Mitosis: spindle evolution and the matrix model

Current spindle models explain “anaphase A” (movement of chromosomes to the poles) in terms of a motility system based solely on microtubules (MTs) and that functions in a manner unique to mitosis. We find both these propositions unlikely. An evolutionary perspective suggests that when the spindle evolved, it should have come to share not only components (e.g., microtubules) of the interphase cell but also the primitive motility systems available, including those using actin and myosin. Other systems also came to be involved in the additional types of motility that now accompany mitosis in extant spindles. The resultant functional redundancy built reliability into this critical and complex process. Such multiple mechanisms are also confusing to those who seek to understand how chromosomes move. Narrowing this commentary down to just anaphase A, we argue that the spindle matrix participates with MTs in anaphase A and that this matrix may contain actin and myosin. The diatom spindle illustrates how such a system could function. This matrix may be motile and work in association with the MT cytoskeleton, as it does with the actin cytoskeleton during cell ruffling and amoeboid movement. Instead of pulling the chromosome polewards, the kinetochore fibre’s role might be to slow polewards movement to allow correct chromosome attachment to the spindle. Perhaps the earliest eukaryotic cell was a cytoplast organised around a radial MT cytoskeleton. For cell division, it separated into two cytoplasts via a spindle of overlapping MTs. Cytokinesis was actin-based cleavage. As chromosomes evolved into individual entities, their interaction with the dividing cytoplast developed into attachment of the kinetochore to radial (cytoplast) MTs. We believe it most likely that cytoplasmic motility systems participated in these events.     

Distribution of phosphorylated spindle-associated proteins in the diatom Stephanopyxis turris

Mitotic spindles isolated from the diatom Stephanopyxis turris become thiophosphorylated in the presence of ATP gamma S at specific locations within the mitotic apparatus, resulting in a stimulation of ATP-dependent spindle elongation in vitro. Here, using indirect immunofluorescence, we compare the staining pattern of an antibody against thiophosphorylated proteins to that of MPM-2, an antibody against mitosis-specific phosphoproteins, in isolated spindles. Both antibodies label spindle poles, kinetochores, and the midzone. Neither antibody exhibits reduced labeling in salt-extracted spindles, although prior salt extraction inhibits thiophosphorylation in ATP gamma S. Furthermore, both antibodies recognize a 205 kd band on immunoblots of spindle extracts. Microtubule-organizing centers and mitotic spindles label brightly with the MPM-2 antibody in intact cells. These results show that functional mitotic spindles isolated from S. turris are phosphorylated both in vivo and in vitro. We discuss the possible role of phosphorylated cytoskeletal proteins in the control of mitotic spindle function.     

Physiological evidence for involvement of a kinesin-related protein during anaphase spindle elongation in diatom central spindles

We have developed a new model system for studying spindle elongation in vitro using the pennate, marine diatom Cylindrotheca fusiformis. C. fusiformis can be grown in bulk to high densities while in log phase growth and synchronized by a simple light/dark regime. Isolated spindles can be attained in quantities sufficient for biochemical analysis and spindle tubulin is approximately 5% of the total protein present. The spindle isolation procedure results in a 10-fold enrichment of diatom tubulin and a calculated 40-fold increase in spindle protein. Isolated spindles or spindles in permeabilized cells can elongate in vitro by the same mechanism and with the same pharmacological sensitivities as described for other anaphase B models (Cande and McDonald, 1986; Masuda et al., 1990). Using this model, in vitro spindle elongation rate profiles were developed for a battery of nucleotide triphosphates and ATP analogs. The relative rates of spindle elongation produced by various nucleotide triphosphates parallel relative rates seen for kinesin-based motility in microtubule gliding assays. Likewise ATP analogs that allow discrimination between myosin-, dynein-, and kinesin-mediated motility produce relative spindle elongation rates characteristic of kinesin motility. Also, isolated spindle fractions are enriched for a kinesin related protein as identified by a peptide antibody against a conserved region of the kinesin superfamily. These data suggest that kinesin-like motility contributes to spindle elongation during anaphase B of mitosis.     

