Dynamics of the actin cytoskeleton,hyphal tip growth and the movement of the two nuclei in the dikaryon ofCoprinus cinereus

We first examined the changes in distribution of F-actin during conjugate division in the apical cells of the dikaryon ofCoprinus cinereus using indirect immunofluorescence microscopy, then followed hyphal tip growth and the movement of the two nuclei in the apical cells using differential interference contrast microscopy (DIC). In apical cells with interphase nuclei, F-actin occurred solely as peripheral plaques, which were distributed along the whole length of the cell and were more concentrated at the tips, where they formed caps. In the early prophase of conjugate division, F-actin was transiently concentrated, as diffused form and plaques, at hyphal regions where the two nuclei sit, and this was accompanied by transient disappearance of the actin cap at the hyphal tip in the majority of cells. The actin cap was also present at the tips of growing clamp cells from late prophase through metaphase and disintegrated during anaphase. In telophase, actin rings formed at the future septa. DIC revealed that, in early prophase, when the F-actin array occurs around the two nuclei and the actin cap is absent at hyphal tips, hyphae kept growing and the second nucleus accelerated its forward movement to catch up with the leading nucleus, which was still moving forward.     

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

BACKGROUND INFORMATION: 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. RESULTS: 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. CONCLUSIONS: 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.     

Multiple forms of actin in Physarum polycephalum

Actin in the acellular slime mold Physarum polycephalum consists of three major forms closely spaced at isoelectric point (IP) 4.7 and a minor form at IP 5.1. Amino acid analysis has shown the IP 5.1 actin to be nearly identical to the 4.7 actins. In actin purified from acetone powder, both actin forms were present. Both forms bound to DNase I and have the same molecular weight of about 43 000 on sodium dodecyl sulfate (SDS) polyacrylamide gels. On 2-D gels of nuclear proteins, both forms of actin were present. The IP 4.7 actins account for 8.6% of total plasmodial protein, and the IP 5.1 form for about 0.7%. In the nucleus the IP 4.7 actins comprise 2.7% of total nuclear protein, and the 5.1 actin about 0.4%. No cell cycle-associated change in the concentration of actins was observed in either total plasmodial extracts or in isolated nuclei. Pulse-labelling experiments have shown that in total plasmodia actin synthesis occurs throughout the cell cycle, with no relative changes in the rate of synthesis. In isolated nuclei labelled during mitosis and early S-phase, there is about twice as much labelled actin as in nuclei labelled prior to mitosis. This result may indicate an increase in the transport of actin into the nucleus.     

The interrelationships of actin and hyphal tip growth in the ascomycete Geotrichum candidum

Geotrichum candidum is unusual among reported hyphal ascomycetes in that its hyphae readily stain with phalloidin to reveal actin concentrated in the Spitzenk?rper (SPK) and plaques associated with the plasma membrane (PM). Loss of SPK actin, but not the PM plaques, following latrunculin B treatment produces tip swelling, consistent with actin restraining tip morphology or localizing vesicle exocytosis. Tip morphogenesis may also involve a spectrin-like protein which concentrates at the apical PM in plaques unassociated with the actin plaques. Branch formation occurs with growth rates initially about 20% those of leading tips, and does not involve a morphologically detectable SPK, nor SPK-like actin ensembles, indicating the dispensibility of this structure in tip growth. Surprisingly, new tubular tips can form in the continued presence of latrunculin, consistent with alternative cellular systems, such as the spectrin-like protein, substituting for actin's critical functions.     

The fate and origin of the nuclear envelope during and after mitosis in Amoeba proteus. I. Synthesis and behavior of phospholipids of the nuclear envelope during the cell life cycle

