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Essentials Endothelial activation initiates multiple processes, including hemostasis and inflammation. The molecules that contribute to these processes are co-stored in secretory granules. How can the cells control release of granule content to allow differentiated responses? Selected agonists recruit an exocytosis-linked actin ring to boost release of a subset of cargo., Summary: Background Endothelial cells harbor specialized storage organelles, Weibel-Palade bodies (WPBs). Exocytosis of WPB content into the vascular lumen initiates primary hemostasis, mediated by von Willebrand factor (VWF), and inflammation, mediated by several proteins including P-selectin. During full fusion, secretion of this large hemostatic protein and smaller pro-inflammatory proteins are thought to be inextricably linked. Objective To determine if secretagogue-dependent differential release of WPB cargo occurs, and whether this is mediated by the formation of an actomyosin ring during exocytosis. Methods We used VWF string analysis, leukocyte rolling assays, ELISA, spinning disk confocal microscopy, high-throughput confocal microscopy and inhibitor and siRNA treatments to demonstrate the existence of cellular machinery that allows differential release of WPB cargo proteins. Results Inhibition of the actomyosin ring differentially effects two processes regulated by WPB exocytosis; it perturbs VWF string formation but has no effect on leukocyte rolling. The efficiency of ring recruitment correlates with VWF release; the ratio of release of VWF to small cargoes decreases when ring recruitment is inhibited. The recruitment of the actin ring is time dependent (fusion events occurring directly after stimulation are less likely to initiate hemostasis than later events) and is activated by protein kinase C (PKC) isoforms. Conclusions Secretagogues differentially recruit the actomyosin ring, thus demonstrating one mechanism by which the prothrombotic effect of endothelial activation can be modulated. This potentially limits thrombosis whilst permitting a normal inflammatory response. These results have implications for the assessment of WPB fusion, cargo-content release and the treatment of patients with von Willebrand disease., (© 2018 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Attractive and repulsive cell guidance is essential for animal life and important in disease. Cell migration toward attractants dominates studies [1-8], but migration away from repellents is important in biology yet relatively little studied [5, 9, 10]. It is widely held that cells initiate migration by protrusion of their front [11-15], yet this has not been explicitly tested for cell guidance because cell margin displacement at opposite ends of the cell has not been distinguished for any cue. We argue that protrusion of the front, retraction of the rear, or both together could in principle break cell symmetry and start migration in response to guidance cues [16]. Here, we find in the Dictyostelium model [6] that an attractant-cAMP-breaks symmetry by causing protrusion of the front of the cell, whereas its repellent analog-8CPT-breaks symmetry by causing retraction of the rear. Protrusion of the front of these cells in response to cAMP starts with local actin filament assembly, while the delayed retraction of the rear is independent of both myosin II polarization and of motor-based contractility. On the contrary, myosin II accumulates locally in the rear of the cell in response to 8CPT, anticipating retraction and required for it, while local actin assembly is delayed and couples to delayed protrusion at the front. These data reveal an important new concept in the understanding of cell guidance., (Copyright © 2018 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

