Reorganization of the subplasmalemmal cytoskeleton in association with exocytosis in rat mast cells

The subplasmalemmal cytoskeleton in mast cells has been studied by scanning electron microscopy of the internal side of the plasma membrane. Rearrangement of the dense subplasmalemmal network of actin filaments took place following cell activation by compound 48/80 and secretion of histamine. The rearrangement was a withdrawal of the subplasmalemmal cytoskeleton from the exocytotic sites and the development of bare, filament-free areas around the sites. In calcium-depleted mast cells we demonstrated a dense network that was difficult to break. Activation of the calcium-depleted cells by compound 48/80 did not induce rearrangement of the network, and in parallel there was no secretion of histamine.     

SUBPLASMALEMMAL MICROFILAMENTS AND MICROTUBULES IN RESTING AND PHAGOCYTIZING CULTIVATED MACROPHAGES

The subplasmalemmal organization of the free and glass-attached surfaces of resting and phagocytizing cultivated macrophages were examined in an attempt to define specific membrane-associated structures related to phagocytosis. From analysis of serial thin sections of oriented cells it was found that the subplasmalemmal region of the attached cell surface has a complex microfilament and microtubule organization relative to the subplasmalemmal area of the free surface. A filamentous network composed of 40–50-Å microfilaments extended for a depth of 400–600 Å from the attached plasma membrane. Immediately subjacent to the filamentous network was a zone of oriented bundles of 40–50-Å microfilaments and a zone of microtubules. Additional microtubules were found to extend from the plasma membrane to the interior of the cell in close association with electron-dense, channellike structures. In contrast, the free aspect of the cultivated macrophage contained only the subplasmalemmal filamentous network. However, after a phagocytic pulse with polystyrene particles (14 µm diam) microtubules and oriented filaments similar to those found on the attached surface were observed surrounding the ingested particles. The observations reported in this paper provide support for the hypothesis that microfilaments and/or microtubules play a role in the translocation of plasma membrane required for the functionally similar processes of phagocytosis and cell attachment to glass.     

Localization and organization of actin in melanophores

Melanophores of the angelfish, Pterophyllum scalare, were studied in an attempt to demonstrate the existence of actin in these cells although microfilaments had previously not been found. By use of a variety of procedures, including immunofluorescence microscopy of intact and detergent-extracted cells, transmission electron microscopy, high voltage electron microscopy of whole-mount preparations, and labeling with heavy meromyosin-subfragment 1, the presence of a loose cortical mesh of actin filaments is demonstrated. In addition, a more parallel array of filaments is detected in microspike- and microvillus-like surface projections. There seem to be no changes in the arrangement of these filaments as a function of the state of pigment distribution. No actin filaments could be found in association with pigment granules or microtubules in more central cell portions. For reasons presently unknown, the preservation of the cortical filament network in lysed cell preparations depends strongly on the presence of an intact microtubular system. The involvement of this subplasmalemmal actin filament network in pigment granule transport remains unclear.     

The cytoskeleton of the resting human blood platelet: structure of the membrane skeleton and its attachment to actin filaments

We used high-resolution EM and immunocytochemistry in combination with different specimen preparation techniques to resolve the ultrastructure of the resting platelet cytoskeleton. The periphery of the cytoskeleton, an electron-dense subplasmalemmal region in thin section electron micrographs, is a tightly woven planar sheet composed of a spectrin-rich network whose interstices contain GPIb/IX-actin-binding protein (ABP) complexes. This membrane skeleton connects to a system of curved actin filaments (F-actin) that emanate from a central oval core of F-actin cross-linked by ABP. The predominant interaction of the radial actin filaments with the membrane skeleton is along their sides, and the strongest connection between the membrane skeleton and F-actin is via ABP-GPIb ligands, although there is evidence for spectrin attaching to the ends of the radial actin filaments as well. Since a mechanical separation of the F-actin cores and radial F-actin-GPIb-ABP complexes from the underlying spectrin-rich skeleton leads to the latter's expansion, it follows that the spectrin-based skeleton of the resting cell may be held in a compressed form by interdigitating GPIb/IX complexes which are immobilized by radial F-actin-ABP anchors.     