Mitosis: new roles for myosin-X and actin at the spindle

Roles for actin and myosin in positioning mitotic spindles in the cell are well established. A recent study of myosin-X function in early Xenopus embryo mitosis now reports that this unconventional myosin is required for pole integrity and normal spindle length by localizing to poles and exerting pulling forces on actin filaments within the spindle.     

Ultraviolet microbeam irradiations of mitotic diatoms: investigation of spindle elongation

Our simple instrumentation for generating a UV-microbeam is described UV microbeam irradiations of the central spindle in the pennate diatom Hantzschia amphioxys have been examined through correlated birefringence light microscopy and TEM. A precise correlation between the region of reduced birefringence and the UV-induced lesion in the microtubules (MTs) of the central spindle is demonstrated. The UV beam appears to dissociate MTs, as MT fragments were rarely encountered. The forces associated with metaphase and anaphase spindles have been studied via localized UV-microbeam irradiation of the central spindle. These spindles were found to be subjected to compressional forces, presumably exerted by stretched or contracting chromosomes. Comparisons are made with the results of other writers. These compressional forces caused the poles of a severed anaphase spindle to move toward each other and the center of the cell. As these poles moved centrally, the larger of the two postirradiational central spindle remnants elongated with a concomitant decrease in the length of the overlap. Metaphase spindles, in contrast, did not elongate nor lose their overlap region. Our interpretation is that the force for anaphase spindle elongation in Hantzschia is generated between half-spindles in the region of MT overlap.     

Development of cortical polarity in mouse eggs: involvement of the meiotic apparatus

Experiments were carried out to determine the origin of cortical polarity in mouse eggs and its possible relation to the meiotic apparatus. Cortices of mature eggs overlying the meiotic apparatus (microvillus-free area) were distinguished by an absence of microvilli and a thickened layer of actin. In contrast, the surfaces of immature oocytes were covered entirely with a dense population of microvilli and were subtended by a uniform layer of actin. When induced to undergo maturation, meiotic spindles formed in the center of immature oocytes and then moved peripherally. Coincident with the cortical localization of the meiotic spindle was the formation of a microvillus-free area, i.e., a loss of microvilli and a thickening of the actin layer associated with this region of the egg cortex. If immature oocytes were incubated in cytochalasin B, meiotic spindles formed; however, they failed to move peripherally and microvillus-free areas did not develop. Oocytes incubated in colchicine did not form meiotic spindles, although the chromosomes condensed and became localized to cortices where microvillus-free areas developed. Cytochalasin B-treated mature eggs maintained intact meiotic spindles and exhibited a disappearance of microvillus-free areas and a reduction in cortical actin. The chromosomes of mature eggs treated with colchicine remained associated with microvillus-free areas despite the disappearance of meiotic spindles. Occasionally, colchicine-treated eggs possessed more than one cortically located mass of chromosomes, each of which was associated with a microvillus-free area. These observations indicate that mechanisms involving the movement of the meiotic spindle to the oocyte cortex and development and maintenance of cortical polarity are cytochalasin B sensitive. Commensurate with the localization of meiotic chromosomes to the egg cortex is the reorganization of cortical actin and the formation of a microvillus-free area.     

Drosophila APC2 and Armadillo participate in tethering mitotic spindles to cortical actin

Proper positioning of mitotic spindles ensures equal allocation of chromosomes to daughter cells. This often involves interactions between spindle and astral microtubules and cortical actin. In yeast and Caenorhabditis elegans, some of the protein machinery that connects spindles and cortex has been identified but, in most animal cells, this process remains mysterious. Here, we report that the tumour suppressor homologue APC2 and its binding partner Armadillo both play roles in spindle anchoring during the syncytial mitoses of early Drosophila embryos. Armadillo, alpha-catenin and APC2 all localize to sites of cortical spindle attachment. APC2-Armadillo complexes often localize with interphase microtubules. Zeste-white 3 kinase, which can phosphorylate Armadillo and APC, is also crucial for spindle positioning and regulates the localization of APC2-Armadillo complexes. Together, these data suggest that APC2, Armadillo and alpha-catenin provide an important link between spindles and cortical actin, and that this link is regulated by Zeste-white 3 kinase.     

