Roles of microfilaments in exocytosis: a new hypothesis

We observed the dynamic changes in the localization of microfilaments during the exocytic secretion of rat parotid and submandibular gland acinar cells, and obtained results which led us to propose a new concept of microfilament function in exocytosis. With the electron microscopy, NBD-Phallacidin (NBD-PL) fluorescence technique and immunohistochemistry for myosin, microfilaments consisting of F-actin and myosin were localized mainly underneath the luminal plasma membrane. Microfilaments were not detectable around the secretory granules which were stored in the cytoplasm, but were clearly observed around them whose membranes were continuous with the luminal plasma membrane. When viewed with NBD-PL and myosin fluorescence, the area of fused granule membranes revealed bright fluorescence in association with the luminal border, so that the luminal membrane undergoing exocytosis appeared like a 'bunch of grapes'. When excess exocytosis was stimulated by isoproterenol (IPR), the number of individual 'grapes' increased dramatically, indicating that the secretory granules are surrounded by microfilaments after the fusion with the luminal membrane. Microfilaments thus continuously undercoat the luminal membrane during exocytosis although the exocytic process involves the dilation and subsequent reduction of the luminal membrane due to the addition and removal of secretory granule membranes. This reduction of the dilated luminal membrane following exocytosis was, however, inhibited when the microfilaments were disrupted by cytochalasin D. Following this treatment, the lumina was expanded extraordinarily and the secretory products remained in the enlarged lumina, showing that the release of secretory products is inhibited when the microfilament function is disturbed. These results indicate that 1) microfilaments are localized mainly underneath the luminal plasma membrane and act as an obstacle to exocytosis when cells are at the resting phase and 2) at the secretory phase microfilaments allow exocytosis by disorganizing their barrier system and then, by encircling the discharged secretory granule membranes, provide forces for the extrusion of secretory products through the action of the acto-myosin contractile system.     

Membrane Phospholipid Redistribution in Cytokinesis： A Theoretical Model

In cell mitosis, cytokinesis is a major deformation process, during which the site of the contractile ring is determined by the biochemical stimulus from asters of the mitotic apparatus, actin and myosin assembly is related to the motion of membrane phospholipids, and local distribution and arrangement of the microfilament cytoskeleton are different at different cytokinesis stages. Based on the Zinemanas-Nir model, a new model is proposed in this study to simulate the entire process by coupling the biochemical stimulus with the mechanical actions. There were three assumptions in this model： the movements of phospholipid proteins are driven by gradients of biochemical stimulus on the membrane surface; the local assembly of actin and myosin filament depends on the amount of phospholipid proteins at the same location; and the surface tension includes membrane tensions due to both the passive deformation of the membrane and the active contraction of actin filament, which is determined by microfilament redistribution and rearrangement. This model could explain the dynamic movement of microfilaments during cytokinesis and predict cell deformation. The calculated results from this model demonstrated that the reorientation of phospholipid proteins and the redistribution and reorientation of microfilaments may play a crucial role in cell division. This model may better represent the cytokinesis process by the introduction of biochemical stimulus.     

Immunofluorescent and ultrastructural studies of polygonal microfilament networks in respreading non-muscle cells

Respreading gerbil fibroma cells (CCL146) have been found to display cytoplasmic actin-based polygonal fiber networks 10 h after replating (stage III of respreading according to Vasiliev & Gelfand, [1]). The networks have been analyzed by immunofluorescence and electron microscopy. The foci, sites of actin, α-actinin and filamin distribution, are condensed meshworks of microfilaments attached to the inner surface of the plasma membrane. The interconnecting fibers, sites of uniform actin distribution and complementary periodicities of α-actinin and myosin, are bundles of parallel microfilaments with periodic dense bodies. Heavy meromyosin (HMM) labelling of the microfilaments in the foci and interconnecting bundles confirm that they contain actin. In addition, approx. 70% of the microfilaments associated with an individual focus have a uniform polarity relative to it (arrowheads pointing away) suggesting that they have their origin there. Our results support earlier conclusions [2] that polygonal networks are structural intermediates responsible for organizing contractile proteins of the cortical microfilament layer into stress fibers.     

