Spindle Actin Research Articles

FAM110A promotes mitotic spindle formation by linking microtubules with actin cytoskeleton

Precise segregation of chromosomes during mitosis requires assembly of a bipolar mitotic spindle followed by correct attachment of microtubules to the kinetochores. This highly spatiotemporally organized process is controlled by various mitotic kinases and molecular motors. We have recently shown that Casein Kinase 1 (CK1) promotes timely progression through mitosis by phosphorylating FAM110A leading to its enrichment at spindle poles. However, the mechanism by which FAM110A exerts its function in mitosis is unknown. Using structure prediction and a set of deletion mutants, we mapped here the interaction of the N- and C-terminal domains of FAM110A with actin and tubulin, respectively. Next, we found that the FAM110A-Δ40-61 mutant deficient in actin binding failed to rescue defects in chromosomal alignment caused by depletion of endogenous FAM110A. Depletion of FAM110A impaired assembly of F-actin in the proximity of spindle poles and was rescued by expression of the wild-type FAM110A, but not the FAM110A-Δ40-61 mutant. Purified FAM110A promoted binding of F-actin to microtubules as well as bundling of actin filaments in vitro. Finally, we found that the inhibition of CK1 impaired spindle actin formation and delayed progression through mitosis. We propose that CK1 and FAM110A promote timely progression through mitosis by mediating the interaction between spindle microtubules and filamentous actin to ensure proper mitotic spindle formation.

F-actin coordinates spindle morphology and function in Drosophila meiosis.

Meiosis is a highly conserved feature of sexual reproduction that ensures germ cells have the correct number of chromosomes prior to fertilization. A subset of microtubules, known as the spindle, are essential for accurate chromosome segregation during meiosis. Building evidence in mammalian systems has recently highlighted the unexpected requirement of the actin cytoskeleton in chromosome segregation; a network of spindle actin filaments appear to regulate many aspects of this process. Here we show that Drosophila oocytes also have a spindle population of actin that appears to regulate the formation of the microtubule spindle and chromosomal movements throughout meiosis. We demonstrate that genetic and pharmacological disruption of the actin cytoskeleton has a significant impact on spindle morphology, dynamics, and chromosome alignment and segregation during maturation and the metaphase-anaphase transition. We further reveal a role for calcium in maintaining the microtubule spindle and spindle actin. Together, our data highlights potential conservation of morphology and mechanism of the spindle actin during meiosis.

WHAMM is essential for spindle formation and spindle actin polymerization in maturing mouse oocytes

ABSTRACT WHAMM (WAS Protein Homolog Associated with Actin, Golgi Membranes, and Microtubules) is involved in Golgi membrane association, microtubule binding, and actin nucleation as a nucleation-promoting factor, which activates the actin-related protein 2/3 complex (the Arp2/3 complex). However, the role of WHAMM in mammalian oocyte maturation is poorly understood. The presence of WHAMM mRNA and protein during all stages of mouse oocyte maturation has been verified. It is mainly co-localized with the actin cage permeating the spindle during mouse oocyte maturation. Through the knockdown of WHAMM, we confirmed that it regulates spindle formation and affects the localization of the microtubule-organizing center (MTOC) during the early stages of spindle formation. Moreover, depletion of WHAMM impaired the formation of the spindle actin and chromosome alignment, which might be the cause of chromosomal aneuploidy and abnormal, asymmetric division. Treatment with brefeldin A (BFA), an inhibitor of vesicle transport from the endoplasmic reticulum (ER) to the Golgi apparatus, induced abnormal and dispersed localization of WHAMM. Taken together, these findings show that WHAMM is an essential component of the actin cytoskeleton machinery and plays a crucial role in oocyte maturation, presumably by controlling the formation of spindles with normal length by activating the formation of the spindle actin via the Arp2/3 complex.

Correction to: Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes.

Chapter 17 "Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes" was previously published non-open access. It has now been changed to open access under a CC BY 4.0 license and the copyright holder has been updated to "The Author(s)."