Changing patterns of plasma membrane-associated filaments during the initial phases of polymorphonuclear leukocyte adherence

By utilizing a combination of several ultrastructural techniques, we have been able to demonstrate differences in filament organization on the adherent plasma membranes of spreading and mobile PMN as well as within the extending lamellipodia. To follow the subplasmalemmal filaments of this small amoeboid cell during these kinetic events, we sheared off the upper portions of cells onto glass and carbon surfaces for 30 s--5 min. The exposed adherent membranes were immediately fixed and processed for high-resolution SEM or TEM. Whole cells were also examined by phase contrast microscopy, SEM, and oriented thin sections. Observed by SEM, the inner surface of nonadherent PMN membranes is free of filaments, but within 30 s of attachment to the substrate a three-dimensional, interlocking network of globular projections and radiating microfilaments--i.e., a subplasmalemmal filament complex--is consistently demonstrable (with or without postfixation in OsO4). Seen by TEM, extending lamellipodia contain a felt of filamentous and finely granular material, distinct from the golbule/filament complex of the adjacent adherent membrane. In the spread cell, this golbule-filament complex covers the entire lower membrane and increases in filament-density over the next 2--3 min. By 3--5 min after plating, as the PMN rounds up before the initiation of amoeboid movements, another pattern emerges--circumferential bands of anastomosing filament bundles in which thick, short filaments resembling myosin are found. This work provides structural evidence on the organization of polymerized contractile elements associated with the plasma membrane during cellular adherence.     

Comparison of the three-dimensional organization of unextracted and Triton-extracted human neutrophilic polymorphonuclear leukocytes

The three-dimensional organization of the cytoplasm of randomly migrating neutrophils was studied by stereo high-voltage electron microscopy. Examination of whole-mount preparations reveals with unusual clarity the structure of the cytoplasmic ground substance and cytoskeletal organization; similar clarity is not observed in conventional sections. An extensive three-dimensional network of fine filaments (microtrabeculae) approximately 7 to 17 nm in diameter extends throughout the cytoplasm and between the two cell cortices; it also comprises the membrane ruffles and filopodia. The granules are dispersed within the lattice and are surrounded by microtrabeculae. The lattice appears to include dense foci from which the microtrabeculae emerge. Triton X-100 dissolves the plasma membrane, most of the granules, and many of the microtrabecular strands and leaves as a more stable structure a cytoskeletal network composed of various filaments and microtubules. Heavy meromyosin-subfragment 1 (S1) decoration discloses actin filaments as the major filamentous component present in membrane ruffles and filopodia. Actin filaments, extending from the leading edge of the cells, are of uniform polarity, with arrowheads pointing towards the cell body. Likewise, the filaments forming the core of filopodia have the barbed end distal. End-to-side associations of actin filaments as well as fine filaments (2--3 nm) which are not decorated with S1 and link actin filaments are observed. The ventral cell cortex includes numerous substrate-associated dense foci with actin filaments radiating from the dense center. Virtually all the microtubules extend from the centrosome. An average of 35 +/- 7 microtubules originate near the pair of centrioles and radiate towards the cell periphery; microtubule fragments are rare. Intermediate filaments form an open network of single filaments in the perinuclear space. Comparison of Triton-extracted and unextracted cells suggest that many of the filamentous strands seen in unextracted cells have as a core a stable actin filament.     

Organization of filaments underneath the plasma membrane of developing chicken skeletal muscle cells in vitro revealed by the freeze-dry and rotary replica method

Summary Cytoskeletal organization and its association with plasma membranes in embryonic chick skeletal muscle cells in vitro was studied by the freeze-drying and rotary-shadowing method of physically ruptured cells. The cytoskeletal filaments underlying the plasma membranes were sparse in myogenic cells at the stage when cells exhibited great lipid fluidity in plasma membranes (fusion competent mononucleated myoblasts and recently fused young myotubes). Myotubes at more advanced stages of development possessed a highly interconnected dense filamentous network just underneath the cell membrane. This subsarcolemmal network was composed predominantly of 8–10 nm filaments; they were identified as actin filaments because of their decoration with myosin subfragment-1. Fine fibrils having a diameter of 3–5 nm were found on the protoplasmic surface of the plasmalemma at both the early and advanced stages of development. They were associated with the subsarcolemmal cytoskeletal filaments. Short 2–5 nm cross-linking filaments were occasionally seen between filaments in the subsarcolemmal network. We conclude that, although the subsarcolemmal cytoskeletal network contains many actin filaments, this domain appears to play some role in preserving the cell shape in the form of the membrane skeleton rather than membrane mobility.     