Microfilaments in Naegleria fowleri amoebae.

Examination by electron microscopy has revealed 2 types of microfilament in the cytoplasm of 3 strains of axenically grown Naegleria fowleri amoebae. Thin, actin-like microfilaments 5-7 nm in diameter are randomly oriented in the nonmotile amoebae, and are concentrated near the plasma membrane. In the actively motile amoebae these microfilaments aggregate to form colateral bundles in close proximity to the plasma membrane. Thick, myosin-like microfilaments 17-19 nm in diameter also occur in the amoebae cytoplasm. The significance of these 2 kinds of microfilament in amoeboid motion is discussed.     

Distribution of actin and tubulin in cells and in glycerinated cell models after treatment with cytochalasin B (CB)

The organization of microfilaments and microtubules in cultured cells before and after the addition of cytochalasin B (CB) was studied both by electron microscopy and immunofluorescence microscopy using antibodies specific for actin, tubulin and tropomyosin. CB induces a rapid disorganization of normal microfilament bundles. Star-like patches of actin and tropomyosin are visualized in immunofluorescence microscopy and dense aggregates of condensed microfilaments are seen in electron microscopy. The integrity of the microtubules is not changed by CB treatment. Addition of CB to glycerinated cells, in contrast to normal cells, does not result in the disorganization of microfilament bundles. CB-treated glycerinated models can still contract upon addition of ATP. Thus the CB-induced rearrangement of microfilament bundles occurs only in vivo and not in glycerinated cell contractility models.     

On the role of microfilaments in cell-shape-mediated growth control of lens epithelial cells

With regard to the fact that, in anchorage-dependent lens epithelial cells, DNA synthesis can be switched on and off by cell flattening and cell rounding, respectively, the state of the microfilaments has been followed by labelling actin with FL-phalloidin during cell-shape alterations. Cell flattening proved to be accompanied by both a structural organization of actin filaments into stress fibres and an enlargement of the area of the cell nucleus. Cell rounding, on the other hand, caused the microfilament bundles to disappear and the area of the nucleus to become smaller. From the time course of the inhibition of DNA synthesis by cytochalasin B, it was inferred that functionally intact microfilaments are required for the entrance of the cells into DNA synthesis but not for the maintenance of ongoing DNA synthesis. The assumption has been made that the tension, generated by microfilaments during cell spreading, will affect the state of the plasma membrane as well as the shape and the structure of the nucleus, which in turn seems to be preparatory for cells to enter the cycle.     

Changes in membrane-microfilament interaction in intercellular adherens junctions upon removal of extracellular Ca2+ ions

EGTA-induced depletion of Ca2+ ions from the culture medium of Madin-Darby bovine kidney epithelial cells results in rapid splitting of adherens-type junctions and the detachment of the vinculin- and actin-containing filament bundle from the cytoplasmic faces of the plasma membrane of the zonula adhaerens. This process was monitored by phase-contrast microscopy, combined with electron microscopy and immunofluorescent localization of the two proteins. It is shown that shortly after extracellular free Ca2+ concentration is lowered to the micromolar range, the actin-containing, junction-associated belt of microfilaments, together with the vinculin-rich junctional plaque material, is irreversibly detached as one structural unit from the plasma membrane, contracts, and is displaced towards the perinuclear cytoplasm where it gradually disintegrates. Other actin- and vinculin-containing structures present in the same cells, notably the focal contacts at the substratum, are not similarly affected by the Ca2+ depletion and retain both the adhesion to the external surface and the association with the plaque and microfilament components. Electron microscopic examination has shown that the membrane domain of the zonulae adhaerentes, unlike that of desmosomes, is not endocytosed after Ca2+ removal and that the displaced actin- and vinculin-containing plaque and filament belt are not associated with a particular membrane. It is further shown that upon restoration of normal Ca2+ levels in the culture medium, new intercellular contacts are established gradually by accretion of both vinculin and actin into new belt-like plaque- and microfilament-containing structures.     