The synthesis and behavior of Amoeba proteus nuclear envelope (NE) phospholipids were studied. Most NE phospholipid synthesis occurs during G2 and little during mitosis or S. (A. proteus has no G1 phase). Autoradiographic observations after implantation of [3-H] choline nuclei into unlabeled cells reveal little turnover of NE phospholipid during interphase but during mitosis all the label is dispersed through the cytoplasm. Beginning at telophase all the label is dispersed through the cytoplasm. Beginning at telophase all the NE phospholipid label returns to the daughter NEs. This observation, along with the finding that no NE phospholipid synthesis occurs during mitosis or S, indicates that no de novo NE phospholipid production is required for newly forming NEs. Similarlyemetine, at concentrations that inhibit 97 percent of protein synthesis, does not prevent the post mitotic formation of NEs, suggesting that previously manufactured proteins are used in making new NEs. If a nucleus containing labeled NE phospholipids is transplanted into an unlabeled nucleate cell and the cell is allowed to grow and divide, the resultant four nuclei are equally labeled. This finding supports, but does not prove (see next paragraph), the conclusion that there probably is no continuity of the A. proteus NE during mitosis. When a phospholipid-labeled nucleus is implanted into a cell in mitosis, the grafted nucleus is not induced to enter mitosis. There is, however, a marked increase in the turnover of that nucleus's NE phospholipids with no apparent breakdown of the NE; this indicated that the mitotic cytoplasm possesses a factor that stimulates NE phospholipid exchange with the cytoplasm. That enhanced turnover is not accompanied by visible structural alteration makes less certain the earlier conclusion that no NE continuity exists during mitosis. Perhaps the most important finding in this study is that there are present, at restricted times in the cell cycle, factors capable of inducing accelerated exchange of structural components without microscopically detectable disruptions of structure.     

Colocalization of cortical microtubules and F-actin in<Emphasis Type="Italic">Dipodascus magnusii</Emphasis> using confocal laser scanning microscopy

Distribution of microtubules and F-actin in aerobically growing cells of Dipodascus magnusii, belonging to the class Saccharomycetes was analyzed using immunofluorescence microscopy and labeling with rhodamine-tagged phalloidin. A conspicuous system of permanent cytoplasmic microtubules was observed in association with multiple nuclei. In elongating cells, helices of cytoplasmic microtubules appeared at the cell cortex. In cells approaching cytokinesis transversely oriented microtubules were revealed at incipient division sites. Confocal laser scanning microscopy showed a continuity of these transverse microtubules with the remaining microtubule network. The actin system of D. magnusii consisted of patches and filaments. Patches were found to accumulate at the tips of growing cells. Bands of fine actin filaments were usually observed before F-actin rings were established. A close cortical association of microtubules with the F-actin ring was documented on individual optical sections of labeled cells. Cells with developing septa showed medial F-actin discs associated at both sides with microtubules. Colocalization of cytoplasmic microtubules with actin filaments at the cortex of dividing cells supports a role of both cytoskeletal components in controlling cell wall growth and septum formation in D. magnusii.     

Effect of brefeldin A and the dynamics of the actin plate on cytokinesis of zygotes in the brown alga,Silvetia babingtonii (Fucales,Phaeophyceae)

In the cytokinesis of brown algae, actin filaments appear like a plate at the intersecting region of microtubules (MTs) that emerge from the centrosomes after mitosis. The function of the actin plate itself is still unknown. To elucidate the relationship between the actin plate, MTs and membrane fusion, without inducing cytoskeleton depolymerization, the effect of brefeldin A (BFA), which prevents the production of vesicles from Golgi bodies, was examined in zygotes of Silvetia babingtonii. The beginning of mitosis was slightly delayed in zygotes under BFA compared with the controls. Almost all zygotes were inhibited for the progression of cytokinesis by BFA treatment. Ultrastructural observations showed that Golgi cisternae became fragmented or curled following continuous treatment with BFA, and the inhibitory status of cytokinesis between zygotes. The next cell cycle started before cytokinesis was completed. Although the appearance of the actin plate was not disturbed by BFA treatment, the behaviour of the actin plate during the transition between the first and second cell cycles could be classified into two patterns: it was either invisible upon the initiation of the next cell cycle, or a portion of it remained even though the next cell cycle had begun. In the latter case, a part of the actin plate seemed to associate with the new partially formed cell partition membrane, and MTs from the centrosomes were bound to it. The actin plate completely disappeared in the next mitosis, then re-emerged in the middle area of the four daughter nuclei. The results of the present study indicated that, under BFA treatment, the actin plate persisted until just before the beginning of the next mitotic phase, when the new, incomplete cell partition membrane was present, and MTs sustained the actin plate until next mitosis.     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Organization of the cytoskeleton in early Drosophila embryos

