Thermoresponsive block copolymer micelles with tunable pyrrolidone-based polymer cores: structure/property correlations and application as drug carriers.

A-B block copolymer micelles comprised of thermoresponsive hydrophilic PNIPAAm (poly(N-isopropylacrylamide)) coronae and hydrophobic PNP (poly(N-acryloyl-2-pyrrolidone)), PMNP (poly(N-acryloyl-5-methoxy-2-pyrrolidone)), or PBNP (poly(N-acryloyl-5-butoxy-2-pyrrolidone)) cores were examined to identify how systematic adjustments to core-segment structure affect micellar physicochemical properties, drug loading efficiency (DLE), and thermoresponsive drug release among these novel systems. Critical micelle concentrations (CMCs) were found to decrease by two orders of magnitude in the order of PNIPAAm-PNP, PNIPAAm-PMNP, and PNIPAAm-PBNP indicating that minor modifications to the pyrrolidone scaffold significantly affect its hydrophobic character. Moreover, the structural modifications were also found to influence micelle size and intermicellar aggregation that occurs above the lower critical solution temperature (LCST). In line with the CMC data, DLE values of doxorubicin-loaded (i.e., DOX-loaded) micelles increase in the order of PNIPAAm-PNP, PNIPAAm-PMNP, and PNIPAAm-PBNP, a trend attributed to enhanced cohesive forces (i.e. London dispersion forces) between DOX and core as the latter becomes more hydrophobic. When heated above the LCST, DOX release decreases in the order of PNIPAAm-PNP, PNIPAAm-PMNP, and PNIPAAm-PBNP suggesting that release processes are impeded by the cohesive forces responsible for efficient encapsulation. Finally, cytotoxicity assays performed above the LCST reveal that DOX-loaded micelles are as cytotoxic as the free drug in formulations where DOX concentrations are equivalent.

Rho overexpression leads to mitosis-associated detachment of cells from epithelial sheets: a link to the mechanism of cancer dissemination.

Dissemination of neoplastic cells from the primary tumor (invasion and metastasis) is a fundamentally dangerous step in multistage carcinogenesis. Recent evidence suggests that Rho GTPase-mediated signaling is linked to dissemination of cells from several different types of human tumors. The Rho family of proteins is typically associated with the regulation of cytoskeletal activity, including actin assembly, microtubule dynamics, and myosin II-dependent contractility of the actin-rich cortex. We examined the effect of overexpression of constitutively active RhoA on islands and monolayers of epithelial cells. Although newly plated cells initially formed small spread islands, there was also a significant population of cells that detached from the substrate, floated in the medium, and then could reattach to the substrate to form new colonies. Detachment of cells from transfected epithelial islands or monolayers occurred in correlation to the plane of cytokinesis after misorientation of the mitotic spindle axis. We suggest that these alterations result from Rho-induced increase of contractility of the cortex of dividing cells, which, during cytokinesis, produces a cell that has budded out of an existing layer of cells. Cell division-mediated detachment of cells from tissue structures may be an important mechanism of tumor dissemination and metastasis.

Rho-dependent formation of epithelial "leader" cells during wound healing.

The motile behavior of epithelial cells located at the edge of a large wound in a monolayer of cultured cells was analyzed. The initial cellular response is alignment of the edge with an accompanying formation of tangential marginal actin bundles within individual cells positioned along the wound edge. Later, coherent out-growths of cell masses occur by the formation of special "leader" cells at the tops of outgrowths and "follower" cells along the sides. Leader cells exhibit profound cytoskeletal reorganization, including disassembly of marginal bundles, the realignment of actin filament bundles, and penetration of microtubules into highly active lamellae. Additionally, cell-cell contacts acquire radial geometry indicative of increased contractile tension. Interestingly, leader cells acquire a cytoskeletal organization and motility typical of fibroblasts. IAR-2 cultures stably transfected with a dominant-negative mutant of RhoA or treated with Rho-kinase inhibitor Y-27632 transformed most edge cells into leader-like cells. Alternatively, transfection of cells with constitutively active RhoA suppressed formation of leaders. Thus, expansion of the epithelial sheet involves functional differentiation into two distinct types of edge cells. The transition between these two patterns is controlled by Rho activity, which in turn controls the dynamic distribution and activity of actin filament bundles, myosin II, and microtubules.

Mechanisms of polarization of the shape of fibroblasts and epitheliocytes: Separation of the roles of microtubules and Rho-dependent actin-myosin contractility.