On the Role of Microfilaments In Cell-Shape-Mediated Growth Control of Lens Epithelial Cells

With regard to the fact that, in anchorage-dependent lens epithelial cells, DNA synthesis can be switched on and off by cell flattening and cell rounding, respectively, the state of the microfilaments has been followed by labelling actin with FL-phalloidin during cell-shape alterations. Cell flattening proved to be accompanied by both a structural organization of actin filaments into stress fibres and an enlargement of the area of the cell nucleus. Cell rounding, on the other hand, caused the microfilament bundles to disappear and the area of the nucleus to become smaller. From the time course of the inhibition of DNA synthesis by cytochalasin B, it was inferred that functionally intact microfilaments are required for the entrance of the cells into DNA synthesis but not for the maintenance of ongoing DNA synthesis. the assumption has been made that the tension, generated by microfilaments during cell spreading, will affect the state of the plasma membrane as well as the shape and the structure of the nucleus, which in turn seems to be preparatory for cells to enter the cycle.     

Cytoskeletal dynamics in rabbit synovial fibroblasts: II. Reformation of stress fibers in cells rounded by treatment with collagenase-inducing agents

Modulation of the synthesis and secretion of extracellular matrix proteins and matrix-degrading metalloproteases by rabbit synovial fibroblasts is an important model system for studying the control of tissue-specific gene expression. Induction of collagenase expression is correlated with changes in cell shape and actin filament distribution, but the role of the cellular cytoskeleton in the sustained synthesis and secretion of metalloproteases has not been closely examined. When cells were allowed to respread after rounding by trypsin or cytochalasin, two known metalloprotease inducers, reformation of stress fibers was observed within 2 h in the presence of serum. In the absence of serum, trypsin-treated cells did not respread substantially, even after 24 h in culture. In contrast, cytochalasin-treated cells recovered almost as rapidly in the absence as in the presence of serum, showing reformation of well-formed microfilament bundles within 30 min of drug removal, especially at the spreading cell edges. High resolution electron-microscopic views of detergent-extracted cytoskeletons confirmed the rapid rebundling of peripheral microfilaments. Acrylamide-treated cells fell between these two extremes, spreading slowly in the absence of serum, but almost as rapidly as cytochalasin-treated cells in its presence. Reestablishment of normal intermediate filament distribution generally lagged slightly behind actin for all treatments, and intermediate filaments always appeared to spread back into the cellular cytoplasm within the confines of the reforming peripheral microfilament bundles. No obvious interaction between these two cytoskeletal elements was observed after any treatment, and no specific role for intermediate filaments in modulating gene expression in these cells is suggested by these results. The serum dependence displayed after trypsin or acrylamide treatment may be due to the disturbances in fibronectin synthesis observed in these cells and is consistent with evidence that both induction and sustained expression of matrix-degrading metalloprotease may involve signals transduced through plasma membrane matrix receptors (integrins).     

Effect of hydrocortisone on cell morphology in C6 cells

Hydrocortisone has been found to induce cell spreading in rat glial C6 cells by 24 hours after its addition. This spreading phenomenon is correlated with an increase in the fraction of the peripheral cytoplasm occupied by microfilaments. Cytochalasin B causes disorganization of microfilaments in the peripheral cytoplasm of the cells. Additionally, it also prevents cell spreading in response to hormonal stimulation. High levels of calcium prevent recovery of normal microfilament organization and cell spreading following removal of cytochalasin B, but have no effect on normal microfilament organization alone. Additionally both the hydrocortisone induced spreading of C6 cells and increases in peripheral microfilaments are shown to be dependent on RNA ans protein synthesis. The levels of protein co-electrophoresing with actin are not effected by hydrocortisone.     

