Dissection of the Ascaris sperm motility machinery identifies key proteins involved in major sperm protein-based amoeboid locomotion

Although Ascaris sperm motility closely resembles that seen in many other types of crawling cells, the lamellipodial dynamics that drive movement result from modulation of a cytoskeleton based on the major sperm protein (MSP) rather than actin. The dynamics of the Ascaris sperm cytoskeleton can be studied in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibers constructed from bundles of MSP filaments. In addition to ATP, MSP, and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins that orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. Here, we identify a fraction of cytosol that is comprised of a small number of proteins but contains all of the soluble components required to assemble fibers. We have purified two of these proteins, designated MSP fiber proteins (MFPs) 1 and 2 and demonstrated by immunolabeling that both are located in the MSP cytoskeleton in cells and in fibers. These proteins had reciprocal effects on fiber assembly in vitro: MFP1 decreased the rate of fiber growth, whereas MFP2 increased the growth rate.     

A unique cytoskeleton associated with crawling in the amoeboid sperm of the nematode, Ascaris suum

Nematode sperm extend pseudopods and pull themselves over substrates. They lack an axoneme or the actin and myosins of other types of motile cells, but their pseudopods contain abundant major sperm protein (MSP), a family of 14-kD polypeptides found exclusively in male gametes. Using high voltage electron microscopy, a unique cytoskeleton was discovered in the pseudopod of in vitro-activated, crawling sperm of the pig intestinal nematode Ascaris suum. It consists of 5-10-nm fuzzy fibers organized into 150-250-nm-thick fiber complexes, which connect to each of the moving pseudopodial membrane projections, villipodia, which in turn make contact with the substrate. Individual fibers in a complex splay out radially from its axis in all directions. The centripetal ends intercalate with fibers from other complexes or terminate in a thickened layer just beneath the pseudopod membrane. Monoclonal antibodies directed against MSP heavily label the fiber complexes as well as individual pseudopodial filaments throughout their length. This represents the first evidence that MSP may be the major filament protein in the Ascaris sperm cytoskeleton. The large fiber complexes can be seen clearly in the pseudopods of live, crawling sperm by computer-enhanced video, differential-interference contrast microscopy, forming with the villipodia at the leading edge of the sperm pseudopod. Even before the pseudopod attaches, the entire cytoskeleton and villipodia move continuously rearwards in unison toward the cell body. During crawling, complexes and villipodia in the pseudopod recede at the same speed as the spermatozoon moves forward, both disappearing at the pseudopod-cell body junction. Sections at this region of high membrane turnover reveal a band of densely packed smooth vesicles with round and tubular profiles, some of which are associated with the pseudopod plasma membrane. The exceptional anatomy, biochemistry, and phenomenology of Ascaris sperm locomotion permit direct study of the involvement of the cytoskeleton in amoeboid motility.     

Preparing to move: assembly of the MSP amoeboid motility apparatus during spermiogenesis in Ascaris

We exploited the rapid, inducible conversion of non-motile Ascaris spermatids into crawling spermatozoa to examine the pattern of assembly of the MSP motility apparatus that powers sperm locomotion. In live sperm, the first detectable motile activity is the extension of spikes and, later, blebs from the cell surface. However, examination of cells by EM revealed that the formation of surface protrusions is preceded by assembly of MSP filament tails on the membranous organelles in the peripheral cytoplasm. These organelle-associated filament meshworks assemble within 30 sec after induction of spermiogenesis and persist until the membranous organelles are sequestered into the cell body when the lamellipod extends. The filopodia-like spikes, which are packed with bundles of filaments, extend and retract rapidly but last only a few seconds before giving way to, or converting into, blebs. Coalescence of these blebs, each supported by a dense mesh of filaments, often initiates lamellipod extension, which culminates in the formation of the robust, dynamic MSP fiber complexes that generate sperm motility. The same membrane phosphoprotein that orchestrates assembly of the fiber complexes at the leading edge of the lamellipod of mature sperm is also found at all sites of filament assembly during spermiogenesis. The orderly progression of steps that leads to construction of a functional motility apparatus illustrates the precise spatio-temporal control of MSP filament assembly in the developing cell and highlights the remarkable similarity in organization and plasticity shared by the MSP cytoskeleton and the actin filament arrays in conventional crawling cells.     