Cultured fibroblasts possess a characteristic polarized phenotype manifested by an elongate cell body with an anterior lamella whose cell edge is divided into protrusion-forming and inactive zones. Disruption of the fibroblast microtubule cytoskeleton leads to an increase in Rho-dependent acto-myosin contractile activity and concomitant loss of structural polarity. The functional relationship of myosin-driven contractile activity to loss of fibroblast anterior-posterior polarity is unknown. To dissect the roles of microtubule assembly and of Rho-dependent contractility on structural polarization of cells, polarized fibroblasts and nonpolarized epitheliocytes were treated with the microtubule-depolymerizing drug, nocodazole, and/or the Rho kinase inhibitor, Y-27632. Fibroblasts incubated with Y-27632 increased their degree of polarization by developing a highly elongate cell body with multiple narrow processes extended from the edges of the cell. Treatment of fibroblasts with nocodazole, alone or in combination with Rho kinase inhibitor, produced discoid or polygonal cells having broad, flattened lamellae that did not form long lamellar extensions. Single cultured epitheliocytes of the IAR-2 line do not display anterior-posterior polarization. When treated with Y-27632, the cells acquired a polarized, elongate shape with narrow protrusions and wide lamellas. Nocodazole alone or in combination with Y-27632 did not change the discoid shape of epitheliocytes, however treatment with Y-27632 produced thinning of the lamellar cytoplasm. We conclude that microtubules provide the necessary framework for polarization of fibroblasts and epitheliocytes, whereas Rho-regulated contractility modulates the degree of polarization of fibroblasts and completely inhibits polarization in epitheliocytes.

Contact interactions between epitheliocytes and fibroblasts: formation of heterotypic cadherin-containing adhesion sites is accompanied by local cytoskeletal reorganization.

Contact interactions between different cell types play a number of important roles in development, for example in cell sorting, tissue organization, and ordered migration of cells. The nature of such heterocellular interactions, in contrast to interactions between cells of the same type, remains largely unknown. In this report, we present experimental data examining the dynamics of heterocellular interactions between epitheliocytes and fibroblasts, which express different cadherin cell adhesion molecules and possess different actin cytoskeletal organizations. Our analysis revealed two striking features of heterocellular contact. First, the active free edge of an epitheliocyte reorganizes its actin cytoskeleton after making contact with a fibroblast. Upon contact with the leading edge of a fibroblast, epitheliocytes disassemble their marginal bundle of actin filaments and reassemble actin filaments into a geometric organization more typical of a fibroblast lamella. Second, epitheliocytes and fibroblasts form cell--cell adhesion structures that have an irregular organization and are associated with components of cell adhesion complexes. The structural organization of these adhesions is more closely related to the type of contacts formed between fibroblasts rather than to those between epitheliocytes. Heterotypic epithelio-fibroblastic contacts, like homotypic contacts between fibroblasts, are transient and do not lead to formation of stable contact interactions. We suggest that heterocellular contact interactions in culture may be regarded as models of how tissue systems consisting of epithelia and mesenchyme interact and become organized in vivo.

Characterization of sea urchin unconventional myosins and analysis of their patterns of expression during early embryogenesis.

Early sea urchin development requires a dynamic reorganization of both the actin cytoskeleton and cytoskeletal interactions with cellular membranes. These events may involve the activities of multiple members of the superfamily of myosin motor proteins. Using RT-PCR with degenerate myosin primers, we identified 11 myosin mRNAs expressed in unfertilized eggs and coelomocytes of the sea urchin Strongylocentrotus purpuratus. Seven of these sea urchin myosins belonged to myosin classes Igamma, II, V, VI, VII, IX, and amoeboid-type I, and the remaining four may be from novel classes. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I were either completely or partially cloned and their molecular structures characterized. Sea urchin myosins-V, -VI, -VII, and amoeboid-type-I shared a high degree of sequence identity with their respective family members from vertebrates and they retained their class-specific structure and domain organization. Analysis of expression of myosin-V, -VI, -VII, and amoeboid-type-I mRNAs during development revealed that each myosin mRNA displayed a distinct temporal pattern of expression, suggesting that myosins might be involved in specific events of early embryogenesis. Interestingly, the onset of gastrulation appeared to be a pivotal point in modulation of myosin mRNA expression. The presence of multiple myosin mRNAs in eggs and embryos provides insight into the potential involvement of multiple specific motor proteins in the actin-dependent events of embryo development.

Myosin-dependent contractile activity of the actin cytoskeleton modulates the spatial organization of cell-cell contacts in cultured epitheliocytes.

The spatial organization of cell-cell adherens junctions is distinct in cultured cells from two different tissue types, specifically, epitheliocytes and fibroblasts. In epitheliocytes, contacts are localized tangentially, along contacting cell edges and in association with circumferential actin bundles. Contacts between fibroblasts are radially oriented; that is, they are perpendicular to the overlapping edges of the cells and are associated with straight bundles of actin filaments. In the present study, we establish that the spatial organization of cell-cell contacts in the epithelial cell line IAR-2 can be converted from the typical tangential pattern to the radial pattern observed in fibroblasts. This transition can be induced by treatment with two agents, phorbol 12-myristate 13-acetate and nocodazole, which have different modes of action. Inhibition of myosin contractility reverses tangential-to-radial conversion of cell-cell contacts. These data suggest that formation of radially aligned contacts depends on modulation of contractility within the actin cytoskeleton through the myosin motor protein. The results open the possibility that modulation of the spatial organization of cell-cell contacts may play important roles in regulating organization and physiological functions of epithelial tissues.