Visualization and Functional Analysis of Spindle Actin and Chromosome Segregation in Mammalian Oocytes.

Chromosome segregation is conserved throughout eukaryotes. In most systems, it is solely driven by a spindle machinery that is assembled from microtubules. We have recently discovered that actin filaments that are embedded inside meiotic spindles (spindle actin) are needed for accurate chromosome segregation in mammalian oocytes. To understand the function of spindle actin in oocyte meiosis, we have developed high-resolution and super-resolution live and immunofluorescence microscopy assays that are described in this chapter.

Centrosomal Actin Assembly Is Required for Proper Mitotic Spindle Formation and Chromosome Congression

SummaryCytoskeletal cross talk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e., during filopodia formation or cilia organization. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle, thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with spindle microtubules forming during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus centrosomal Arp2/3 complex activity plays a role in the maintenance of genomic integrity during mitosis.

Centrosomal Actin Assembly is Required for Proper Mitotic Spindle Formation and Chromosome Congression

Cytoskeletal crosstalk between actin filaments and microtubules is a common mechanism governing the assembly of cellular structures, i.e. during filopodia formation or cilia organisation. However, potential actin-microtubule interactions during mammalian cell divisions are less well understood. At mitotic entry, centrosomes propagate the formation of the mitotic spindle thereby aligning individual chromosomes to the metaphase plate, a process coined chromosome congression. Here, we identify actin filament assembly spatially defined at centrosomes contemporaneously with forming spindle microtubules during prometaphase. We show that pharmacological Arp2/3 complex inhibition as well as overexpression of the Arp2/3 complex inhibitory protein Arpin decreased spindle actin formation. As a consequence, mitotic spindle formation is impaired, which resulted in disorganized chromosome congression and ultimately mitotic defects in non-transformed cells. Thus, centrosomal Arp2/3 complex activity plays a role for maintenance of genomic integrity during mitosis.

Actin protects mammalian eggs against chromosome segregation errors.

Chromosome segregation is driven by a spindle that is made of microtubules but is generally thought to be independent of actin. Here, we report an unexpected actin-dependent mechanism that drives the accurate alignment and segregation of chromosomes in mammalian eggs. Prominent actin filaments permeated the microtubule spindle in eggs of several mammalian species, including humans. Disrupting actin in mouse eggs led to significantly increased numbers of misaligned chromosomes as well as lagging chromosomes during meiosis I and II. We found that actin drives accurate chromosome segregation by promoting the formation of functional kinetochore fibers, the microtubule bundles that align and segregate the chromosomes. Thus, actin is essential to prevent chromosome segregation errors in eggs, which are a leading cause of miscarriages, infertility, and Down syndrome.

Jasplakinolide, an actin stabilizing agent, alters anaphase chromosome movements in crane‐fly spermatocytes

We added jasplakinolide to anaphase crane-fly spermatocytes and determined its effects on chromosome movement. Previous work showed that the actin depolymerizing agents cytochalasin D or latrunculin B blocked or slowed chromosome movements. We studied the effects of jasplakinolide, a compound that stabilizes actin filaments. Jasplakinolide had the same effect on movements of each half- bivalent in a separating pair of half-bivalents, but different half-bivalent pairs in the same cell often responded differently, even when the concentrations of jasplakinolide varied by a factor of two. Jasplakinolide had no effect on about 20% of the pairs, but otherwise caused movements to slow, or to stop, or, rarely, to accelerate. When cells were kept in jasplakinolide, stopped pairs eventually resumed movement; slowed pairs did not change their speeds. Confocal microscopy indicated that neither the distributions of spindle actin filaments nor the distributions of spindle microtubules were altered by the jasplakinolide. It is possible that jasplakinolide binds to spindle actin and blocks critical binding sites, but we suggest that jasplakinolide affects anaphase chromosome movement by preventing actin-filament depolymerization that is necessary for anaphase to proceed. Overall, our data indicate that actin is involved in one of the redundant mechanisms cells use to move chromosomes.