Structural organization of ovarian follicle cells in the cotton bug (Dysdercus intermedius) and the honeybee (Apis mellifera)

Summary The somatic epithelia of Dysdercus and Apis follicles were analyzed by electron microscopy, and the patterns of F-actin and microtubules were studied by fluorescence microscopy. The epithelia in both species differ considerably in shape and in the organization of the cytoskeleton. During previtellogenic stages, the epithelium consists of columnar-shaped cells with small (Dysdercus) or no (Apis) lateral intercellular spaces. During vitellogenesis, the follicle cells round up; the intercellular spaces increase in size in Dysdercus follicles, whereas in Apis follicles they remain small. Along the basal surface of the follicle cells, there are conspicuous parallel bundles of microfilaments perpendicular to the anteroposterior axis of the follicles. In the honeybee, these microfilament bundles are present in long filopodia, most of which are embedded in thickenings of the basement membrane and extend over the surfaces of neighbouring cells. In the cotton bug, the basal surface of the follicle cells is thrown into parallel folds. The microfilament bundles are located just underneath the cell membrane where the folds contact the basement membrane. In the polar regions of the Dysdercus follicle, the epithelial cells become flat and adhere to each other without forming intercellular spaces. The basement membrane is particularly thick in the polar areas; this has also been observed in Apis follicles around the intercellular bridge connecting oocyte and nurse cells.     

Relation between cytoskeleton,hypo-osmotic treatment and volume regulation in Ehrlich ascites tumor cells

Summary Pretreatment with cytochalasin B, which is known to disrupt microfilaments, significantly inhibits regulatory volume decrease (RVD) in Ehrlich ascites tumor cells, suggesting that an intact microfilament network is a prerequisite for a normal RVD response. Colchicine, which is known to disrupt microtubules, has no significant effect on RVD. Ehrlich cells have a cortical three-dimensional, orthogonal F-actin filament network which makes the cells look completely black in light microscopy following immunogold/silver staining using anti-actin antibodies. After addition of cytochalasin B, the stained cells get lighter with black dots localized to the plasma membrane and appearance of multiple knobby protrusions at cell periphery. Also, a significant decrease in the staining of the cells is seen after 15 min of RVD in hypotonic medium. This microfilament reorganization appears during RVD in the presence of external Ca²⁺ or Ca²⁺-ionophore A23187. It is, however, abolished in the absence of extracellular calcium, with or without prior depletion of intracellular Ca²⁺ stores. An effect of increased calcium influx might therefore be considered. The microfilament reorganization during RVD is abolished by the calmodulin antagonists pimozide and trifluoperazine, suggesting the involvement of calmodulin in the process. The microfilament reorganization is also prevented by addition of quinine. This quinine inhibition is overcome by addition of the K⁺ ionophore valinomycin.     

Spreading of vascular endothelial cells in culture: Spatial reorganization of cytoplasmic fibers and organelles

The three-dimensional organization and fine structure of cytoplasmic components within whole non-embedded bovine aortic endothelial cells were examined during their attachment and spreading in tissue culture. Cells were cultured directly on Formvar-coated gold grids, fixed in glutaraldehyde and osmium tetroxide, critical point dried and examined by transmission electron microscopy (TEM) using stereoscopic methods, and by scanning electron microscopy (SEM). Reorganization of cytoplasmic structures during cell spreading occurred in four sequential stages: (1) spreading of the plasma membrane and unstructured cytoplasmic matrix; (2) spreading of cytoplasmic fiber systems (microtubules, microfilament bundles and microtrabecular system); (3) alignment of microfilament bundles and formation of radial tracts of microtubules; and (4) centripetal movement of organelles along radial tracts. These stages observed by TEM correlated with progressive degrees of cell flattening as visualized by SEM. These studies demonstrate that a characteristic reorganization of intracellular fiber systems and organelles accompanies the spreading of endothelial cells in culture.     

Isolation of microvillar microfilaments and associated transmembrane complex from ascites tumor cell microvilli