For decades, ever growing data on myosin II provides strong evidence that interaction of myosin-II-motor-domain with actin filaments within cells retracts the cell rear during actin-based cell migration. Now it is clear myosin II motor-activity is not the sole force involved. Alternative force-generating mechanisms within cells clearly also exist to power cell rear retraction during actin-based cell migration. Given that nematode sperm cells migrate without actin and without cytoskeletal motor proteins it is perhaps not surprising other types of force power cell rear retraction in actin-based systems. Here, cell rear retraction driven by actin filament depolymerisation, actin filament crosslinking, cell front protrusion and possibly apparent membrane tension and their importance relative to myosin II-motor-based contractility are discussed., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background Information: Cell-cell adhesion and contraction play an essential role in the maintenance of geometric shape and polarisation of epithelial cells. However, the molecular regulation of contraction during cell elongation leading to epithelial polarisation and acquisition of geometric cell shape is not clear., Results: Upon induction of cell-cell adhesion, we find that human keratinocytes acquire specific geometric shapes favouring hexagons, by re-modelling junction length/orientation and thus neighbour allocation. Acquisition of geometric shape correlates temporally with epithelial polarisation, as shown by an increase in lateral height. ROCK1 and ROCK2 are important regulators of myosin II contraction, but their specific role in epithelial cell shape has not been addressed. Depletion of ROCK proteins interferes with the correct proportion of hexagonal cell shapes and full elongation of lateral domain. Interestingly, ROCK proteins are not essential for maintenance of circumferential thin bundles, the main contractile epithelial F-actin pool. Instead, ROCK1 or ROCK2 regulates thin bundle contraction and positioning along the lateral domain, an important event for the stabilisation of the elongating lateral domain. Mechanistically, E-cadherin clustering specifically leads to ROCK1/ROCK2-dependent inactivation of myosin phosphatase and phosphorylation of myosin regulatory light chain. These events correlate temporally with the increase in lateral height and thin bundle compaction towards junctions., Conclusion: We conclude that ROCK proteins are necessary for acquisition of elongated and geometric cell shape, two key events for epithelial differentiation., (Copyright © 2012 Soçiété Francaise des Microscopies and Société de Biologie Cellulaire de France.)

It is well known that actin can associate with intracellular membranes to drive endocytosis and the rocketing motion of bacteria, virions and some organelles and to regulate synaptic vesicle plasticity. Actin also has been extensively reported to be involved at several steps of exocytosis; however, it has typically been described as functioning either within the actin cortex or by providing actin tracks for organelle transport. Increasingly, actin filament coats or rings have been directly localized on the surface of the exocytic organelle. Here, we suggest a common mechanism for actin-based regulation of large secretory granules whereby organelle-associated actomyosin II contraction either directly expels secretory content or stabilizes the exocytosing organelle., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

In migrating cells, the relative importance of myosin II contractility for cell rear retraction varies [1-12]. However, in myosin II-inhibited polarizing cells, actin organization is compromised [13-18]; thus it remains unclear whether myosin II is simply required for correct actin arrangement or also directly drives rear retraction [9]. Ascaris sperm cells lack actin and associated motors, and depolymerization of major sperm protein is instead thought to pull the cell rear forward [19, 20]. Opposing views exist on whether actin could also have this function [19, 20] and has not been directly experimentally sought. We probe function at high temporal resolution in polarizing fibroblasts that establish migration by forming the cell rear first [9, 15, 21]. We show that in cells with correctly organized actin, that actin filament depolymerization directly drives retraction of the rear margin to polarize cells and spatially accounts for most cell rear retraction during established migration. Myosin II contractility is required early, to form aligned actin bundles that are needed for polarization, and also later to maintain bundle length that ensures directed protrusion at the cell front. Our data imply a new mechanism: actin depolymerization-based force retracts the cell rear to polarize cells with no direct contribution from myosin II contractility., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The study of actin in regulated exocytosis has a long history with many different results in numerous systems. A major limitation on identifying precise mechanisms has been the paucity of experimental systems in which actin function has been directly assessed alongside granule content release at distinct steps of exocytosis of a single secretory organelle with sufficient spatiotemporal resolution. Using dual-color confocal microscopy and correlative electron microscopy in human endothelial cells, we visually distinguished two sequential steps of secretagogue-stimulated exocytosis: fusion of individual secretory granules (Weibel-Palade bodies [WPBs]) and subsequent expulsion of von Willebrand factor (VWF) content. Based on our observations, we conclude that for fusion, WPBs are released from cellular sites of actin anchorage. However, once fused, a dynamic ring of actin filaments and myosin II forms around the granule, and actomyosin II contractility squeezes VWF content out into the extracellular environment. This study therefore demonstrates how discrete actin cytoskeleton functions within a single cellular system explain actin filament-based prevention and promotion of specific exocytic steps during regulated secretion.