Dephosphorylation of Major Sperm Protein (MSP) Fiber Protein 3 by Protein Phosphatase 2A during Cell Body Retraction in the MSP-based Amoeboid Motility of Ascaris Sperm

The crawling movement of nematode sperm requires coordination of leading edge protrusion with cell body retraction, both of which are powered by modulation of a cytoskeleton based on major sperm protein (MSP) filaments. We used a cell-free in vitro motility system in which both protrusion and retraction can be reconstituted, to identify two proteins involved in cell body retraction. Pharmacological and depletion-add back assays showed that retraction was triggered by a putative protein phosphatase 2A (PP2A, a Ser/Thr phosphatase activated by tyrosine dephosphorylation). Immunofluorescence showed that PP2A was present in the cell body and was concentrated at the base of the lamellipod where the force for retraction is generated. PP2A targeted MSP fiber protein 3 (MFP3), a protein unique to nematode sperm that binds to the MSP filaments in the motility apparatus. Dephosphorylation of MFP3 caused its release from the cytoskeleton and generated filament disassembly. Our results suggest that interaction between PP2A and MFP3 leads to local disassembly of the MSP cytoskeleton at the base of the lamellipod in sperm that in turn pulls the trailing cell body forward.     

Capping Protein Terminates but Does Not Initiate Chemoattractant-induced Actin Assembly in Dictyostelium

The first step in the directed movement of cells toward a chemotactic source involves the extension of pseudopods initiated by the focal nucleation and polymerization of actin at the leading edge of the cell. We have previously isolated a chemoattractant-regulated barbed-end capping activity from Dictyostelium that is uniquely associated with capping protein, also known as cap32/34. Although uncapping of barbed ends by capping protein has been proposed as a mechanism for the generation of free barbed ends after stimulation, in vitro and in situ analysis of the association of capping protein with the actin cytoskeleton after stimulation reveals that capping protein enters, but does not exit, the cytoskeleton during the initiation of actin polymerization. Increased association of capping protein with regions of the cell containing free barbed ends as visualized by exogenous rhodamine-labeled G-actin is also observed after stimulation. An approximate threefold increase in the number of filaments with free barbed ends is accompanied by increases in absolute filament number, whereas the average filament length remains constant. Therefore, a mechanism in which preexisting filaments are uncapped by capping protein, in response to stimulation leading to the generation of free barbed ends and filament elongation, is not supported. A model for actin assembly after stimulation, whereby free barbed ends are generated by either filament severing or de novo nucleation is proposed. In this model, exposure of free barbed ends results in actin assembly, followed by entry of free capping protein into the actin cytoskeleton, which acts to terminate, not initiate, the actin polymerization transient.     

Centripetal flow and directed reassembly of the major sperm protein (MSP) cytoskeleton in the amoeboid sperm of the nematode, Ascaris suum.

The cytoskeleton of the amoeboid spermatozoa of Ascaris suum consists of major sperm protein (MSP) filaments arranged into long, branched fiber complexes that span the length of the pseudopod and treadmill rearward continuously due to assembly and disassembly at opposite ends of the complexes (Sepsenwol et al., Journal of Cell Biology 108:55-66, (1989)). Examination by video-enhanced microscopy showed that this cytoskeletal flow is tightly coupled to sperm locomotion. The fiber complexes treadmilled rearward at the same rate (10-50 microns/min) as the cell crawled forward. Only fiber complexes with their plasmalemmal ends within a limited sector along the leading edge of the pseudopod underwent continuous assembly. Thus, the location of this sector, which occupies about 50% of the pseudopod perimeter, determined the direction of sperm locomotion. Treatment of sperm with agents that lower intracellular pH, such as weak acids and protonophores, caused the fiber complexes to disassemble completely in 4-5 sec. Removal of these compounds resulted in reassembly of the cytoskeleton in a pattern that mimicked treadmilling in intact sperm. The fiber complexes were reconstructed by assembly at their plasmalemmal ends so that within 30-60 sec the entire filament system reformed and the cell resumed locomotion. Both cytoskeletal reassembly and treadmilling required exogenous HCO3-. These results suggest that variation in intracellular pH may help regulate cytoskeletal treadmilling and thereby play a significant role in sperm locomotion.     