The association of microvillar microfilaments with the microvillar membrane actin-containing transmembrane complex of MAT-C1 13762 ascites tumor cell microvilli has been investigated by differential centrifugation, gel electrophoresis and electron microscopy of detergent extracts of the isolated microvilli. Several methods have been used to reduce breakdown and solubilization of the microfilament core actin during the detergent extractions for preparation of microvillar core microfilaments. Gel electrophoresis of differential centrifugation fractions demonstrated that over 70% of the total microvillus actin could be pelleted with microfilament cores at 10 000 g under extraction conditions which reduce filament breakdown. Transmission electron microscopy (TEM) of all of the core preparations showed arrays of microfilaments and small microfilament bundles. The major protein components of the microfilament cores, observed by sodium dodecyl sulfate (SDS) electrophoresis, were actin and alpha-actinin. Among the less prominent polypeptide components was a 58 000 Dalton polypeptide (58 K), previously identified as a member of the MAT-Cl transmembrane complex. This three-component complex contains, in addition to 58 K, actin associated directly and stably with a cell surface glycoprotein (Carraway, CAC, Jung, G & Carraway, K L, Proc. natl acad. sci. US 80 (1983) 430). Evidence that the apparent association of complex with the microfilament core was not due simply to co-sedimentation was provided by myosin affinity precipitation. These results provide further evidence that the transmembrane complex is a site for the interaction of microfilaments with the microvillar plasma membrane.     

Formation of bundles of microfilaments during spreading of fibroblasts on the substrate

Electron microscopy was used to study the sites of formation of bundles of parallel microfilaments in the early stages of spreading of normal mouse embryo fibroblasts on the substrate. Bundles of microfilaments were not found in suspended cells. Contact of the surface of spherical cells with the substrate was not sufficient for the formation of bundles: these bundles were not seen near the under surface of cells that were already attached to the substrate but had not yet developed cytoplasmic outgrowths at their periphery. Peripheral cytoplasmic outgrowths (microspikes and lamellar processes) attached to the substrate were found to be the only sites of localization of the first bundles of microfilaments seen in the spreading cells. It is suggested that surface and/or cytoplasm of the newly-formed peripheral cytoplasmic outgrowth may have some special properties necessary for the initiation of the development of microfilament bundles.     

src64 and tec29 are required for microfilament contraction during Drosophila cellularization

Formation of the Drosophila cellular blastoderm involves both membrane invagination and cytoskeletal regulation. Mutations in src64 and tec29 reveal a novel role for these genes in controlling contraction of the actin-myosin microfilament ring during this process. Although membrane invagination still proceeds in mutant embryos, its depth is not uniform, and basal closure of the cells does not occur during late cellularization. Double-mutant analysis between scraps, a mutation in anillin that eliminates microfilament rings, and bottleneck suggests that microfilaments can still contract even though they are not organized into rings. However, the failure of rings to contract in the src64 bottleneck double mutant suggests that src64 is required for microfilament ring contraction even in the absence of Bottleneck protein. Our results suggest that src64-dependent microfilament ring contraction is resisted by Bottleneck to create tension and coordinate membrane invagination during early cellularization. The absence of Bottleneck during late cellularization allows src64-dependent microfilament ring constriction to drive basal closure.     

Microtubule and microfilament rearrangements during capping of concanavalin A receptors on cultured ovarian granulosa cells

Thin-section electron microscope analysis of rat and rabbit-cultured granulosa cells treated with concanavalin A (Con A) at 37 degrees C revealed coordinated changes in the cytoplasmic disposition of microfilaments, thick filaments, and microtubules during cap formation and internalization of lectin-receptor complexes. Con A-receptor clustering is accompanied by an accumulation of subplasmalemmal microfilaments which assemble into a loosely woven ring as patches of receptor move centrally on the cell surface. Periodic densities appear in the microfilament ring which becomes reduced in diameter as patches coalesce to form a single central cap. Microtubules and thick filaments emerge associated with the capped membrane. Capping is followed by endocytosis of the con A-receptor complexes. During this process, the microfilament ring is displaced basally into the cytoplasm and endocytic vesicles are transported to the paranuclear Golgi complex along microtubules and thick filaments. Eventually, these vesicles aggregate near the cell center where they are embedded in a dense meshwork of thick filaments. Freeze-fracture analysis of Con A-capped granulosa cells revealed no alteration in the arrangement of peripheral intramembrane particles but large, smooth domains were conspicuous in the capped region of the plasma membrane. The data are discussed with reference to the participation of microtubules and microfilaments in the capping process.     