The cytoskeleton of early, non-cellularized Drosophila embryos has been examined by indirect immunofluorescence techniques, using whole mounts to visualize the cortical cytoplasm and sections to visualize the interior. Before the completion of outward nuclear migration at nuclear cycle 10, both actin filaments and microtubules are concentrated in a uniform surface layer a few micrometers deep, while a network of microtubules surrounds each of the nuclei in the embryo interior. These two filament-rich regions in the early embryo correspond to special regions of cytoplasm that tend to exclude cytoplasmic particles in light micrographs of histological sections. After the nuclei in the interior migrate to the cell surface and form the syncytial blastoderm, each nucleus is seen to be surrounded by its own domain of filament-rich cytoplasm, into which the cytoskeletal proteins of the original surface layer have presumably been incorporated. At interphase, the microtubules seem to be organized from the centrosome directly above each nucleus, extending to a depth of at least 40 microns throughout the cortical region of cytoplasm (the periplasm). During this stage of the cell cycle, there is also an actin "cap" underlying the plasma membrane immediately above each nucleus. As each nucleus enters mitosis, the centrosome splits and the microtubules are rearranged to form a mitotic spindle. The actin underlying the plasma membrane spreads out, and closely spaced adjacent spindles become separated by transient membrane furrows that are associated with a continuous actin filament-rich layer. Thus, each nucleus in the syncytial blastoderm is surrounded by its own individualized region of the cytoplasm, despite the fact that it shares a single cytoplasmic compartment with thousands of other nuclei.     

The central role of septa in the basidiomycete Schizophyllum commune hyphal morphogenesis

The purpose of the present research was to observe in the filamentous basidiomycete Schizophyllum commune, the connection between the nuclear division and polymerization of the contractile actin ring with subsequent formation of septa in living hyphae. The filamentous actin was visualized using Lifeact-mCherry and the nuclei with EGFP tagged histone 2B (H2B). Time-lapse fluorescence microscopy confirmed that in monokaryotic and dikaryotic hyphae, the first signs of the contractile actin ring occur at the site of the nuclear division, in one to two minutes after division. At this stage, the telophase nuclei have moved tens of micrometers from the division site. The actin ring is replaced by the septum in six minutes. The apical cells treated with filamentous actin disrupting drug latrunculin A, had swollen tips but the cells were longer than in control samples due to the absence of the actin rings. The nuclear pairing and association with clamp cell development as well as the clamp cell fusion with the subapical cell was disrupted in latrunculin-treated dikaryotic hyphae, indicating that actin filaments are involved in these processes, also regulated by the A and B mating-type genes. This suggests that the actin cytoskeleton may indirectly be a target for mating-type genes.     

Maternal effect mutations of the sponge locus affect actin cytoskeletal rearrangements in Drosophila melanogaster embryos

In the syncytial blastoderm stage of Drosophila embryogenesis, dome-shaped actin "caps" are observed above the interphase nuclei. During mitosis, this actin rearranges to participate in the formation of pseudocleavage furrows, transient membranous invaginations between dividing nuclei. Embryos laid by homozygous sponge mothers lack these characteristic actin structures, but retain other actin associated structures and processes. Our results indicate that the sponge product is specifically required for the formation of actin caps and metaphase furrows. The specificity of the sponge phenotype permits dissection of both the process of actin cap formation and the functions of actin caps and metaphase furrows. Our data demonstrate that the distribution of actin binding protein 13D2 is unaffected in sponge embryos and suggest that 13D2 is upstream of actin in cortical cap assembly. Although actin caps and metaphase furrows have been implicated in maintaining the fidelity of nuclear division and the positions of nuclei within the cortex, our observations indicate that these structures are dispensible during the early syncytial blastoderm cell cycles. A later requirement for actin metaphase furrows in preventing the nucleation of mitotic spindles between inappropriate centrosomes is observed. Furthermore, the formation of actin caps and metaphase furrows is not a prerequisite for the formation of the hexagonal array of actin instrumental in the conversion of the syncytial embryo into a cellular blastoderm.     

Peripheral nuclear matrix actin forms perinuclear shells

Perinuclear actin shells have been reported in a variety of organisms. The shells have been identified by staining perinuclear material with fluorescently-labelled phalloidin, but have not been localized to a specific subcellular compartment at the ultrastructural level. We show here that the shells of 3T3 cells lie in the peripheral nuclear matrix. Nuclear shells and matrix actin in other parts of the nucleus are not usually detected by immunohistochemical staining because they are inaccessible to antibodies or to phalloidin. Immunohistochemical detection of nuclear actin is only possible during its deposition at the end of mitosis, or in interphase nuclei that have been extracted with detergent, digested with nucleases and washed with high salt buffers. J. Cell. Biochem. 70:240–251, 1998. © 1998 Wiley-Liss, Inc.     