Nematode sperm motility: nonpolar filament polymerization mediated by end-tracking motors

In nematode sperm cell motility, major sperm protein (MSP) filament assembly results in dynamic membrane protrusions in a manner that closely resembles actin-based motility in other eukaryotic cells. Paradoxically, whereas actin-based motility is driven by addition of ATP-bound actin subunits onto actin filament plus-ends located at the cell membrane, MSP dimers assemble from solution into nonpolar filaments that lack a nucleotide binding site. Thus, filament polarity and on-filament ATP hydrolysis, although essential for actin-based motility, appear to be unnecessary for membrane protrusions by MSP. As a potential resolution to this paradox, we propose a model for MSP filament assembly and force generation by MSP filament end-tracking proteins. In this model, ATP hydrolysis drives affinity-modulated, processive interactions between membrane-associated proteins and elongating filament ends. However, in contrast to the "actoclampin" model for actin filament end-tracking motors, ATP activates the tracking protein (or a soluble cofactor) rather than the MSP subunits themselves (in contrast to activation of actin subunits by ATP binding). The MSP end-tracking model predicts properties that are consistent with several key observations of MSP-based motility, including persistent membrane attachment, polymerization of filament ends at the membrane with depolymerization of free-filament ends away from the membrane, as well as a saturating dependence of polymerization rate on the concentration of non-MSP soluble cytoplasmic components.     

Dynamin2 and cortactin regulate actin assembly and filament organization

The GTPase dynamin is required for endocytic vesicle formation. Dynamin has also been implicated in regulating the actin cytoskeleton, but the mechanism by which it does so is unclear. Through interactions via its proline-rich domain (PRD), dynamin binds several proteins, including cortactin, profilin, syndapin, and murine Abp1, that regulate the actin cytoskeleton. We investigated the interaction of dynamin2 and cortactin in regulating actin assembly in vivo and in vitro. When expressed in cultured cells, a dynamin2 mutant with decreased affinity for GTP decreased actin dynamics within the cortical actin network. Expressed mutants of cortactin that have decreased binding of Arp2/3 complex or dynamin2 also decreased actin dynamics. Dynamin2 influenced actin nucleation by purified Arp2/3 complex and cortactin in vitro in a biphasic manner. Low concentrations of dynamin2 enhanced actin nucleation by Arp2/3 complex and cortactin, and high concentrations were inhibitory. Dynamin2 promoted the association of actin filaments nucleated by Arp2/3 complex and cortactin with phosphatidylinositol 4,5-bisphosphate (PIP2)-containing lipid vesicles. GTP hydrolysis altered the organization of the filaments and the lipid vesicles. We conclude that dynamin2, through an interaction with cortactin, regulates actin assembly and actin filament organization at membranes.     

Cytoskeleton and vesicle mobility in astrocytes

Exocytotic vesicles in astrocytes are increasingly viewed as essential in astrocyte-to-neuron communication in the brain. In neurons and excitable secretory cells, delivery of vesicles to the plasma membrane for exocytosis involves an interaction with the cytoskeleton, in particular microtubules and actin filaments. Whether cytoskeletal elements affect vesicle mobility in astrocytes is unknown. We labeled single vesicles with fluorescent atrial natriuretic peptide and monitored their mobility in rat astrocytes with depolymerized microtubules, actin, and intermediate filaments and in mouse astrocytes deficient in the intermediate filament proteins glial fibrillary acidic protein and vimentin. In astrocytes, as in neurons, microtubules participated in directional vesicle mobility, and actin filaments played an important role in this process. Depolymerization of intermediate filaments strongly affected vesicle trafficking and in their absence the fraction of vesicles with directional mobility was reduced.     

