Spatial organization and isotubulin composition of microtubules in epidermal tendon cells of Artemia franciscana

Epidermally derived tendon cells attach the exoskeleton (cuticle) of the Branchiopod crustacean, Artemia franciscana, to underlying muscle in the hindgut, while the structurally similar transalar tendon (epithelial) cells, which also arise from the epidermis and are polarized, connect dorsal and ventral exopodite surfaces. To establish these latter attachments the transalar tendon cells interact with cuticles on opposite sides of the exopodite by way of their apical surfaces and with one another via basal regions, or the cuticle attachments may be mediated through linkages with phagocytic storage cells found in the hemolymph. In some cases, phyllopod tendon cells attach directly to muscle cells. Tendon cells in the hindgut of Artemia possess microtubule bundles, as do the transalar cells, and they extend from the basal myotendinal junction to the apical domain located near the cuticle. The bundled microtubules intermingle with thin filaments reminiscent of microfilaments, but intermediate filament-like structures are absent. Microtubule bundles converging at apical cell surfaces contact structures termed apical invaginations, composed of cytoplasmic membrane infoldings associated with electron-dense material. Intracuticular rods protrude from apical invaginations, either into the cuticle during intermolt or the molting fluid in premolt. Confocal microscopy of immunofluorescently stained samples revealed tyrosinated, detyrosinated, and acetylated tubulins, the first time posttranslationally modified isoforms of this protein have been demonstrated in crustacean tendon cells. Microfilaments, as shown by staining with phalloidin, coincided spatially with microtubule bundles. Artemia tendon cells clearly represent an interesting system for study of cytoskeleton organization within the context of cytoplasmic polarity and the results in this article indicate functional cooperation of microtubules and microfilaments. These cytoskeletal elements, either acting independently or in concert, may transmit tension from muscle to cuticle in the hindgut and resist compression when connecting exopodite cuticular surfaces.     

Actomyosin contractility and microtubules drive apical constriction in Xenopus bottle cells

Cell shape changes are critical for morphogenetic events such as gastrulation, neurulation, and organogenesis. However, the cell biology driving cell shape changes is poorly understood, especially in vertebrates. The beginning of Xenopus laevis gastrulation is marked by the apical constriction of bottle cells in the dorsal marginal zone, which bends the tissue and creates a crevice at the blastopore lip. We found that bottle cells contribute significantly to gastrulation, as their shape change can generate the force required for initial blastopore formation. As actin and myosin are often implicated in contraction, we examined their localization and function in bottle cells. F-actin and activated myosin accumulate apically in bottle cells, and actin and myosin inhibitors either prevent or severely perturb bottle cell formation, showing that actomyosin contractility is required for apical constriction. Microtubules were localized in apicobasally directed arrays in bottle cells, emanating from the apical surface. Surprisingly, apical constriction was inhibited in the presence of nocodazole but not taxol, suggesting that intact, but not dynamic, microtubules are required for apical constriction. Our results indicate that actomyosin contractility is required for bottle cell morphogenesis and further suggest a novel and unpredicted role for microtubules during apical constriction.     

Exoskeleton anchoring to tendon cells and muscles in molting isopod crustaceans

Specialized mechanical connection between exoskeleton and underlying muscles in arthropods is a complex network of interconnected matrix constituents, junctions and associated cytoskeletal elements, which provides prominent mechanical attachment of the epidermis to the cuticle and transmits muscle tensions to the exoskeleton. This linkage involves anchoring of the complex extracellular matrix composing the cuticle to the apical membrane of tendon cells and linking of tendon cells to muscles basally. The ultrastructural arhitecture of these attachment complexes during molting is an important issue in relation to integument integrity maintenance in the course of cuticle replacement and in relation to movement ability. The aim of this work was to determine the ultrastructural organization of exoskeleton - muscles attachment complexes in the molting terrestrial isopod crustaceans, in the stage when integumental epithelium is covered by both, the newly forming cuticle and the old detached cuticle. We show that the old exoskeleton is extensively mechanically connected to the underlying epithelium in the regions of muscle attachment sites by massive arrays of fibers in adult premolt Ligia italica and in prehatching embryos and premolt marsupial mancas of Porcellio scaber. Fibers expand from the tendon cells, traverse the new cuticle and ecdysal space and protrude into the distal layers of the detached cuticle. They likely serve as final anchoring sites before exuviation and may be involved in animal movements in this stage. Tendon cells in the prehatching embryo and in marsupial mancas display a substantial apicobasally oriented transcellular arrays of microtubules, evidently engaged in myotendinous junctions and in apical anchoring of the cuticular matrix. The structural framework of musculoskeletal linkage is basically established in described intramarsupial developmental stages, suggesting its involvement in animal motility within the marsupium.     