Directed cell migration requires the breaking of cell symmetry to generate a cell front and a cell rear along an axis approximately aligned with the direction of locomotion. In most cell types, regulated actin polymerization promotes initial cell front formation and its subsequent persistent protrusion, whereas myosin II-based forces are required to initially create and then maintain the cell rear. Molecular models for cell migration have focused extensively on cell protrusion, and the breaking of cell symmetry is almost universally portrayed with the cell front forming first. Although data supports this model for cells moving towards chemo-attractants, in the absence of any guidance cue, cell symmetry is broken by the cells constitutively forming the cell rear first. This allows an alternative model for triggering cell migration starting with retraction at the back of the cell. In this model, actomyosin II activity within the cell body and prospective cell rear occurs before a spatial bias in actin polymerization at the cell front. Creating the cell rear first may be a useful tool employed by a wide-range of migrating cell types, particularly when moving away from repellent cues.

Actomyosin II filament assemblies in cells are required for shaping the cell body and forming the cell rear during morphological polarization and triggering of migration. However, precise steps in myosin II-based mechanisms are unknown in this event; one reason is due to lack of information on the organization of the actin filament substrate for myosin II. Whilst muscle sarcomeric-like contraction drives cell tension in stationary nonmuscle cells, alternative nonsarcomeric modes of myosin II force-generation power forwards movement of the cell body in already migrating cells. Which one contributes to initial cell shape change has not previously been experimentally sought in any polarizing cell. Sarcomeric and nonsarcomeric-based force require completely different types of organization and filament polarity in the actin substrate for myosin II, and these can only currently be distinguished by labour-intensive submicron analysis and electron microscopy. For the first time in any polarizing cell using such analysis we have identified that oriented actomyosin II filament bundles, required for fibroblast polarization, are nonsarcomeric and are organized with graded filament polarity. As this actin organization is similar to the organization in already migrating fibroblasts, we conclude that graded filament polarity is a pivotal myosin II substrate coordinating initial cell shape change and triggering of migration.

In migrating fibroblasts actomyosin II bundles are graded polarity (GP) bundles, a distinct organization to stress fibers. GP bundles are important for powering cell migration, yet have an unknown mechanism of formation. Electron microscopy and the fate of photobleached marks show actin filaments undergoing retrograde flow in filopodia, and the lamellipodium are structurally and dynamically linked with stationary GP bundles within the lamella. An individual filopodium initially protrudes, but then becomes separated from the tip of the lamellipodium and seeds the formation of a new GP bundle within the lamella. In individual live cells expressing both GFP-myosin II and RFP-actin, myosin II puncta localize to the base of an individual filopodium an average 28 s before the filopodium seeds the formation of a new GP bundle. Associated myosin II is stationary with respect to the substratum in new GP bundles. Inhibition of myosin II motor activity in live cells blocks appearance of new GP bundles in the lamella, without inhibition of cell protrusion in the same timescale. We conclude retrograde F-actin flow and myosin II activity within the leading cell edge delivers F-actin to the lamella to seed the formation of new GP bundles.

How formation of the front and rear of a cell are coordinated during cell polarization in migrating cells is not well understood. Time-lapse microscopy of live primary chick embryo heart fibroblasts expressing GFP-actin show that, prior to cell polarization, polymerized actin in the cell body reorganizes to form oriented actin-filament bundles spanning the entire cell body. Within an average of 5 minutes of oriented actin bundles forming, localized cell-edge retraction initiates at either the side or at one end of the newly formed bundles and then elaborates around the nearest end of the bundles to form the cell rear, the first visual break in cell symmetry. Localized net protrusion occurs at the opposing end of the bundles to form the cell front and lags formation of the rear of the cell. Consequently, cells acquire full polarity and start to migrate in the direction of the long axis of the bundles, as previously documented for already migrating cells. When ADF/cofilin family protein activity or actin-filament disassembly is specifically blocked during cell polarization, reorganization of polymerized actin to form oriented actin-filament bundles in the cell body fails, and formation of the cell rear and front is inhibited. We conclude that formation of oriented actin-filament bundles in the cell body requires ADF/cofilin family proteins, and is an early event needed to coordinate the spatial location of the cell rear and front during fibroblast polarization.