Myosin mRNA accumulation and myofibrillogenesis at the myotendinous junction of stretched muscle fibers

Myofiber growth and myofibril assembly at the myotendinous junction (MTJ) of stretch-hypertrophied rabbit skeletal muscle was studied by in situ hybridization, immunofluorescence, and electron microscopy. In situ hybridization identified higher levels of myosin heavy chain (MHC) mRNA at the MTJ of fibers stretched for 4 d. Electron microscopy at the MTJ of these lengthening fibers revealed a large cytoplasmic space devoid of myofibrils, but containing polysomes, sarcoplasmic reticulum and T-membranes, mitochondria, Golgi complexes, and nascent filament assemblies. Tallies from electron micrographs indicate that myofibril assembly in stretched fibers followed a set sequence of events. (a) In stretched fiber ends almost the entire sarcolemmal membrane was electron dense but only a portion had attached myofibrils. Vinculin, detected by immunofluorescence, was greatly increased at the MTJ membrane of stretched muscles. (b) Thin filaments were anchored to the sarcolemma at the electron dense sites. (c) Thick filaments associated with these thin filaments in an unregistered manner. (d) Z-bodies splice into thin filaments and subsequently thin and thick filaments fall into sarcomeric register. Thus, the MTJ is a site of mRNA accumulation which sets up regional protein synthesis and myofibril assembly. Stretched muscles also lengthen by the addition of myotubes at their ends. After 6 d of stretch these myotubes make up the majority of fibers at the muscle ends. Essentially all these myotubes repeat the developmental program of primary myotubes and express slow MHC. MHC mRNA distribution in myotubes is disorganized as is the distribution of their myofibrils.     

Structure of MFP2 and its function in enhancing MSP polymerization in Ascaris sperm amoeboid motility

The simplicity and specialization of the cell motility machinery of Ascaris sperm provides a powerful system in which to probe the basic molecular mechanism of amoeboid cell motility. Although Ascaris sperm locomotion closely resembles that seen in many other types of crawling cell, movement is generated by modulation of a cytoskeleton based on the major sperm protein (MSP) rather than the actin present in other cell types. The Ascaris motility machinery can be studied conveniently in a cell-free in vitro system based on the movement of plasma membrane vesicles by fibres constructed from bundles of MSP filaments. In addition to ATP, MSP and a plasma membrane protein, reconstitution of MSP motility in this cell-free extract requires cytosolic proteins to orchestrate the site-specific assembly and bundling of MSP filaments that generates locomotion. One of these proteins, MFP2, accelerates the rate of movement in this assay. Here, we describe crystal structures of two isoforms of MFP2 and show that both are constructed from two domains that have the same fold based on a novel, compact beta sheet arrangement. Patterns of conservation observed in a structure-based analysis of MFP2 sequences from different nematode species identified regions that may be putative functional interfaces involved both in interactions between MFP2 domains and also with other components of the sperm motility machinery. Analysis of the growth of fibres in vitro in the presence of added MFP2 indicated that MFP2 increases the rate of locomotion by enhancing the effective rate of MSP filament polymerization. This observation, together with the structural data, suggests that MFP2 may function in a manner analogous to formins in actin-based motility.     

The Caenorhabditis Elegans Spe-6 Gene Is Required for Major Sperm Protein Assembly and Shows Second Site Non-Complementation with an Unlinked Deficiency

Caenorhabditis elegans spermatozoa move by crawling. Their motility requires thin cytoskeletal filaments assembled from a unique cytoskeletal protein, the major sperm protein (MSP). During normal sperm development the MSP is segregated to developing sperm by assembly into filaments that form a paracrystalline array in a transient organelle, the fibrous body-membranous organelle. Mutations in the spe-6 gene cause sterility because they lead to defective primary spermatocytes that do not form spermatids. In these mutant spermatocytes the MSP fails to assemble into fibrous body filaments. Instead, the unassembled MSP distributes throughout the cytoplasm and nucleus. Thus, the spe-6 gene product is necessary for normal MSP localization and assembly during sperm development. In addition to their MSP assembly defect, spe-6 mutant spermatocytes arrest meiosis at diakinesis although their spindle pole bodies still replicate and separate. This results in spermatocytes with four half-spindles surrounding condensed, but unsegregated, chromosomes. All four spe-6 alleles, as well as a chromosome III deficiency that deletes the spe-6 gene, fail to complement two small overlapping chromosome IV deficiencies, eDf18 and eDf19. This non-allele-specific second site non-complementation suggests a concentration-dependent interaction between the spe-6 gene product and products of the gene(s) under eDf18 and eDf19, which include a cluster of sperm-specific genes. Since MSP filament assembly is highly concentration-dependent in vitro, the non-complementation might be expected if the sperm-specific gene products under eDf18 and eDf19 were needed together with the spe-6 gene product to promote MSP assembly.     