Dynein-dependent movements of the mitotic spindle in Saccharomyces cerevisiae Do not require filamentous actin

In budding yeast, the mitotic spindle is positioned in the neck between the mother and the bud so that both cells inherit one nucleus. The movement of the mitotic spindle into the neck can be divided into two phases: (1) Kip3p-dependent movement of the nucleus to the neck and alignment of the short spindle, followed by (2) dynein-dependent movement of the spindle into the neck and oscillation of the elongating spindle within the neck. Actin has been hypothesized to be involved in all these movements. To test this hypothesis, we disrupted the actin cytoskeleton with the use of mutations and latrunculin A (latrunculin). We assayed nuclear segregation in synchronized cell populations and observed spindle movements in individual living cells. In synchronized cell populations, no actin cytoskeletal mutant segregated nuclei as poorly as cells lacking dynein function. Furthermore, nuclei segregated efficiently in latrunculin-treated cells. Individual living cell analysis revealed that the preanaphase spindle was mispositioned and misaligned in latrunculin-treated cells and that astral microtubules were misoriented, confirming a role for filamentous actin in the early, Kip3p-dependent phase of spindle positioning. Surprisingly, mispositioned and misaligned mitotic spindles moved into the neck in the absence of filamentous actin, albeit less efficiently. Finally, dynein-dependent sliding of astral microtubules along the cortex and oscillation of the elongating mitotic spindle in the neck occurred in the absence of filamentous actin.     

The role of tubulin polymerization during spindle elongation in vitro

We describe the effect of exogenous tubulin on reactivation of anaphase spindle elongation in isolated diatom spindles. In the absence of tubulin, spindle elongation is limited to the equivalent of the microtubule overlap zone, but in the presence of tubulin spindle elongation is several times the length of the overlap zone. Biotinylated neurotubulin is incorporated into the overlap zone and around the poles. Before spindles have elongated by the equivalent of the overlap zone, there are two regions of incorporated tubulin flanking this zone. After further elongation, there is one broad zone of incorporated tubulin in the spindle midzone. Spindle elongation and the pattern of tubulin incorporation into the midzone, but not the poles, are ATP-dependent and vanadate-sensitive. These results suggest that tubulin adds onto the ends of microtubules in the overlap zone, which then slide through the midzone as the spindle elongates.     

The distribution of cytoplasmic microtubules throughout the cell cycle of the centric diatom Stephanopyxis turris: their role in nuclear migration and positioning the mitotic spindle during cytokinesis

The cell cycle of the marine centric diatom Stephanopyxis turris consists of a series of spatially and temporally well-ordered events. We have used immunofluorescence microscopy to examine the role of cytoplasmic microtubules in these events. At interphase, microtubules radiate out from the microtubule-organizing center, forming a network around the nucleus and extending much of the length and breadth of the cell. As the cell enters mitosis, this network breaks down and a highly ordered mitotic spindle is formed. Peripheral microtubule bundles radiate out from each spindle pole and swing out and away from the central spindle during anaphase. Treatment of synchronized cells with 2.5 X 10(-8) M Nocodazole reversibly inhibited nuclear migration concurrent with the disappearance of the extensive cytoplasmic microtubule arrays associated with migrating nuclei. Microtubule arrays and mitotic spindles that reformed after the drug was washed out appeared normal. In contrast, cells treated with 5.0 X 10(-8) M Nocodazole were not able to complete nuclear migration after the drug was washed out and the mitotic spindles that formed were multipolar. Normal and multipolar spindles that were displaced toward one end of the cell by the drug treatment had no effect on the plane of division during cytokinesis. The cleavage furrow always bisected the cell regardless of the position of the mitotic spindle, resulting in binucleate/anucleate daughter cells. This suggests that in S. turris, unlike animal cells, the location of the plane of division is cortically determined before mitosis.     