The microtubule cytoskeleton in hyphae ofUromyces phaseoli germlings: Its relationship to the region of nucleation and to the F-actin cytoskeleton

Summary The microtubule and F-actin cytoskeleton of nondifferentiated germlings ofUromyces phaseoli was studied using immunofluorescence methodologies. The microtubules were oriented mostly parallel to the longitudinal axis of the hypha. Microtubule depolymerizing agents, such as cold, demecolcine, griseofulvin and nocodazole, were effective in destroying the microtubule network, but not the F-actin system. Repolymerization of microtubules, following release from these agents, occurred first in the hyphal apices and not near the nuclei or spindle pole bodies. It was concluded that the microtubule nucleating region in such fungal cells is located in the apical regions. Enhanced microtubule arrays were visualized following incubation of the cells in taxol, an agent known to favor microtubule polymerization.     

Positioning and capture of cell surface-associated microtubules in epithelial tendon cells that differentiate in primary embryonic Drosophila cell cultures

Using primary embryonic Drosophila cell cultures, we have investigated the assembly of transcellular microtubule bundles in epidermal tendon cells. Muscles attach to the tendon cells of previously undescribed epidermal balls that form shortly after culture initiation. Basal capture of microtubule ends in cultured tendon cells is confined to discrete sites that occupy a relatively small proportion of the basal cell surface. These capturing sites are associated with hemiadherens junctions that link the ends of muscle cells to tendon cell bases. In vivo, muscle attachment and microtubule capture occur across the entire cell base. The cultured tendon cells reveal that the basal ends of their microtubules can be precisely targeted to small, pre-existing, structurally well-defined cortical capturing sites. However, a search and capture targeting procedure, such as that undertaken by kinetochore microtubules, cannot fully account for the precision of microtubule capture and positioning in tendon cells. We propose that cross-linkage of microtubules is also required to zip them into apicobasally oriented alignment, progressing from captured basal plus ends to apical minus ends. This involves repositioning of apical minus ends before they become anchored to an apical set of hemiadherens junctions. The proposal is consistent with our finding that hemiadherens junctions assemble at tendon cell bases before they do so at cell apices in both cultures and embryos. It is argued that control of microtubule positioning in the challenging spatial situations found in vitro involves the same procedures as those that operate in vivo.     

Organization of the cytoskeleton in the coenocytic algaVaucheria longicaulis var.macounii: an experimental study