Coupling actin dynamics and membrane dynamics during endocytosis.

A convergence of cellular, genetic and biochemical studies supports the hypothesis that the actin cytoskeleton is coupled to endocytic processes, but the roles played by actin filaments during endocytosis are not yet clear. Recent studies have identified several proteins that may functionally link the endocytic machinery with actin filament dynamics. Three of these proteins, Abp1p, Pan1p and cortactin, are activators of actin assembly nucleated by the Arp2/3 complex, a key regulator of actin assembly in vivo. Two others, intersectin and syndapin, bind N-WASp, a potent activator of actin assembly via the Arp2/3 complex. All of these proteins also bind components of the endocytic machinery, and thus, could coordinately regulate actin assembly and trafficking events. Hip1R, an F-actin-binding protein that associates with clathrin-coated vesicles, may physically link endocytic vesicles to actin filaments. The GTPase dynamin is implicated in modulating actin filaments at specialized actin-rich structures of the cell cortex, suggesting that dynamin may regulate the organization of cortical actin filaments as well as regulate actin dynamics during endocytosis. Finally, myosin VI may generate actin-dependent forces for membrane invagination or vesicle movement during the early stages of endocytosis.     

Actin filament uncapping localizes to ruffling lamellae and rocketing vesicles

Regulated actin filament assembly is critical for eukaryotic cell physiology. Actin filaments are polar structures, and those with free high affinity or barbed ends are crucial for actin dynamics and cell motility. Actin filament barbed-end-capping proteins inhibit filament elongation after binding, and their regulated disassociation is proposed to provide a source of free filament ends to drive processes dependent on actin polymerization. To examine whether dissociation of actin filament capping proteins occurs with the correct spatio-temporal constraints to contribute to regulated actin assembly in live cells, I measured the dissociation of an actin capping protein, gelsolin, from actin in cells using a variation of fluorescence resonance energy transfer (FRET). Uncapping was found to occur in cells at sites of active actin assembly, including protruding lamellae and rocketing vesicles, with the correct spatio-temporal properties to provide sites of actin filament polymerization during protrusion. These observations are consistent with models where uncapping of existing filaments provides sites of actin filament elongation.     

A Ser/Thr kinase required for membrane-associated assembly of the major sperm protein motility apparatus in the amoeboid sperm of Ascaris

Leading edge protrusion in the amoeboid sperm of Ascaris suum is driven by the localized assembly of the major sperm protein (MSP) cytoskeleton in the same way that actin assembly powers protrusion in other types of crawling cell. Reconstitution of this process in vitro led to the identification of two accessory proteins required for MSP polymerization: an integral membrane phosphoprotein, MSP polymerization-organizing protein (MPOP), and a cytosolic component, MSP fiber protein 2 (MFP2). Here, we identify and characterize a 34-kDa cytosolic protein, MSP polymerization-activating kinase (MPAK) that links the activities of MPOP and MFP2. Depletion/add-back assays of sperm extracts showed that MPAK, which is a member of the casein kinase 1 family of Ser/Thr protein kinases, is required for motility. MPOP and MPAK comigrated by native gel electrophoresis, coimmunoprecipitated, and colocalized by immunofluorescence, indicating that MPOP binds to and recruits MPAK to the membrane surface. MPAK, in turn, phosphorylated MFP2 on threonine residues, resulting in incorporation of MFP2 into the cytoskeleton. Beads coated with MPAK assembled a surrounding cloud of MSP filaments when incubated in MPAK-depleted sperm extract, but only when supplemented with detergent-solubilized MPOP. Our results suggest that interactions involving MPOP, MPAK, and MFP2 focus MSP polymerization to the plasma membrane at the leading edge of the cell thereby generating protrusion and minimizing nonproductive filament formation elsewhere.     