Interactions between actin filaments and between actin filaments and membranes in quick-frozen and deeply etched hair cells of the chick ear

Replicas of the apical surface of hair cells of the inner ear (vestibular organ) were examined after quick freezing and rotary shadowing. With this technique we illustrate two previously undescribed ways in which the actin filaments in the stereocilia and in the cuticular plate are attached to the plasma membrane. First, in each stereocilium there are threadlike connectors running from the actin filament bundle to the limiting membrane. Second, many of the actin filaments in the cuticular plate are connected to the apical cell membrane by tiny branched connecting units like a "crow's foot." Where these "feet" contact the membrane there is a small swelling. These branched "feet" extend mainly from the ends of the actin filaments but some connect the lateral surfaces of the actin filaments as well. Actin filaments in the cuticular plate are also connected to each other by finer filaments, 3 nm in thickness and 74 +/- 14 nm in length. Interestingly, these 3-nm filaments (which measure 4 nm in replicas) connect actin filaments not only of the same polarity but of opposite polarities as documented by examining replicas of the cuticular plate which had been decorated with subfragment 1 (S1) of myosin. At the apicolateral margins of the cell we find two populations of actin filaments, one just beneath the tight junction as a network, the other at the level of the zonula adherens as a ring. The latter which is quite substantial is composed of actin filaments that run parallel to each other; adjacent filaments often show opposite polarities, as evidenced by S1 decoration. The filaments making up this ring are connected together by the 3-nm connectors. Because of the polarity of the filaments this ring may be a "contractile" ring; the implications of this is discussed.     

Cytoskeletal involvement during hypo-osmotic swelling and volume regulation in cultured chick cardiac myocytes

The membrane skeleton in spherical cardiac myocytes subjected to hypo-osmotic challenge was examined by laser scanning confocal microscopy. A distinct cortical layer intimately localized under the plasmalemma was revealed for spectrin and actin (including filamentous actin and alpha-sarcomeric actin). Desmin filaments were abundant and in close contact with the plasmalemma. During swelling and subsequent regulatory volume decrease (RVD) the structural integrity of these cytoskeletal elements remained intact, and the close association between actin and plasmalemma persisted as confirmed by double immunolabeling. Subplasmalemmal beta-tubulin labeling was sparse. Hypo-osmotic conditions disrupted the microtubules and depolymerized tubulin. Neither pretreatment with taxol nor with colchicine, resulted in any effect on cell volume regulation. The present results show that actin, desmin, and spectrin contribute to a subplasmalemmal cytoskeletal network in spherical cardiac myocytes, and that this membrane skeleton remains structurally intact during swelling and RVD. It is suggested that the integrity of this membrane skeleton is important for stabilization of the plasmalemma and the membrane-integrated proteins during hypo-osmotic challenge, and that it may participate in the regulation of the cell volume.     

Connections of intermediate filaments with the nuclear lamina and the cell periphery

We investigated the relationship between intermediate filaments (IFs) and other detergent- and nuclease-resistant filamentous structures of cultured liver epithelial cells (T51B cell line) using whole mount unembedded preparations which were sequentially extracted with Triton X-100 and nucleases. Immunogold labelling and stereoscopic observation facilitated the examination of each filamentous structure and their three-dimensional relationships to each other. After solubilizing phospholipid, nucleic acid and soluble cellular protein, the resulting cytoskeleton preparation consisted of a network of cytokeratin and vimentin IFs linked by 3 nm filaments. The IFs were anchored to and determined the position of the nuclear lamina filaments (NLF) network and the centrioles. The NLF was composed of the nuclear lamina filaments measuring 3-6 nm in diameter which radiated from and anchored to the skeleton of the nuclear pores. The IFs located in the nuclear region appeared to be interwoven with the NLF. At the cell surface, the IFs seemed to be attached to the putative actin filament network. They formed a focally interrupted plexus-like structure at the cell periphery. Fragments of vimentin filaments were found among the filamentous network located at the cell surface, and some filaments terminated blindly there.     