The ability of epithelial cells to polarize requires cell-cell adhesion mediated by cadherin receptors. During cell-cell contact, the mechanism via which a flat, spread cell shape is changed into a tall, cuboidal epithelial morphology is not known. We found that cadherin-dependent adhesion modulates actin dynamics by triggering changes in actin organization both locally at junctions and within the rest of the cell. Upon induction of cell-cell contacts, two spatial actin populations are distinguishable: junctional actin and peripheral thin bundles. With time, the relative position of these two populations changes and becomes indistinguishable to form a cortical actin ring that is characteristic of mature, fully polarized epithelial cells. Junctional actin and thin actin bundles differ in their actin dynamics and mechanism of formation, and interestingly, have distinct roles during epithelial polarization. Whereas junctional actin stabilizes clustered cadherin receptors at cell-cell contacts, contraction of peripheral actin bundle is essential for an increase in the maximum height at the lateral domain during polarization (cuboidal morphology). Thus, both junctional actin and thin bundles are necessary, and cooperate with each other to generate a polarized epithelial morphology.

The role of myosin II in mitosis is generally thought to be restricted to cytokinesis. We present surprising new evidence that cortical myosin II is also required for spindle assembly in cells. Drug- or RNAi-mediated disruption of myosin II in cells interferes with normal spindle assembly and positioning. Time-lapse movies reveal that these treatments block the separation and positioning of duplicated centrosomes after nuclear envelope breakdown (NEBD), thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. Immobilization of cortical movement with tetravalent lectins produces similar spindle defects to myosin II disruption and suggests that myosin II activity is required within the cortex. Latex beads bound to the cell surface move in a myosin II-dependent manner in the direction of the separating asters. We propose that after NEBD, completion of centrosome separation and positioning around chromosomes depends on astral microtubule connections to a moving cell cortex.

To migrate, normally a cell must establish morphological polarity and continuously protrude a single lamellipodium, polarized in the direction of migration. We have previously shown that actin filament disassembly is necessary for protrusion of the lamellipodium during fibroblast migration. As ADF/cofilin (AC) proteins are essential for the catalysis of filament disassembly in cells, we assessed their role in polarized lamellipodium protrusion in migrating fibroblasts. We compared the spatial distribution of AC and the inactive, phosphorylated AC (pAC) in migrating cells. AC, but not pAC, localized to the lamellipodium. To investigate a role for AC in cell polarity, we increased the proportion of pAC in migrating fibroblasts by overexpressing constitutively active (CA) LIM kinase 1. In 87% of cells expressing CA LIM kinase, cell polarity was abolished. In such cells, the single polarized lamellipodium was replaced by multiple nonpolarized lamellipodia, which, in contrast to nonexpressing migrating cells, stained for pAC. Cell polarity was rescued by coexpressing an active, nonphosphorylatable Xenopus AC (CA XAC) with the CA LIMK. Furthermore, overexpressing a pseudophosphorylated (less active) XAC by itself also abolished cell polarity. We conclude that locally maintaining ADF/cofilin in the active, nonphosphorylated state within the lamellipodium is necessary to maintain polarized protrusion during cell migration.

Modulating the concentration of the actin-binding protein Ena/Vasp within the lamellipodium of a migrating fibroblast results in marked changes in lamellipodium behaviour and actin network organization at the cell's leading edge. This can explain a cell motility paradox.

Lamellipodium protrusion is linked to actin filament disassembly in migrating fibroblasts [Cramer, 1999: Curr. Biol. 9:1095-1105]. To further study this relationship, we have identified a method to specifically and sensitively detect G-actin in distinct spatial locations in motile cells using deoxyribonuclease I (DNase I). Although DNase I can bind both G- and F-actin in vitro [Mannherz et al., 1980: Eur. J. Biochem. 95:377-385], when cells were fixed in formaldehyde and permeabilized in detergent, fluorescently-labelled DNase I specifically stained G-actin and not F-actin. 92-98% of actin molecules were stably retained in cells during fixation and permeabilization. Further, increasing or decreasing cellular G-actin concentration by treating live cells with latrunculin-A or jasplakinolide, respectively, caused a respective increase and decrease in DNase I cell-staining intensity as expected. These changes in DNase I fluorescence intensity accurately reflected increases and decreases in cellular G-actin concentration independently measured in lysates prepared from drug-treated live cells (regression coefficient = 0.98). This shows that DNase I cell-staining is very sensitive using this method. Applying this method, we found that the ratio of G-/F-actin is lower in both the lamellipodium and in a broad band immediately behind the lamellipodium in migrating compared to non-migrating fibroblasts. Thus, we predict that protrusion of the lamellipodium in migrating fibroblasts requires tight coupling to filament disassembly at least in part because G-actin is relatively limited within and behind the lamellipodium. This is the first report to directly demonstrate high sensitivity of cell-staining for any G-actin probe and this, together with the ready commercial accessibility of fluorescently-labelled DNase I, make it a simple, convenient, and sensitive tool for cell-staining of G-actin., (Copyright 2002 Wiley-Liss, Inc.)