Summary The organization of the cytoskeleton of vegetative filaments ofVaucheria longicaulis Hoppaugh var.macounii Blum is investigated by fluorescence microscopy using monoclonal anti -tubulin antibodies and fluorescein (FITC)-labelled phalloidin. Confocal laser scanning microscopy observations give further information on the distribution of the cytoskeletal elements. Phalloidin labelling reveals F-actin bundles in the cortical cytoplasm of both fixed and unfixed vegetative filaments of this alga. In addition a more diffuse fluorescent component, seen at higher magnification to be made up of thinner F-actin bundles, can also be detected in unfixed cells. The distribution of the F-actin bundles resemble that of filamentous structures observed with differential interference contrast (DIC) microscopy in living cells. These structures seem to correspond to the microtubule associated reticulum (MAR) described in literature and overall the evidence suggests that actin and MAR elements are co-distributed. F-actin bundles are always found in association with focal masses (foci) of phalloidin-positive material. Foci are also observed by DIC microscopy associated with the cytoplasmic filamentous structures in living cells.Depolymerization of F-actin with cytochalasin D and the subsequent repolymerization that occurs on transfer ofVaucheria vegetative filaments to cytochalasin-free medium suggest that these foci are involved in the organization of the F-actin array. Immunofluorescence for -tubulin reveals microtubule bundles that are shorter in length and straighter in configuration than microfilament bundles. Microtubule bundles are associated with spot-like focal structures that, in many instances, show a close relationship with respect to nuclei. Oryzalin and cold temperature cause the depolymerization of the microtubule bundles and suggest, in conjunction with repolymerization studies, that these fluorescent spots associated with the ends of the microtubule bundles are involved in their organization; hence, they represent microtubule organizing centres or MTOCs. The importance of both microfilament and microtubule bundle focal regions is discussed with respect to the apical growth exhibited by the vegetative filaments of this alga.     

The organization of F-actin in root tip cells ofAdiantum capillus veneris throughout the cell cycle

Summary The patterns of F-actin in relation to microtubule (Mt) organization in dividing root tip cells ofAdiantum capillus veneris were studied with rhodamine-phalloidin (RP) labelling and tubulin immunofluorescence. Interphase cells display a well organized network of cortical/subcortical, endoplasmic and perinuclear actin filaments (AFs), not particularly related to the interphase Mt arrays. The cortical AFs seem to persist during the cell cycle while the large subcortical AF bundles disappear by preprophase/prophase and reappear after cytokinesis is completed. In some but not all of the preprophase cells the cortical AFs tend to form a band (AF-PPB) coincident with the preprophase band of Mts (Mt-PPB). In metaphase and anaphase cells AFs are localized in the cell cortex, around the spindle and inside it coincidently with kinetochore Mt bundles. During cytokinesis AFs are consistently found in the phragmoplast. In oryzalin treated cells neither Mt-PPBs, spindles and phragmoplasts exist, nor such F-actin structures can be observed. In cells recovering from oryzalin, AF-PPBs, AF kinetochore bundles and AF phragmoplasts reform. They show the same pattern with the reinstating respective Mt arrays. In contrast, in cells treated with cytochalasin B (CB), AFs disappear but all categories of Mt arrays form normally.These observations show that F-actin organization in root tip cells ofA. capillus veneris differs from that of root tip cells of flowering plants examined so far. In addition, Mts seem to be crucial for F-actin organization as far as it concerns the PPB, the mitotic spindle, and the phragmoplast.Abbreviations AF actin filament - CB cytochalasin B - MBS m-male-imidobenzoyl-N-hydroxysuccinimide ester - MSB microtubule stabilizing buffer - Mt microtubule - PBS phosphate buffered saline - PPB preprophase band - RP rhodamine phalloidin     

The myosin ATPase inhibitor 2,3-butanedione-2-monoxime disorganizes microtubules as well as F-actin in Saccharomyces cerevisiae

Interactions between microtubules and filamentous actin (F-actin) are essential to many cellular processes, but their mechanisms are poorly understood. We investigated possible roles of the myosin family of proteins in the interactions between filamentous actin (F-actin) and microtubules of budding yeast Saccharomyces cerevisiae with the general myosin ATPase inhibitor 2,3-butanedione-2-monoxime (BDM). The growth of S. cerevisiae was completely inhibited by BDM at 20 mmol/L and the effect of BDM on cell growth was reversible. In more than 80% of BDM-treated budding yeast cells, the polarized distribution of F-actin was lost and fewer F-actin dots were observed. When cells were synchronized in G₁ with α-factor and released in the presence of BDM, cell number did not increase and cells were mainly arrested in G₁ DNA content without any bud, suggesting that myosin activity is required for new bud formation and the start of a new cell cycle. More than 10% of the BDM-treated cells also revealed defects in nuclear migration to the bud neck as well as in nuclear shape. Consistent with these defects, the orientation of mitotic spindles was random in the 57% of cells treated with 20 mmol/L BDM and immunostained with anti-tubulin antibody. Furthermore, microtubule structures were completely disorganized in most of the cells incubated in 50 mmol/L BDM, while similar amounts of tubulin proteins were present in both BDM-treated and untreated cells. These results show that the general myosin inhibitor BDM disorganizes microtubule structures as well as F-actin, and suggest that BDM-sensitive myosin activities are necessary for the interaction of F-actin and microtubules to coordinate polarized bud growth and the shape and migration of the nucleus in S. cerevisiae. This revised version was published online in July 2006 with corrections to the Cover Date.     