Immunocytochemical localization of fodrin and ankyrin in bovine chromaffin cells in vitro.

We raised antibodies to brain fodrin and erythrocyte ankyrin and examined the distribution of the antigens in cultured bovine chromaffin cells by immunocytochemical techniques. Immunofluorescence microscopy of whole cells showed intense labeling for both proteins, but fine localization could not be determined. In contrast, in cell specimens mechanically unroofed before fixation, the distribution of the two proteins revealed an apparent difference in the ventral plasma membrane: immunofluorescence for fodrin was dense and mostly even, whereas that for ankyrin appeared as scattered dots. Immunogold electron microscopy of the unroofed cells showed that labeling for fodrin was localized in a network of thin filaments, the diameter of which was 2-3 nm at the thinnest portion. Ankyrin labeling was mostly associated with filaments 5-10 nm in diameter. Notably, labeling for both fodrin and ankyrin was found over the coated membrane. The present results indicate that fodrin and ankyrin in the chromaffin cell do not constitute a submembranous network as spectrin and ankyrin do in the erythrocyte; whereas fodrin is closely associated with the plasma membrane, ankyrin is mostly linked to the cytoskeleton. The existence of both proteins in the coated region implies that they are functionally related to exocytosis and/or to ensuing membrane retrieval in the chromaffin cell.     

Immunolocalization of a novel, cytoskeleton-associated polypeptide of Mr 230,000 daltons (p230)

Antibodies were raised against a cytoskeleton-associated, nonphosphorylated, 230,000-dalton bovine lens polypeptide (designated p230), and rendered monospecific by using a novel immunoaffinity technique. In immunofluorescence and electron microscopy of cultured fibroblasts, as well as of various other cells (endothelial, epithelial, lenticular, monocytes, neuroblastoma cells) and tissues (human kidney and liver), p230 was localized as a distinct subplasmalemmal layer in the peripheral cytoplasm of the cells. It constituted less than 0.3% of the total cellular protein in cultured fibroblasts and was not extractable with Triton X-100. In detergent-extracted cytoskeletal preparations of cultured fibroblasts, p230 remained as an elaborate peripheral network that showed a distribution distinctly different from that of the major cytoskeletal structures, stress fibers, cortical myosin, vinculin, and intermediate filaments (IF). The distribution was not dependent on the presence of intact stress fibers or microtubules, as shown by double-fluorescence microscopy of cells exposed to cytochalasin B or cultured in the presence of monensin and of cold-treated cells. Upon demecolcine-induced reorganization of intermediate filaments, however, the localization of p230 was rapidly altered to a dense plaque underneath the perinuclear aggregate of intermediate filaments. On the other hand, p230 seemed to colocalize with the detergent-resistant cell surface lamina, visualized in fluorescence microscopy with fluorochrome-coupled wheat germ agglutinin-lectin. The results suggest that p230 is part of a cell surface- and cytoskeleton-associated subplasmalemmal structure that may play an important role in cell surface-cytoskeleton interaction in various cells both in vitro and in vivo.     

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.     

Intracellular calcium gradients in cultured human uterine smooth muscle: a functionally important subplasmalemmal space

The plasma membrane contains the key elements for the control of coupling excitation to contraction in smooth muscle. The superficial calcium buffer barrier, initially proposed by van Breemen for vascular smooth muscle, may participate in the regulation of calcium entry in other smooth muscle types. To investigate the relationship between the sarcoplasmic reticulum (SR) and the plasma membrane in myometrial smooth muscle cells, we performed experiments using videofluorescence imaging and cell-attached electrophysiology. The cell-attached patch was used as a reporter for the free calcium in the subplasmalemmal space by monitoring openings of the Maxi-K channel. Calcium green-1 was used to simultaneously monitor changes of the deep cytosolic calcium concentrations. The cell with the patch attached was stimulated via an intercellular calcium wave from an adjacent cell. In this fashion, release of SR calcium was accomplished with minimal disturbance of the plasma membrane and the subplasmalemmal space of the cell studied. With physiological bathing solution, six of seven calcium waves activated Maxi-K channels. Surprisingly, the Maxi-K channels began opening 6.3 +/- 4.7s (range 2.6-15.0s) after the wave passed the pipette location. When plasma membrane calcium fluxes were inhibited with 100 microM lanthanum, no Maxi-K channel openings were observed in six of seven experiments. These results are best explained by a subplasmalemmal space in which the calcium concentration is largely controlled by store-operated channels. These results suggest the superficial buffer barrier as merely one aspect of subplasmalemmal regulation of calcium dynamics, and emphasize the importance of store-operated calcium channels during dynamic calcium changes.     