Background: Simple epithelia encase developing embryos and organs. Although these epithelia consist of only one or two layers of cells, they must provide tight barriers for the tissues that they envelop. Apoptosis occurring within these simple epithelia could compromise this barrier. How, then, does an epithelium remove apoptotic cells without disrupting its function as a barrier?, Results: We show that apoptotic cells are extruded from a simple epithelium by the concerted contraction of their neighbors. A ring of actin and myosin forms both within the apoptotic cell and in the cells surrounding it, and contraction of the ring formed in the live neighbors is required for apoptotic cell extrusion, as injection of a Rho GTPase inhibitor into these cells completely blocks extrusion. Addition of apoptotic MDCK cells to an intact monolayer induces the formation of actin cables in the cells contacted, suggesting that the signal to form the cable comes from the dying cell. The signal is produced very early in the apoptotic process, before procaspase activation, cell shrinkage, or phosphatidylserine exposure. Remarkably, electrical resistance studies show that epithelial barrier function is maintained, even when large numbers of dying cells are being extruded., Conclusions: We propose that apoptotic cell extrusion is important for the preservation of epithelial barrier function during cell death. Our results suggest that an early signal from the dying cell activates Rho in live neighbors to extrude the apoptotic cell out of the epithelium.

Background: In motile cells, protrusion of the lamellipodium (a type of cell margin) requires assembly of actin monomers into actin filaments at the tip of the lamellipodium. The importance of actin-filament disassembly in this process is less well understood, and is assessed here using the actin drug jasplakinolide, which has two known activities - inhibition of filament disassembly and induction of an increase in actin polymer., Results: In cells the two activities of jasplakinolide were found to be separable; 1 microM jasplakinolide could permeate cells, bind cellular filamentous actin (F-actin) and inhibit filament disassembly within 3.5 minutes, but significant increase in actin polymer was not detected until 60 minutes of treatment. In live, permeabilised cells, jasplakinolide did not inhibit filament assembly from supplied, purified actin monomers. In migrating chick fibroblasts, lamellipodium protrusion was blocked within 1-5 minutes of treatment with 1 microM jasplakinolide, without any perturbation of actin organisation. In non-migrating chick fibroblasts, there was a delay in the onset of jasplakinolide-induced inhibition of lamellipodium protrusion, during which lamellipodium length increased linearly with no increase in protrusion rate. Motility of the bacterium Listeria in infected PtK2 cells was reduced 2.3-fold within 3 minutes of treatment with 1 microM jasplakinolide., Conclusions: Actin-filament disassembly is tightly coupled to lamellipodium protrusion in migrating chick fibroblasts and motility of Listeria in PtK2 cells. One simple interpretation of these data is a situation whereby ongoing actin-filament assembly uses free actin monomer derived from filament disassembly, in preference to stored monomer.