Plant mitochondria move on F-actin, but their positioning in the cortical cytoplasm depends on both F-actin and microtubules

Mitochondrion movement and positioning was studied in elongating cultured cells of tobacco (Nicotiana tabacum L.), containing mitochondria-localized green fluorescent protein. In these cells mitochondria are either actively moving in strands of cytoplasm transversing or bordering the vacuole, or immobile positioned in the cortical layer of cytoplasm. Depletion of the cell's ATP stock with the uncoupling agent DNP shows that the movement is much more energy demanding than the positioning. The active movement is F-actin based. It is inhibited by the actin filament disrupting drug latrunculin B, the myosin ATPase inhibitor 2,3-butanedione 2-monoxime and the sulphydryl-modifying agent N-ethylmaleimide. The microtubule disrupting drug oryzalin did not affect the movement of mitochondria itself, but it slightly stimulated the recruitment of cytoplasmic strands, along which mitochondria travel. The immobile mitochondria are often positioned along parallel lines, transverse or oblique to the cell axis, in the cortical cytoplasm of elongated cells. This positioning is mainly microtubule based. After complete disruption of the F-actin, the mitochondria parked themselves into conspicuous parallel arrays transverse or oblique to the cell axis or clustered around chloroplasts and around patches and strands of endoplasmic reticulum. Oryzalin inhibited all positioning of the mitochondria in parallel arrays.     

Mid1ip1b modulates apical reorientation of non-centrosomal microtubule organizing center in epithelial cells

In most kinds of animal cells, the centrosome serves as the main microtubule organizing center (MTOC) that nucleates microtubule arrays throughout the cytoplasm to maintain cell structure, cell division and intracellular transport. Whereas in epithelial cells, non-centrosomal MTOCs are established in the apical domain for generating asymmetric microtubule fibers and cilia in epithelial cells for the organ morphogenesis during embryonic development. However, the mechanism by which MTOCs localize to the apical domain in epithelial cells remains largely unknown. Here, we show that Mid1ip1b has a close interaction with γ-tubulin protein, the central component of MTOC, and modulates lumen opening of the neural tube, gut, intestine, and kidney of zebrafish. Knockdown or dominant negative effect of Mid1ip1b resulted in failure of lumen formation of the organs as aforementioned. Moreover, the non-centrosomal MTOCs were unable to orientate to the apical domain in Mid1ip1b knockdown epithelial cells, and the centrosomal MTOCs were inaccurately placed in the apical domain, resulting in defective formation of asymmetric microtubules and misplacement of cilia in the apical domain. These data uncover a molecule that controls the proper localization of MTOCs in the apical domain in epithelial cells for organ morphogenesis during embryonic development.     