Effects of latrunculin B on the actin cytoskeleton and hyphal growth in Phytophthora infestans

The actin cytoskeleton is conserved in all eukaryotes, but its functions vary among different organisms. In oomycetes, the function of the actin cytoskeleton has received relatively little attention. We have performed a bioinformatics study and show that oomycete actin genes fall within a distinct clade that is divergent from plant, fungal and vertebrate actin genes. To obtain a better understanding of the functions of the actin cytoskeleton in hyphal growth of oomycetes, we studied the actin organization in Phytophthora infestans hyphae and the consequences of treatment with the actin depolymerising drug latrunculin B (latB). This revealed that latB treatment causes a concentration dependent inhibition of colony expansion and aberrant hyphal growth. The most obvious aberrations observed upon treatment with 0.1 μM latB were increased hyphal branching and irregular tube diameters whereas at higher concentrations latB (0.5 and 1 μM) tips of expanding hyphae changed into balloon-like shapes. This aberrant growth correlated with changes in the organization of the actin cytoskeleton. In untreated hyphae, staining with fluorescently tagged phalloidin revealed two populations of actin filaments: long, axially oriented actin filament cables and cortical actin filament plaques. Two hyphal subtypes were recognized, one containing only plaques and the other containing both cables and plaques. In the latter, some hyphae had an apical zone without actin filament plaques. Upon latB treatment, the proportion of hyphae without actin filament cables increased and there were more hyphae with a short apical zone without actin filament plaques. In general, actin filament plaques were more resilient against actin depolymerisation than actin filament cables. Besides disturbing hyphal growth and actin organization, actin depolymerisation also affected the positioning of nuclei. In the presence of latB, the distance between nuclei and the hyphal tip decreased, suggesting that the actin cytoskeleton plays a role in preventing the movement of nuclei towards the hyphal tip.     

Unique actin and microtubule arrays co-ordinate the differentiation of microspores to mature pollen in Nicotiana tabacum

The complex cellular events that occur during development of the male gametophyte of higher plants suggest a role for the cytoskeleton. This investigation has revealed that unique microtubule arrays mediate events that occur during microspore development; both actin and microtubule arrays have important roles during the asymmetrical microspore mitosis and unique actin arrays mediate events that occur during early pollen development. Migration of the nucleus to the generative pole during cellular polarization of the microspore is mediated by a microtubule cage that encloses the nucleus. Nuclear position at the generative pole is maintained by an actin net that tethers it to the pole prior to the asymmetrical mitosis. During entry into mitosis, the microtubule cage becomes modified and transforms into the asymmetrical mitotic spindle. Actin is localized within the region of the mitotic spindle and in the phragmoplast. Following mitosis, actin networks enclose first the generative cell and then the vegetative nucleus. These actin networks function during migration of the generative cell and vegetative nucleus toward the centre of the pollen grain. Mature pollen contains a dense cortical actin meshwork and a disc-shaped microtubule array enclosing the generative cell. The functional importance of the unique actin and microtubule arrays is verified by their targeted disruption with specific cytoskeletal inhibitors, which disrupt normal development and cellular morphology. In summary, these data provide evidence that the co-ordinated reorganization of unique actin and microtubule arrays is an essential determinant of microspore and pollen development.     

Fission Yeast Pob1p, Which Is Homologous to Budding Yeast Boi Proteins and Exhibits Subcellular Localization Close to Actin Patches, Is Essential for Cell Elongation and Separation

The fission yeast pob1 gene encodes a protein of 871 amino acids carrying an SH3 domain, a SAM domain, and a PH domain. Gene disruption and construction of a temperature-sensitive pob1 mutant indicated that pob1 is essential for cell growth. Loss of its function leads to quick cessation of cellular elongation. Pob1p is homologous to two functionally redundant Saccharomyces cerevisiae proteins, Boi1p and Boi2p, which are necessary for cell growth and relevant to bud formation. Overexpression of pob1 inhibits cell growth, causing the host cells to become round and swollen. In growing cells, Pob1p locates at cell tips during interphase and translocates near the division plane at cytokinesis. Thus, this protein exhibits intracellular dynamics similar to F-actin patches. However, Pob1p constitutes a layer, rather than patches, at growing cell tips. It generates two split discs flanking the septum at cytokinesis. The pob1-defective cells no longer elongate but swell gradually at the middle, eventually assuming a lemon-like morphology. Analysis using the pob1-ts allele revealed that Pob1p is also essential for cell separation. We speculate that Pob1p is located on growing plasma membrane, possibly through the function of actin patches, and may recruit proteins required for the synthesis of cell wall.     