The actin microfilament network within elongating pollen tubes of the gymnospermPicea abies (Norway spruce)

Summary Actin microfilaments form a dense network within pollen tubes of the gymnosperm Norway spruce (Picea abies). Microfilaments emanate from within the pollen grain and form long, branching arrays passing through the aperture and down the length of the pollen tube to the tip. Pollen tubes are densely packed with large amyloplasts, which are surrounded by branching microfilament bundles. The vegetative nucleus is suspended within the elongating pollen tube within a complex array of microfilaments oriented both parallel to and perpendicular with the growing axis. Microfilament bundles branch out along the nuclear surface, and some filaments terminate on or emanate from the surface. Microfilaments in the pollen tube tip form a 6 m thick, dense, uniform layer beneath the plasma membrane. This layer ensheathes an actin depleted core which contains cytoplasm and organelles, including small amyloplasts, and extends back 36 m from the tip. Behind the core region, the distinct actin layer is absent as microfilaments are present throughout the pollen tube. Organelle zonation is not always maintained in these conifer pollen tubes. Large amyloplasts will fill the pollen tube up to the growing tip, while the distinct layer of microfilaments and cytoplasm beneath the plasma membrane is maintained. The distinctive microfilament arrangement in the pollen tube tips of this conifer is similar to that seen in tip growth in fungi, ferns and mosses, but has not been reported previously in seed plants.     

Cytoarchitecture of Kirsten sarcoma virus-transformed rat kidney fibroblasts: butyrate-induced reorganization within the actin microfilament network

Murine sarcoma virus-transformed rat fibroblasts (KNRK cells) undergo marked cytoarchitectural reorganization during in vitro exposure to sodium-n-butyrate (NaB) resulting in restoration of (1) a more typical fibroblastoid morphology, (2) proper cell-to-cell orientation, and (3) substratum adherence. Augmented cell spreading, involving greater than 90% of the population, was a function of culture density and time of exposure to NaB (2 mM final concentration). Induced cell spreading reflected a 2.5- to 3.0-fold increase in both total cellular actin content and deposition of actin into the detergent-resistant cytoskeleton. Cytoskeletal actin deposition in response to NaB was accompanied by the formation of occasionally dense, parallel alignments of F-actin-containing microfilaments and by a dramatic increase in the size and incidence of actin-enriched membrane ruffles. Long-term NaB-treated cells exhibited parallel orientations of microfilaments similar to those found in untransformed fibroblasts. Increased cytoskeletal actin occurred within 24 hr of NaB exposure, correlating with the initial reorganization of actin-containing microfilaments detected microscopically, and reflected concomitant 3-fold increases in cellular alpha-actinin and fibronectin content. In contrast, the amount of vimentin, tropomyosin, and tubulin in NaB-treated cells was significantly decreased. NaB-induced morphologic restructuring of sarcoma virus-transformed fibroblasts, thus, impacts on all three basic cytoskeletal systems. Selective increases, however, were evident in particular cytoskeletal proteins (actin, alpha-actinin, fibronectin) implicated in microfilament networking and cell spreading.     

Molecular events during membrane fusion. A study of exocytosis in rat peritoneal mast cells

We have used thin section and freeze-fracture electron microscopy to study membrane changes occurring during exocytosis in rat peritoneal mast cells. By labeling degranulating mast cells with ferritin-conjugated lectins and anti-immunoglobulin antibodies, we demonstrate that these ligands do not bind to areas of plasma membrane or granule membrane which have fused with, or are interacting with, granule membrane. Moreover, intramembrane particles are also largely absent from both protoplasmic and external fracture faces of plasma and granule membranes in regions where these membranes appear to be interacting. Both the externally applied ligands and intramembrane particles are sometimes concentrated at the edges of fusion sites. The results indicate that membrane proteins are displaced laterally into adjacent membrane regions before the fusion process and that fusion occurs between protein-depleted lipid bilayers. The finding of protein-depleted blebs in regions of plasma and granule membrane interaction raises the interesting possibility that blebbing may be a process for exposing the granule contents to the extracellular space and for the elimination of excess lipid while conserving membrane proteins.