Direct observation of mitotic spindle elongation in vitro

Successful reactivation in vitro of anaphase B has recently been achieved with mitotic spindles isolated from the diatom Stephanopyxis turris. When a population of isolated spindles was studied indirectly by using immunofluorescence, nearly all of them were found to have elongated; however, when studied directly by using video microscopy, only a small proportion of spindles elongated. We report here conditions that allow nearly all of the spindles to elongate when observed directly with video microscopy. These direct observations validate previous ones made using indirect immunofluorescence. In addition, we find that the isolated spindles elongate with a linear rate, that the elongation is unchanged after the chromatin surrounding the spindles is digested with DNase I, and that during elongation a phase-dense matrix may accumulate in the spindle midzone.     

Disruption of astral microtubule contact with the cell cortex activates a Bub1, Bub3, and Mad3-dependent checkpoint in fission yeast

In animal and yeast cells, the mitotic spindle is aligned perpendicularly to the axis of cell division. This ensures that sister chromatids are separated to opposite sides of the cytokinetic actomyosin ring. In fission yeast, spindle rotation is dependent upon the interaction of astral microtubules with the cortical actin cytoskeleton. In this article, we show that addition of Latrunculin A, which prevents spindle rotation, delays the separation of sister chromatids and anaphase promoting complex-mediated destruction of spindle-associated Securin and Cyclin B. Moreover, we find that whereas sister kinetochore pairs normally congress to the spindle midzone before anaphase onset, this congression is disrupted when astral microtubule contact with the actin cytoskeleton is disturbed. By analyzing the timing of kinetochore separation, we find that this anaphase delay requires the Bub3, Mad3, and Bub1 but not the Mad1 or Mad2 spindle assembly checkpoint proteins. In agreement with this, we find that Bub1 remains associated with kinetochores when spindles are mispositioned. These data indicate that, in fission yeast, astral microtubule contact with the medial cell cortex is monitored by a subset of spindle assembly checkpoint proteins. We propose that this checkpoint ensures spindles are properly oriented before anaphase takes place.     

Adducin-1 is essential for mitotic spindle assembly through its interaction with myosin-X

Mitotic spindles are microtubule-based structures, but increasing evidence indicates that filamentous actin (F-actin) and F-actin–based motors are components of these structures. ADD1 (adducin-1) is an actin-binding protein that has been shown to play important roles in the stabilization of the membrane cortical cytoskeleton and cell–cell adhesions. In this study, we show that ADD1 associates with mitotic spindles and is crucial for proper spindle assembly and mitotic progression. Phosphorylation of ADD1 at Ser12 and Ser355 by cyclin-dependent kinase 1 enables ADD1 to bind to myosin-X (Myo10) and therefore to associate with mitotic spindles. ADD1 depletion resulted in distorted, elongated, and multipolar spindles, accompanied by aberrant chromosomal alignment. Remarkably, the mitotic defects caused by ADD1 depletion were rescued by reexpression of ADD1 but not of an ADD1 mutant defective in Myo10 binding. Together, our findings unveil a novel function for ADD1 in mitotic spindle assembly through its interaction with Myo10.     

AMPK regulates mitotic spindle orientation through phosphorylation of myosin regulatory light chain

The proper orientation of the mitotic spindle is essential for mitosis; however, how these events unfold at the molecular level is not well understood. AMP-activated protein kinase (AMPK) regulates energy homeostasis in eukaryotes, and AMPK-null Drosophila mutants have spindle defects. We show that threonine(172) phosphorylated AMPK localizes to the mitotic spindle poles and increases when cells enter mitosis. AMPK depletion causes a mitotic delay with misoriented spindles relative to the normal division plane and a reduced number and length of astral microtubules. AMPK-depleted cells contain mitotic actin bundles, which prevent astral microtubule-actin cortex attachments. Since myosin regulatory light chain (MRLC) is an AMPK downstream target and mediates actin function, we investigated whether AMPK signals through MRLC to control spindle orientation. Mitotic levels of serine(19) phosphorylated MRLC (pMRLC(ser19)) and spindle pole-associated pMRLC(ser19) are abolished when AMPK function is compromised, indicating that AMPK is essential for pMRLC(ser19) spindle pole activity. Phosphorylation of AMPK and MRLC in the mitotic spindle is dependent upon calcium/calmodulin-dependent protein kinase kinase (CamKK) activity in LKB1-deficient cells, suggesting that CamKK regulates this pathway when LKB1 function is compromised. Taken together, these data indicate that AMPK mediates spindle pole-associated pMRLC(ser19) to control spindle orientation via regulation of actin cortex-astral microtubule attachments.     