Quick-freeze,deep-etch visualization of exocytosis in anterior pituitary secretory cells: localization and possible roles of actin and annexin II

The exocytotic process in the anterior pituitary secretory cells was studied using quick-freeze deep-etch electron microscopy, fluorescein-isothiocyanate-phalloidin staining, heavy meromyosin decoration, and immuno-electron microscopy. The subcortical actin filaments are distributed unevenly in the peripheral cytoplasm. Few secretory granules are seen beneath the plasma membrane in the region where the peripheral cytoplasm is occupied by numerous subcortical actin filaments. On the contrary, in the region free of the subcortical actin filaments, many secretory granules lie in contact with the plasma membrane. Thus, the subcortical actin filaments may control the approach of the secretory granules to the plasma membrane in these cells. The granule and plasma membranes that lie in close proximity are linked by intervening strands. Unfused portions of both membranes remain linked by these strands during membrane fusion and opening. These strands may be involved in membrane contact, fusion and opening during exocytosis. Annexin II (calpactin I) has been demonstrated immunocytochemically to be localized at the contact sites between the granule and plasma membranes, and is therefore a possible component of the intervening strands. Membrane fusion starts within focal regions of both membranes less than 50 nm in diameter. The plasma membrane shows inward depressions toward the underlying granules immediately before fusion. The disappearance of intramembranous particles from the exocytotic site of the membrane has not been observed.     

Cytoskeletal reorganization of human platelets after stimulation revealed by the quick-freeze deep-etch technique

We studied the cytoskeletal reorganization of saponized human platelets after stimulation by using the quick-freeze deep-etch technique, and examined the localization of myosin in thrombin-treated platelets by immunocytochemistry at the electron microscopic level. In unstimulated saponized platelets we observed cross-bridges between: adjoining microtubules, adjoining actin filaments, microtubules and actin filaments, and actin filaments and plasma membranes. After activation with 1 U/ml thrombin for 3 min, massive arrays of actin filaments with mixed polarity were found in the cytoplasm. Two types of cross-bridges between actin filaments were observed: short cross-bridges (11 +/- 2 nm), just like those observed in the resting platelets, and longer ones (22 +/- 3 nm). Actin filaments were linked with the plasma membrane via fine short filaments and sometimes ended on the membrane. Actin filaments and microtubules frequently ran close to the membrane organelles. We also found that actin filaments were associated by end-on attachments with some organelles. Decoration with subfragment 1 of myosin revealed that all the actin filaments associated end-on with the membrane pointed away in their polarity. Immunocytochemical study revealed that myosin was present in the saponin-extracted cytoskeleton after activation and that myosin was localized on the filamentous network. The results suggest that myosin forms a gel with actin filaments in activated platelets. Close associations between actin filaments and organelles in activated platelets suggests that contraction of this actomyosin gel could bring about the observed centralization of organelles.     

The cytoskeleton of cryofixed Purkinje cells of the chicken cerebellum

Summary Cerebella of 3- to 6-week-old chickens were cryofixed in a nitrogen-cooled propane jet, deep-etched and rotary-shadowed. The use of a brief perfusion of 0.32 M sucrose improved the quality of the cryofixation and allowed the study of the deeper layers of the cerebellar cortex. It is reported that the cytoskeleton of the Purkinje cells (PC) shows distinct domains and composition of filamentous structures in the different regions of the cell cytoplasm, such as the perikaryon, the cytoplasm of dendrites and the axoplasm. The perikaryon is occupied by a meshwork of fine filaments, 4–7 nm in diameter, that extends from the nuclear outer membrane to the cell membrane. In this zone the cell organelles (e.g., endoplasmic reticulum, mitochondria) adopt a circular arrangement around the nucleus. All structures are anchored by microfilaments to the cytoplasmic network. The dendrites show a dense cytoplasmic network including bundles of microtubules, neurofilaments and microfilaments. Numerous aggregated globular components are attached to this cytoskeleton. The cytoskeleton of the dendritic spines shows axially oriented 10-nm bundles of filaments, which are interconnected and anchored also to the cell membrane and the components of the agranular endoplasmic reticulum by cross-linkers. As described in peripheral nerves, the axoplasm of axons in the central nervous system exhibits predominantly neurofilaments and microtubules aligned along the axis of the neuntes in a three-dimensional arrangement and interconnected by cross-linker filaments and filamentous structures.     