Three-dimensional localization and redistribution of F-actin in higher plant mitosis and cell plate formation

The distribution of F-actin cables in dividing endosperm cells of a higher plant, Haemanthus, was visualized with the immunogold-silver-enhanced method and compared with the arrangement of immunogold-stained microtubules in the same cells. The three-dimensional distribution of F-actin cables and microtubules during mitosis and cell plate formation was analyzed using ultrathin optical sectioning of whole mounts in polarized light video microscopy. F-actin cables form a loose irregular network in the interphase cytoplasm. Much of this network remains outside of the spindle during mitosis. A few F-actin cables were detected within the spindle. Their pronounced rearrangement during mitosis appears to be related to the presence and growth of microtubule arrays. During prometaphase, actin cables located on the spindle surface and those present within the spindle tend to arrange parallel to the long axis of the spindle. Cables outside the spindle do not reorient, except those at the polar region, where they appear to be compressed by the elongating spindle. Beginning with mid-anaphase, shorter actin cables oriented in various directions accumulate at the equator. Some of them are incorporated into the phragmoplast and cell plate and are gradually fragmented as the cell plate is formed and ages. Actin cables adjacent to microtubule arrays often show a regular punctate staining pattern. Such a pattern is seldom observed in the peripheral cytoplasm, which contains few microtubules. The rearrangement of F-actin cables mimicks the behavior of spindle inclusions, such as starch grains, mitochondria, etc., implying that F-actin is redistributed passively by microtubule growth or microtubule-related transport. Thus F-actin or actomyosin-based motility does not appear to be directly involved in mitosis and cytokinesis in higher plants.     

The cytoskeleton of chick retinal pigment epithelial cells in situ

Summary Gelatin-coated slides were used to obtain en face preparations of retinal pigment epithelium (RPE) from 6-to 21-day-old chick embryos in order to study the distribution of F-actin in microfilaments (MF) and the MF-associated proteins, myosin, tropomyosin,-actinin and vinculin in situ at different stages of development by fluorescence microscopy. The epithelial sheets were fixed in formaldehyde and then extracted in a solution containing 0.1% Triton X-100. NBD-Phallacidin was used to visualize the F-actin in MF, and antisera against myosin, tropomyosin,-actinin and vinculin were used to determine the distribution of these four MF-associated proteins. F-actin, myosin, tropomyosin,-actinin and vinculin were present in cortical rings around the apical ends of the RPE cells throughout this period of development. Of these proteins, only F-actin was identified in the apical processes of RPE cells. The increase in the amount of F-actin could be followed as the length and the number of apical processes increased with age and maturation of RPE cells. F-actin was first detected in numerous short apical processes on the surface of each RPE cell on day 12. From day 12 to day 17, they were at an intermediate stage of elongation and from day 17 onward all of the RPE cells had long F-actin-containing apical processes. These results indicate that the F-actin-containing MF assemble much later in the apical processes than in the cortical rings. Also the cortical rings and apical processes of RPE cells resemble those in absorptive intestinal cells in that the cortical rings in both cell types contain MF associated with myosin, tropomyosin,-actinin and vinculin while the MF in the apical processes and microvilli lack these MF associated proteins, and both of these structures lack talin. In addition to apical processes and cortical rings, stained fibers were also observed at a level below the cortical rings. The simple and highly reproducible en face method described is useful for determining changes in the organization of cytoskeletal components and other macromolecules in RPE cells and other epithelial cells in situ.     

Abundance of actin filaments in the preprophase band and mitotic spindle of brick1 Zea mays mutant

The preprophase band and mitotic spindle of dividing protodermal cells of wild-type Zea mays leaves include few actin filaments. Surprisingly, abundant actin filaments were observed in the above arrays, in dividing protodermal cells in the leaves of the brick1 mutant. The same abundance was observed in the spindle of Taxol-treated brick1 mitotic protodermal cells. Apart from the above difference, the relevant arrays displayed normal microtubule organization in both wild type and mutant cells, as far as can be discerned by immunofluorescence microscopy. Accordingly, the abundance of actin filaments in the preprophase band and spindle of brick1 mitotic cells seems not to influence the structure of the above arrays and might be a non-functional “side-effect” of defective F-actin organization in this mutant.     

Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinoderm embryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.     