The mechanism of anaphase spindle elongation: uncoupling of tubulin incorporation and microtubule sliding during in vitro spindle reactivation

To study tubulin polymerization and microtubule sliding during spindle elongation in vitro, we developed a method of uncoupling the two processes. When isolated diatom spindles were incubated with biotinylated tubulin (biot-tb) without ATP, biot-tb was incorporated into two regions flanking the zone of microtubule overlap, but the spindles did not elongate. After biot-tb was removed, spindle elongation was initiated by addition of ATP. The incorporated biot-tb was found in the midzone between the original half-spindles. The extent and rate of elongation were increased by preincubation in biot-tb. Serial section reconstruction of spindles elongating in tubulin and ATP showed that the average length of half-spindle microtubules increased due to growth of microtubules from the ends of native microtubules. The characteristic packing pattern between antiparallel microtubules was retained even in the "new" overlap region. Our results suggest that the forces required for spindle elongation are generated by enzymes in the overlap zone that mediate the sliding apart of antiparallel microtubules, and that tubulin polymerization does not contribute to force generation. Changes in the extent of microtubule overlap during spindle elongation were affected by tubulin and ATP concentration in the incubation medium. Spindles continued to elongate even after the overlap zone was composed entirely of newly polymerized microtubules, suggesting that the enzyme responsible for microtubule translocation either is bound to a matrix in the spindle midzone, or else can move on one microtubule toward the spindle midzone and push another microtubule of opposite polarity toward the pole.     

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     

Myosin localization during meiosis I of crane-fly spermatocytes gives indications about its role in division

We showed previously that in crane-fly spermatocytes myosin is required for tubulin flux [Silverman-Gavrila and Forer, 2000a: J Cell Sci 113:597-609], and for normal anaphase chromosome movement and contractile ring contraction [Silverman-Gavrila and Forer, 2001: Cell Motil Cytoskeleton 50:180-197]. Neither the identity nor the distribution of myosin(s) were known. In the present work, we used immunofluorescence and confocal microscopy to study myosin during meiosis-I of crane-fly spermatocytes compared to tubulin, actin, and skeletor, a spindle matrix protein, in order to further understand how myosin might function during cell division. Antibodies to myosin II regulatory light chain and myosin II heavy chain gave similar staining patterns, both dependent on stage: myosin is associated with nuclei, asters, centrosomes, chromosomes, spindle microtubules, midbody microtubules, and contractile rings. Myosin and actin colocalization along kinetochore fibers from prometaphase to anaphase are consistent with suggestions that acto-myosin forces in these stages propel kinetochore fibres poleward and trigger tubulin flux in kinetochore fibres, contributing in this way to poleward chromosome movement. Myosin and actin colocalization at the cell equator in cytokinesis, similar to studies in other cells [e.g., Fujiwara and Pollard, 1978: J Cell Biol 77:182-195], supports a role of actin-myosin interactions in contractile ring function. Myosin and skeletor colocalization in prometaphase spindles is consistent with a role of these proteins in spindle formation. After microtubules or actin were disrupted, myosin remained in spindles and contractile rings, suggesting that the presence of myosin in these structures does not require the continued presence of microtubules or actin. BDM (2,3 butanedione, 2 monoxime) treatment that inhibits chromosome movement and cytokinesis also altered myosin distributions in anaphase spindles and contractile rings, consistent with the physiological effects, suggesting also that myosin needs to be active in order to be properly distributed.