The golgi comprises a paired stack that is separated at G2 by modulation of the actin cytoskeleton through Abi and Scar/WAVE

During the cell cycle, the Golgi, like other organelles, has to be duplicated in mass and number to ensure its correct segregation between the two daughter cells. It remains unclear, however, when and how this occurs. Here we show that in Drosophila S2 cells, the Golgi likely duplicates in mass to form a paired structure during G1/S phase and remains so until G2 when the two stacks separate, ready for entry into mitosis. We show that pairing requires an intact actin cytoskeleton which in turn depends on Abi/Scar but not WASP. This actin-dependent pairing is not limited to flies but also occurs in mammalian cells. We further show that preventing the Golgi stack separation at G2 blocks entry into mitosis, suggesting that this paired organization is part of the mitotic checkpoint, similar to what has been proposed in mammalian cells.     

Cell division site placement and asymmetric growth in mycobacteria

Mycobacteria are members of the actinomycetes that grow by tip extension and lack apparent homologues of the known cell division regulators found in other rod-shaped bacteria. Previous work using static microscopy on dividing mycobacteria led to the hypothesis that these cells can grow and divide asymmetrically, and at a wide range of sizes, in contrast to the cell growth and division patterns observed in the model rod-shaped organisms. In this study, we test this hypothesis using live-cell time-lapse imaging of dividing Mycobacterium smegmatis labelled with fluorescent PBP1a, to probe peptidoglycan synthesis and label the cell septum. We demonstrate that the new septum is placed accurately at mid-cell, and that the asymmetric division observed is a result of differential growth from the cell tips, with a more than 2-fold difference in growth rate between fast and slow growing poles. We also show that the division site is not selected at a characteristic cell length, suggesting this is not an important cue during the mycobacterial cell cycle.     

Relationship between organization of the actin cytoskeleton and the cell cycle in normal and adenovirus-infected rat cells.

Flow cytometry and staining with 7-nitrobenz-2-oxa-1,3-diazole-phallacidin were used to investigate organization of the actin cytoskeleton in rat embryo cells at different stages of normal and adenovirus E1A-induced cell cycles. In uninfected cells in G0-G1 and S phases, actin was predominantly in the form of stress fibers. In G2, this organization changed to peripheral rings of thin filaments, while during mitosis, actin had a diffuse distribution. Infection of quiescent rat cells by adenovirus caused them to enter the cell cycle and replicate DNA and also caused disruption of stress fibers. Rapid disappearance of stress fibers and the appearance of peripheral rings of actin filaments began from 13 h after infection and closely followed synthesis of the E1A proteins. Infected cells began S phase at about 24 h after infection, and cells in G2 and mitosis were seen from 30 to 50 h. Thus, disruption of the actin cytoskeleton is an early effect of E1A and not an indirect consequence of the entry of infected cells into the cell cycle.     

Function of the dense plaques during mitosis in Trypanosoma cruzi

During mitosis in Trypanosoma cruzi ten dense plaques originate from the single, large nucleolus present in interphase and the preliminary phase of mitosis. Each plaque becomes associated with two microtubular bundles which end at the poles of the dividing nucleus. After an equilibrium phase in which the plaques are located near the nuclear equator, each plaque divides into two half-plaques. Each half-plaque migrates to a pole in the next stage. Although the composition of the plaques is unknown, they are associated with chromatin fibers. It is concluded that plaques act as kinetochores of higher organisms and provide a mechanical device for equal distribution of chromatin to daughter nuclei.     

Interkinetic nuclear migration: a mysterious process in search of a function

During interkinetic nuclear migration (INM), the nuclei in many epithelial cells migrate between the apical and basal surfaces, coordinating with the cell cycle, and undergoing cytokinesis at the apical surface. INM is observed in a wide variety of tissues and species. Recent advances in time-lapse microscopy have provided clues about the mechanisms and functions of INM. Whether actin or microtubules are responsible for nuclear migration is controversial. How mitosis is initiated during INM is poorly understood, as is the relationship between the cell cycle and nuclear movement. It is possible that the disagreements stem from differences in the tissues being studied, since epithelia undergoing INM vary greatly in terms of cell height and cell fates. In this review we examine the reports addressing the mode and mechanisms that regulate INM and suggest possible functions for this dramatic event.