The Association of Myosin IB with Actin Waves in Dictyostelium Requires Both the Plasma Membrane-Binding Site and Actin-Binding Region in the Myosin Tail

F-actin structures and their distribution are important determinants of the dynamic shapes and functions of eukaryotic cells. Actin waves are F-actin formations that move along the ventral cell membrane driven by actin polymerization. Dictyostelium myosin IB is associated with actin waves but its role in the wave is unknown. Myosin IB is a monomeric, non-filamentous myosin with a globular head that binds to F-actin and has motor activity, and a non-helical tail comprising a basic region, a glycine-proline-glutamine-rich region and an SH3-domain. The basic region binds to acidic phospholipids in the plasma membrane through a short basic-hydrophobic site and the Gly-Pro-Gln region binds F-actin. In the current work we found that both the basic-hydrophobic site in the basic region and the Gly-Pro-Gln region of the tail are required for the association of myosin IB with actin waves. This is the first evidence that the Gly-Pro-Gln region is required for localization of myosin IB to a specific actin structure in situ. The head is not required for myosin IB association with actin waves but binding of the head to F-actin strengthens the association of myosin IB with waves and stabilizes waves. Neither the SH3-domain nor motor activity is required for association of myosin IB with actin waves. We conclude that myosin IB contributes to anchoring actin waves to the plasma membranes by binding of the basic-hydrophobic site to acidic phospholipids in the plasma membrane and binding of the Gly-Pro-Gln region to F-actin in the wave.     

Glomus tumor cells: evaluation of smooth muscle and endothelial cell properties

Six cases of glomus tumor in superficial soft tissues were investigated immunohistochemically for the presence of different types of intermediate filaments, myosin, laminin, a basal lamina glycoprotein, and the endothelial cell markers, factor VIII-related antigen (FVIIIR:Ag) and Ulex europaeus I lectin (UEA I) binding sites. The tumor cells appeared to contain only vimentin, the fibroblast-type of intermediate filament protein. They were also positive for myosin, and were invested by laminin-positive basal lamina-like material, but did not express endothelial cell markers. Ultrastructural studies revealed prominent arrays of both intermediate filaments and microfilaments, the latter resembling the myofilament bundles seen in smooth muscle cells. The results show that glomus tumor cells resemble smooth muscle cells in their content of myosin and in some ultrastructural features. In their lack of desmin, however, they differ from most types of smooth muscle cell, although they are similar in this respect to some vascular smooth muscle cells.     

Rho-dependent control of anillin behavior during cytokinesis

Anillin is a conserved protein required for cytokinesis but its molecular function is unclear. Anillin accumulation at the cleavage furrow is Rho guanine nucleotide exchange factor (GEF)(Pbl)-dependent but may also be mediated by known anillin interactions with F-actin and myosin II, which are under RhoGEF(Pbl)-dependent control themselves. Microscopy of Drosophila melanogaster S2 cells reveal here that although myosin II and F-actin do contribute, equatorial anillin localization persists in their absence. Using latrunculin A, the inhibitor of F-actin assembly, we uncovered a separate RhoGEF(Pbl)-dependent pathway that, at the normal time of furrowing, allows stable filamentous structures containing anillin, Rho1, and septins to form directly at the equatorial plasma membrane. These structures associate with microtubule (MT) ends and can still form after MT depolymerization, although they are delocalized under such conditions. Thus, a novel RhoGEF(Pbl)-dependent input promotes the simultaneous association of anillin with the plasma membrane, septins, and MTs, independently of F-actin. We propose that such interactions occur dynamically and transiently to promote furrow stability.     

Role for actin filament turnover and a myosin II motor in cytoskeleton-driven disassembly of the epithelial apical junctional complex