Force arising from myosin activity drives a number of different types of motility in eukaryotic cells. Outside of muscle tissue, the precise mechanism of myosin-based cell motility is for the most part theoretical. A large part of the problem is that, aside from cell surface features such as lamellipodia and microvilli, relatively little is known about the structural organization of potential actin substrates for myosin in non-muscle motile cells. Several groups [Cramer, Siebert and Mitchison (1997) J. Cell Biol. 136, 1287-1305; Guild, Connelly, Shaw and Tilney (1997) J. Cell Biol. 138, 783-797; Svitkina, Verkhovsky, McQuade and Borisy (1997) J. Cell Biol. 139, 397-415] have begun to address this issue by determining actin organization throughout entire non-muscle motile cells. These studies reveal that a single motile cell comprises up to four distinct structural groups of actin organization, distinguished by differences in actin filament polarity: alternating, uniform, mixed or graded. The relative abundance and spatial location in cells of a particular actin organization varies with cell type. The existence in non-muscle motile cells of alternating-polarity actin filament bundles, the organization of muscle sarcomeres, provides direct structural evidence that some forms of motility in non-muscle cells are based on sarcomeric contraction, a recurring theory in the literature since the early days of muscle research. In this scenario, as in muscle sarcomeres, myosin generates isometric force, which is ideally suited to driving symmetrical types of motility, e.g. healing of circular wounds in coherent groups of cells. In contrast, uniform-polarity actin filament bundles and oriented meshworks in cells allow oriented movement of myosin, potentially over relatively long distances. In this simple 'transport-based' scenario, the direction in which myosin generates force is inherently polarized, and is well placed for driving asymmetrical or polarized types of motility, e.g. as expected for long-range transport of membrane organelles. In the more complex situation of cell locomotion, the predominant actin organization detected in locomoting fish keratocytes and locomoting primary heart fibroblasts excludes sarcomeric contraction force from having a major role in pulling these cell types forward during locomotion. Instead Svitkina et al. propose that 'dynamic network contraction' of a weakly adherent uniform-polarity actin filament meshwork is the basis of keratocyte locomotion. For fibroblast locomotion, however, Cramer et al. prefer a transport mechanism based on graded-polarity actin filament bundles.

In motile, eukaryotic cells, a variety of cell-associated material (collectively termed here as 'particles') continuously flows, relative to the substratum, from the front to the back of the extreme margin of the cell (termed the 'lamellipodium'). This retrograde particle flow, occurs both over the surface of, and inside the lamellipodium. Force to drive retrograde particle flow in lamellipodia is dependent on actin filaments, but the precise mechanism of force generation, and function of the flow is generally not well understood. Actin filaments themselves, in lamellipodia of most motile cell types studied also flow retrograde relative to the substratum. This actin flow, in Aplysia bag cell neuronal growth cones, is known to be driven by activity of a myosin. In these growth cones, retrograde flow of cell surface-attached particles is coupled to retrograde actin flow. In Aplysia, force from retrograde actin flow may limit certain types of growth cone motility. In other motile cell types, such as keratocytes and fibroblasts, the mechanism of retrograde particle flow and function of retrograde actin flow in lamellipodia is poorly understood. For these cell types, recent data provide a basis for proposing alternative actin-based mechanisms to drive retrograde particle flow in lamellipodia. One mechanism is based on activity of a putative pointed end- directed actin motor, and the other on tension-driven surface lipid flow. Here I will review recent advances that have been made in determining the molecular mechanism of force generation to drive retrograde particle flow relative to the substratum in lamellipodia of motile cells. I will address the function of retrograde actin flow in lamellipodia, and apparent differences between Aplysia and other motile cell types.

We have determined the structural organization and dynamic behavior of actin filaments in entire primary locomoting heart fibroblasts by S1 decoration, serial section EM, and photoactivation of fluorescence. As expected, actin filaments in the lamellipodium of these cells have uniform polarity with barbed ends facing forward. In the lamella, cell body, and tail there are two observable types of actin filament organization. A less abundant type is located on the inner surface of the plasma membrane and is composed of short, overlapping actin bundles (0.25-2.5 microm) that repeatedly alternate in polarity from uniform barbed ends forward to uniform pointed ends forward. This type of organization is similar to the organization we show for actin filament bundles (stress fibers) in nonlocomoting cells (PtK2 cells) and to the known organization of muscle sarcomeres. The more abundant type of actin filament organization in locomoting heart fibroblasts is mostly ventrally located and is composed of long, overlapping bundles (average 13 microm, but can reach up to about 30 microm) which span the length of the cell. This more abundant type has a novel graded polarity organization. In each actin bundle, polarity gradually changes along the length of the bundle. Actual actin filament polarity at any given point in the bundle is determined by position in the cell; the closer to the front of the cell the more barbed ends of actin filaments face forward. By photoactivation marking in locomoting heart fibroblasts, as expected in the lamellipodium, actin filaments flow rearward with respect to substrate. In the lamella, all marked and observed actin filaments remain stationary with respect to substrate as the fibroblast locomotes. In the cell body of locomoting fibroblasts there are two dynamic populations of actin filaments: one remains stationary and the other moves forward with respect to substrate at the rate of the cell body. This is the first time that the structural organization and dynamics of actin filaments have been determined in an entire locomoting cell. The organization, dynamics, and relative abundance of graded polarity actin filament bundles have important implications for the generation of motile force during primary heart fibroblast locomotion.