The effect of calcium activation of skinned fiber bundles on the structure of Limulus thick filaments

Here we present evidence that strongly suggests that the well-documented phenomenon of A-band shortening in Limulus telson muscle is activation dependent and reflects fragmentation of thick filaments at their ends. Calcium activation of detergent-skinned fiber bundles of Limulus telson muscle results in large decreases in A-band (from 5.1 to 3.3 microns) and thick filament (from 4.1 to 3.3 microns) lengths and the release of filament end fragments. In activated fibers, maintained stretched beyond overlap of thick and thin filaments, these end fragments are translocated to varying depths within the I-bands. Here they are closely associated with fine filamentous structures that also span the gap between A- and I-bands and attach to the distal one-third of the thick filaments. End-fragments are rarely, if ever, present in similarly stretched and skinned, but unstimulated fibers, although fine "gap filaments" persist. Negatively stained thick filaments, separated from skinned, calcium-activated, fiber bundles, allowed to shorten freely, are significantly shorter than those obtained from unstimulated fibers, but are identical to the latter with respect to both the surface helical array of myosin heads and diameters. Many end-fragments are present on grids containing thick filaments from activated fibers; few, if any, on those from unstimulated fibers. SDS-PAGE shows no evidence of proteolysis due to activation and demonstrates the presence of polypeptides with very high molecular weights in the preparations. We suggest that thick filament shortening is a direct result of activation in Limulus telson muscle and that it occurs largely by breakage within a defined distal region of each polar half of the filament. It is possible that at least some of the fine "gap filaments" are composed of a titin-like protein. They may move the activation-produced, fragmented ends of thick filaments to which they attach, into the I-bands by elastic recoil, in highly stretched fibers.     

Critical contact residues that mediate polymerization of nematode major sperm protein

The polymerization of protein filaments provides the motive force in a variety of cellular processes involving cell motility and intracellular transport. Regulated assembly and disassembly of the major sperm protein (MSP) underlies amoeboid movement in nematode sperm, and offers an attractive model system for characterizing the biomechanical properties of filament formation and force generation. To that end, structure-function studies of MSP from the nematode Caenorhabditis elegans have been performed. Recombinant MSP was purified from Escherichia coli using a novel affinity chromatography technique, and filament assembly was assessed by in vitro polymerization in the presence of polyethylene glycol. Prior molecular studies and structure from X-ray crystallography have implicated specific residues in protein-protein interactions necessary for filament assembly. Purified MSP containing substitutions in these residues fails to form filaments in vitro. Short peptides based on predicted sites of interaction also effectively disrupt MSP polymerization. These results confirm the structural determination of intermolecular contacts and demonstrate the importance of these residues in MSP assembly.     

Microtubules as platforms for assaying actin polymerization in vivo

The actin cytoskeleton is continuously remodeled through cycles of actin filament assembly and disassembly. Filaments are born through nucleation and shaped into supramolecular structures with various essential functions. These range from contractile and protrusive assemblies in muscle and non-muscle cells to actin filament comets propelling vesicles or pathogens through the cytosol. Although nucleation has been extensively studied using purified proteins in vitro, dissection of the process in cells is complicated by the abundance and molecular complexity of actin filament arrays. We here describe the ectopic nucleation of actin filaments on the surface of microtubules, free of endogenous actin and interfering membrane or lipid. All major mechanisms of actin filament nucleation were recapitulated, including filament assembly induced by Arp2/3 complex, formin and Spir. This novel approach allows systematic dissection of actin nucleation in the cytosol of live cells, its genetic re-engineering as well as screening for new modifiers of the process.     

Ena/VASP proteins capture actin filament barbed ends

Ena/VASP (vasodialator-stimulated protein) proteins regulate many actin-dependent events, including formation of protrusive structures, fibroblast migration, neurite extension, cell-cell adhesion, and Listeria pathogenesis. In vitro, Ena/VASP activities on actin are complex and varied. They promote actin assembly, protect filaments from cappers, bundle filaments, and inhibit filament branching. To determine the mechanisms by which Ena/VASP proteins regulate actin dynamics at barbed ends, we monitored individual actin filaments growing in the presence of VASP and profilin using total internal reflection fluorescence microscopy. Filament growth was unchanged by VASP, but filaments grew faster in profilin-actin and VASP than with profilin-actin alone. Actin filaments were captured directly by VASP-coated surfaces via interactions with growing barbed ends. End-attached filaments transiently paused but resumed growth after becoming bound to the surface via a filament side attachment. Thus, Ena/VASP proteins promote actin assembly by interacting directly with actin filament barbed ends, recruiting profilin-actin, and blocking capping.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.