Disassembly of the epithelial apical junctional complex (AJC), composed of the tight junction (TJ) and adherens junction (AJ), is important for normal tissue remodeling and pathogen-induced disruption of epithelial barriers. Using a calcium depletion model in T84 epithelial cells, we previously found that disassembly of the AJC results in endocytosis of AJ/TJ proteins. In the present study, we investigated the role of the actin cytoskeleton in disassembly and internalization of the AJC. Calcium depletion induced reorganization of apical F-actin into contractile rings. Internalized AJ/TJ proteins colocalized with these rings. Both depolymerization and stabilization of F-actin inhibited ring formation and disassembly of the AJC, suggesting a role for actin filament turnover. Actin reorganization was accompanied by activation (dephosphorylation) of cofilin-1 and its translocation to the F-actin rings. In addition, Arp3 and cortactin colocalized with these rings. F-actin reorganization and disassembly of the AJC were blocked by blebbistatin, an inhibitor of nonmuscle myosin II. Myosin IIA was expressed in T84 cells and colocalized with F-actin rings. We conclude that disassembly of the AJC in calcium-depleted cells is driven by reorganization of apical F-actin. Mechanisms of such reorganization involve cofilin-1-dependent depolymerization and Arp2/3-assisted repolymerization of actin filaments as well as myosin IIA-mediated contraction.     

Gonococcal invasion into epithelial cells depends on both cell polarity and ezrin

Neisseria gonorrhoeae (GC) establishes infection in women from the cervix, lined with heterogeneous epithelial cells from non-polarized stratified at the ectocervix to polarized columnar at the endocervix. We have previously shown that GC differentially colonize and transmigrate across the ecto and endocervical epithelia. However, whether and how GC invade into heterogeneous cervical epithelial cells is unknown. This study examined GC entry of epithelial cells with various properties, using human cervical tissue explant and non-polarized/polarized epithelial cell line models. While adhering to non-polarized and polarized epithelial cells at similar levels, GC invaded into non-polarized more efficiently than polarized epithelial cells. The enhanced GC invasion in non-polarized epithelial cells was associated with increased ezrin phosphorylation, F-actin and ezrin recruitment to GC adherent sites, and the elongation of GC-associated microvilli. Inhibition of ezrin phosphorylation inhibited F-actin and ezrin recruitment and microvilli elongation, leading to a reduction in GC invasion. The reduced GC invasion in polarized epithelial cells was associated with non-muscle myosin II-mediated F-actin disassembly and microvilli denudation at GC adherence sites. Surprisingly, intraepithelial GC were only detected inside epithelial cells shedding from the cervix by immunofluorescence microscopy, but not significantly in the ectocervical and the endocervical regions. We observed similar ezrin and F-actin recruitment in exfoliated cervical epithelial cells but not in those that remained in the ectocervical epithelium, as the luminal layer of ectocervical epithelial cells expressed ten-fold lower levels of ezrin than those beneath. However, GC inoculation induced F-actin reduction and myosin recruitment in the endocervix, similar to what was seen in polarized epithelial cells. Collectively, our results suggest that while GC invade non-polarized epithelial cells through ezrin-driven microvilli elongation, the apical polarization of ezrin and F-actin inhibits GC entry into polarized epithelial cells.     

Myosin XI drives polarized growth by vesicle focusing and local enrichment of F-actin in Physcomitrium patens

In tip-growing plant cells, growth results from myosin XI and F-actin-mediated deposition of cell wall polysaccharides contained in secretory vesicles. Previous evidence showed that myosin XI anticipates F-actin accumulation at the cell’s tip, suggesting a mechanism where vesicle clustering via myosin XI increases F-actin polymerization. To evaluate this model, we used a conditional loss-of-function strategy by generating moss (Physcomitrium patens) plants harboring a myosin XI temperature-sensitive allele. We found that loss of myosin XI function alters tip cell morphology, vacuolar homeostasis, and cell viability but not following F-actin depolymerization. Importantly, our conditional loss-of-function analysis shows that myosin XI focuses and directs vesicles at the tip of the cell, which induces formin-dependent F-actin polymerization, increasing F-actin’s local concentration. Our findings support the role of myosin XI in vesicle focusing, possibly via clustering and F-actin organization, necessary for tip growth, and deepen our understanding of additional myosin XI functions.

Vesicle clustering by the molecular motor myosin XI enhances actin polymerization-dependent motility and polarized vesicle accumulation in tip-growing cells.