INHIBITION OF PHAGOCYTOSIS AND PLASMA MEMBRANE MOBILITY OF THE CULTIVATED MACROPHAGE BY CYTOCHALASIN B : Role of Subplasmalemmal Microfilaments

Functional and morphologic effects of cytochalasin B on the cultivated macrophage were examined to determine the basis for plasma membrane movements of the type required for endocytosis and/or spreading on a substratum. Inhibition of phagocytosis and changes in cell shape by cytochalasin B exhibited nearly identical dose-response curves requiring 2–5 x 10^-6 M and 1–2 x 10^-5 M cytochalasin B to inhibit these functions by 50% and 100%, respectively. In contrast, hexose transport was ten times more sensitive to the drug requiring 2–3 x 10^-7 M cytochalasin B to achieve 50% inhibition of 2-deoxyglucose uptake. Inhibition of phagocytosis and changes in cell shape could not be explained solely by drug effects on hexose transport. Analysis of serial thin sections showed that cytochalasin B doses inhibitory for hexose transport had no effect on distribution or organization of either of the two subplasmalemmal microfilament types. However, cytochalasin B concentrations (2.0 x 10^-5 M) that inhibited phagocytosis and altered cell shape disorganized and/or disrupted oriented bundles of 40–50-Å subplasmalemmal microfilaments, but had no effect on the microfilamentous network. Comparative dose-response studies showing positive correlations among cytochalasin B effects on phagocytosis, changes in cell shape, and alterations in oriented subplasmalemmal microfilament bundles provide additional support for the hypothesis that microfilamentous structures play a role in translocation of plasma membrane required for endocytosis and cell motility.     

Subcellular motility: A correlated light and electron microscopic study using cultured cells

To assess the possible role of filaments in subcellular motility, particular cultured cells were studied by light and electron microscopy. Motile cell margins always contained meshworks of ~50 Å diam. filaments. Organelles moved within cytoplasm occupied by a meshwork of 50–100 Å filaments and microtubules. When cells were treated with cytochalasin B, movements of cell margins stopped, but organelle movements continued; electron microscopically, while subplasmalemmal filaments had disappeared, subcortical filaments and microtubules remained. When cells were treated with hypertonic medium, organelle movements ceased but marginal movements continued; electron microscopically, although cell margins contained normal filament arrays, few subcortical filaments remained. It is concluded that while cell margins are moved by a meshwork of filaments, organelle movement is mediated by a subcortical meshwork of filaments and microtubules.     

Exocytosis in neuroendocrine cells: new tasks for actin

Most secretory cells undergoing calcium-regulated exocytosis in response to cell surface receptor stimulation display a dense subplasmalemmal actin network, which is remodeled during the exocytotic process. This review summarizes new insights into the role of the cortical actin cytoskeleton in exocytosis. Many earlier findings support the actin-physical-barrier model whereby transient depolymerization of cortical actin filaments permits vesicles to gain access to their appropriate docking and fusion sites at the plasma membrane. On the other hand, data from our laboratory and others now indicate that actin polymerization also plays a positive role in the exocytotic process. Here, we discuss the potential functions attributed to the actin cytoskeleton at each major step of the exocytotic process, including recruitment, docking and fusion of secretory granules with the plasma membrane. Moreover, we present actin-binding proteins, which are likely to link actin organization to calcium signals along the exocytotic pathway. The results cited in this review are derived primarily from investigations of the adrenal medullary chromaffin cell, a cell model that is since many years a source of information concerning the molecular machinery underlying exocytosis.