Calyculin A, an enhancer of myosin, speeds up anaphase chromosome movement.

Actin and myosin inhibitors often blocked anaphase movements in insect spermatocytes in previous experiments. Here we treat cells with an enhancer of myosin, Calyculin A, which inhibits myosin-light-chain phosphatase from dephosphorylating myosin; myosin thus is hyperactivated. Calyculin A causes anaphase crane-fly spermatocyte chromosomes to accelerate poleward; after they reach the poles they often move back toward the equator. When added during metaphase, chromosomes at anaphase move faster than normal. Calyculin A causes prometaphase chromosomes to move rapidly up and back along the spindle axis, and to rotate. Immunofluorescence staining with an antibody against phosphorylated myosin regulatory light chain (p-squash) indicated increased phosphorylation of cleavage furrow myosin compared to control cells, indicating that calyculin A indeed increased myosin phosphorylation. To test whether the Calyculin A effects are due to myosin phosphatase or to type 2 phosphatases, we treated cells with okadaic acid, which inhibits protein phosphatase 2A at concentrations similar to Calyculin A but requires much higher concentrations to inhibit myosin phosphatase. Okadaic acid had no effect on chromosome movement. Backward movements did not require myosin or actin since they were not affected by 2,3-butanedione monoxime or LatruculinB. Calyculin A affects the distribution and organization of spindle microtubules, spindle actin, cortical actin and putative spindle matrix proteins skeletor and titin, as visualized using immunofluorescence. We discuss how accelerated and backwards movements might arise.

Visualization of actin cytoskeletal dynamics during the cell cycle in tobacco (Nicotiana tabacum L. cv Bright Yellow) cells

The actin cytoskeleton forms distinct actin arrays which fulfil their functions during cell cycle progression. Reorganization of the actin cytoskeleton occurs during transition from one actin array to another. Although actin arrays have been well described during cell cycle progression, the dynamic organization of the actin cytoskeleton during actin array transition remains to be dissected. In the present study, a GFP (green fluorescent protein)-mTalin (mouse talin) fusion gene was introduced into suspension-cultured tobacco BY-2 (Nicotiana tabacum L. cv Bright Yellow) cells by a calli-cocultivation transformation method to visualize the reorganization of the actin cytoskeleton in vivo during the progression of the cell cycle. Typical actin structures were indicated by GFP-mTalin, such as the pre-prophase actin band, mitotic spindle actin filament cage and phragmoplast actin arrays. In addition, dynamic organization of actin filaments was observed during the progression of the cell from metaphase to anaphase. In late metaphase, spindle actin filaments gradually shrank to the equatorial plane along both the long and short axes. Soon after the separation of sister chromosomes, actin filaments aligned in parallel at the cell division plane, forming a cylinder-like structure. During the formation of the cell plate, one cylinder-like structure changed into two cylinder-like structures: the typical actin arrays of the phragmoplast. However, the two actin arrays remained overlapping at the margin of the centrally growing cell plate, forming an actin wreath. When the cell plate matured further, an actin filament network attached to the cell plate was formed. Our results clearly describe the dynamic organization of the actin cytoskeleton during mitosis and cytokinesis of a plant cell. This demonstrates that GFP-mTalin-transformed tobacco BY-2 cells are a valuable tool to study actin cytoskeleton functions in the plant cell cycle.

Phallacidin stains the mitotic spindle of the diatom Pinnularia spp.

Mitotic spindles of the diatom Pinnularia viritiformis stained with the fluorescent actin-labelling reagent bodipyphallacidin revealed actin among the chromosomes and extending along the spindle to the poles. This is the first report of actin's presence within spindles of this ecologically important group of organisms. Since diatom mitoses have a number of marked differences compared to that of many other eukaryotes (Int Rev Cytol 128 (1991) 63; Cell 14 (1978) 455), the present observations substantially extend the diversity of mitotic spindle types in which there is evidence of spindle actin.