We have studied two types of cell motility directed toward the cell center: retraction of the cell margin and rearward flow of small cytoplasmic nodules during mitotic cell rounding in Potoroo tridactylis kidney (PtK2) cells by time-lapse video microscopy, drug treatments, and photoactivation of fluorescence. Nodules flow rearward on thin, actin-rich fibers (retraction fibers) exposed as the cell margin retracts. Retraction of the cell margin and rearward flow of nodules require intact actin filaments, but are insensitive to an inhibitor of myosin function (butanedione monoxime). Using photoactivation of fluorescence marking, we have determined that actin filaments in the majority of retraction fibers remain stationary while the cell margin retracts and nodules flow rearward. The pointed ends of retraction fiber actin filaments face the cell center. We argue that nodule motility is driven by a novel actin-based force that perhaps also partially contributes to retraction of the cell margin during cell rounding at mitosis.

We have investigated a role for myosin in postmitotic Potoroo tridactylis kidney (PtK2) cell spreading by inhibitor studies, time-lapse video microscopy, and immunofluorescence. We have also determined the spatial organization and polarity of actin filaments in postmitotic spreading cells. We show that butanedione monoxime (BDM), a known inhibitor of muscle myosin II, inhibits nonmuscle myosin II and myosin V adenosine triphosphatases. BDM reversibly inhibits PtK2 postmitotic cell spreading. Listeria motility is not affected by this drug. Electron microscopy studies show that some actin filaments in spreading edges are part of actin bundles that are also found in long, thin, structures that are connected to spreading edges and substrate (retraction fibers), and that 90% of this actin is oriented with barbed ends in the direction of spreading. The remaining actin in spreading edges has a more random orientation and spatial arrangement. Myosin II is associated with actin polymer in spreading cell edges, but not retraction fibers. Myosin II is excluded from lamellipodia that protrude from the cell edge at the end of spreading. We suggest that spreading involves myosin, possibly myosin II.

Force arising from actin polymerization and myosin activity drives a number of different actin-based cell movements. Several new reports support previous data suggesting that actin polymerization drives lamellipodial protrusion and bacterial propulsion, and one report describes a more indirect role for actin assembly in axonal elongation. The major new findings of the past year concerning possible motility roles for myosin describe myosin-driven protrusion of cell margins.

PC12 cells, a cell line derived from a rat pheochromocytoma, have both regulated and constitutive secretory pathways. Regulated secretion occurs via large dense core granules, which are related to chromaffin granules and are abundant in these cells. In addition, PC12 cells also contain small electron-lucent vesicles, whose numbers increase in response to nerve growth factor and which may be related to cholinergic synaptic vesicles. These could characterize a second regulated secretory pathway. We have investigated the trafficking of protein markers for both these organelles. We have purified and characterized the large dense core granules from these cells using sequential velocity and equilibrium gradients. We demonstrate the copurification of the major PC12 soluble regulated secretory protein (secretogranin II) with this organelle. As a marker for the synaptic vesicle-like organelles in this system, we have used the integral membrane glycoprotein p38 or synaptophysin. We show that the p38-enriched fraction of PC12 cells comigrates with rat brain synaptic vesicles on an equilibrium gradient. We also demonstrate that p38 purifies away from the dense core granules; less than 5% of this protein is found in our dense granule fraction. Finally we show that p38 does not pass through the dense granule fraction in pulse-chase experiments. These results rule out the possibility of p38 reaching the small clear vesicles via mature dense granules and imply that these cells may have two independently derived regulated pathways.