Evidence that actin and myosin are involved in the poleward flux of tubulin in metaphase kinetochore microtubules of crane-fly spermatocytes.

We studied the effects of various drugs on the poleward flux of tubulin in kinetochore microtubules in metaphase-I crane-fly spermatocytes. We used as a measure of tubulin flux a 'gap' in acetylation of kinetochore microtubules immediately poleward from the kinetochore; the 'gap' is caused by a time lag between incorporation of new tubulin subunits at the kinetochore and subsequent acetylation of those subunits as they flux to the pole. We confirmed that the 'gap' is due to flux by showing that the 'gap' disappeared when cells were treated briefly with the anti-tubulin drug nocodazole, which decreases microtubule dynamics. The 'gap' disappeared when cells were treated for 10 minutes with anti-actin drugs (cytochalasin D, latrunculin B, swinholide A), or with the anti-myosin drug 2,3-butanedione 2-monoxime. The 'gap' did not disappear when cells were treated with the actin stabilizing drug jasplakinolide. We studied whether these drugs altered spindle actin. We used fluorescent phalloidin to visualize spermatocyte F-actin, which was associated with kinetochore spindle fibers as well as the cell cortex, the contractile ring and finger-like protrusions at the poles. Spindle F-actin was no longer seen after cells were treated with cytochalasin D, swinholide A or a high concentration of latrunculin B, whereas a low concentration of latrunculin B, which did not completely remove the 'gap', caused reduced staining of spindle actin. Neither 2,3-butanedione 2-monoxime nor jasplakinolide altered spindle actin. These data suggest that an actomyosin mechanism drives the metaphase poleward tubulin flux.

F-Actin organization during the cell cycle of Sphacelaria rigidula (Phaeophyceae)

The organization of F-actin in vegetative cells of the brown alga Sphacelaria rigidula was studied after staining with a modified rhodaminephalloidin (Rh-Ph) protocol. The interphase vegetative cells display a well-organized F-actin cytoskeleton, consisting of cortical, endoplasmic and perinuclear arrays of actin filaments (AFs). The organization of these AFs changes slightly during mitosis, while they almost disappear at cytokinesis. The perinuclear AF population becomes more obvious during prophase, especially at the poles. At metaphase, an actin spindle is organized, co-localized with the microtubule spindle, while at anaphase an interzonal AF population appears, which persists at early telophase. At advanced telophase the image changes to a rather diffuse actin meshwork in the mid-area between the daughter nuclei. During post-telophase-early cytokinesis this AF system becomes gradually disassembled and a conspicuous actin plate is formed on the cytokinetic plane. This plate becomes more compact with the progress of cytokinesis, and seems to overcoat the patches of the forming diaphragm, and finally the daughter plasmalemmata. Treatment with cytochalasin B disturbs mitosis, not allowing the cells to proceed to anaphase, and prevents cytokinesis. These observations show that AFs are ubiquitous cytoskeletal elements of the vegetative cells of brown algae. The probable role of AFs during the cell cycle and particularly the unique actin configuration involved in cytokinesis are discussed.

Actin in sperm tails and meiotic spindles.

IN many cases cellular motility seems to be due to interactions between actin and myosin1–8. Actin (or actin-like material) has also been identified in other cellular systems which exhibit motility9–14 and actin-like and myosin-like proteins have been claimed to be isolated from mitochondria15 and chloroplasts16. While it is not proved that motility in these systems is due to actin and myosin, actin-myosin interactions are probably involved. Flagellar (ciliary) motions and chromosome movements, on the other hand, have generally been attributed to microtubules17–19, and thus there seemed to be two different kinds of motile systems; one based on actin-myosin interactions and the other on microtubules. We have found, however, actin-like filaments in both sperm tails (flagellae) and meiotic spindles of crane flies. Although we are well aware that we have demonstrated merely the presence of actin and have by no means proven that the flagellar and spindle actin has anything to do with motility, we nonetheless suggest that motility in all motile systems in higher cells is based on actin-myosin interactions.