Analysis of actin filament bundle dynamics during contact formation in live epithelial cells.

The actin cytoskeleton is an integral component of the cell-cell adherens junction complex. We used fluorescence labeling of actin filaments and time-lapse laser scanning confocal microscopy to investigate the functional relationship between the organization of the actin cytoskeleton and formation of adherens junctions in live epithelial cells. Rhodamine-phalloidin was loaded into cultured cells by wounding epithelial monolayers in the presence of fluorescent analog. Rhodamine-phalloidin was incorporated into the actin filaments in stress fibers, circumferential bundles, and marginal bundles. Cells containing labeled actin filaments appeared physiologically normal since the rates of migration, rates of pseudopodial protrusion/retraction, ability to form contacts, and sensitivity to cytochalasin B were equivalent to non-loaded, control epithelial cells. Marginal actin bundles initially formed as bow-shaped bundles that were observed to straighten as the bundles flowed rearward and away from the free cell edge. When lamellae from adjacent cells made contact, rearward flow of marginal bundles ceased and the bundles started to disassemble with higher frequency. Next, we observed the formation of arc-like bundles at the edges of contacting cells, a position suggestive of a role in lateral expansion of the contact. During later stages of contact formation, new actin bundles assembled along the length of the expanding cell-cell boundary. These newly formed bundles are likely to participate in the establishment of the initial cadherin/actin cytoskeleton linkage and eventually form the circumferential bundles at the cell-cell adherens junction. Additionally, indirect immunolocalization studies characterized the location of myosin-II. A model is presented describing the function of the spatial and temporal dynamics of actin filament bundles and myosin-II activity in the formation of adherens junctions.

Disassembly of actin filaments leads to increased rate and frequency of mitochondrial movement along microtubules.

In activated sea urchin coelomocytes, cytoplasmic organelles move along distinct actin and microtubule dependent pathways, actin-based motility is driven by an unconventional myosin, and microtubule disassembly does not effect actin-dependent organelle motility [D'Andrea et al., 1994: J. Cell Sci. 107:2081-2094]. Given the growing evidence for potential interactions between components of the actin and microtubule cytoskeletons, we examined the effect of actin filament disassembly on the movement of mitochondria along microtubules in activated coelomocytes. Coelomocytes treated with cytochalasin B (CB), to disrupt actin filaments, exhibited a thinning of the cytoplasm, enhanced lateral undulation of microtubules, and ceased centripetal cortical flow of actin. Interestingly, the loss of actin filaments resulted in a approximately 1.5-fold increase in the average velocity of outward and inward moving mitochondria and increased the frequency of centripetal movement. To test if enhanced motility along microtubules was a consequence of decreased actin-myosin interaction, coelomocytes were treated with 2,3-butanedione monoxime (BDM), a potent inhibitor of myosin activity [Cramer and Mitchison, 1995: J. Cell Biol. 131:179-189]. BDM inhibited all types of actin-based motility observed in these cells including retrograde cortical flow, protrusion and retraction of the cell edge, and movement of intracellular organelles. Surprisingly, BDM treatment stopped the movement of mitochondria in CB-exposed cells, suggesting that BDM can also act as an inhibitor of organelle movement along microtubules. Collectively, these data demonstrated that microtubule-dependent mitochondrial motility and microtubule movement were sensitive to changes in the assembly state of the actin cytoskeleton.

Dynamics of contacts between lamellae of fibroblasts: essential role of the actin cytoskeleton.

We investigated actin cytoskeletal and adhesion molecule dynamics during collisions of leading lamellae of nontransformed and oncogene-transformed fibroblasts. By using real-time video microscopy, it was found that during lamellar collision there was considerable overlapping of leading lamellae followed by subsequent retraction. Overlapping of nontransformed fibroblasts was accompanied by formation of beta-catenin-positive contact structures organized into strands oriented parallel to the long axis of the cell that were associated with bundles of actin filaments. Maintenance of such cell-cell contact structures critically depended on the contractility of actin cytoskeleton, as inhibition of contractility with serum-free medium or 2,3-butanedione 2-monoxime (BDM) resulted in loss of strand formation. Strand formation was recovered when cells in serum-free medium were incubated with the microtubule inhibitor nocodazole, which is known to increase contractility. Oncogene-transformed fibroblasts reacted to collisions with responses similar to nontransformed fibroblasts but did not develop well-organized cell-cell contacts. A model is presented to describe how differences in the organization of the actin cytoskeleton could account for the structurally distinct responses to cell-cell contact by polarized fibroblastic cells versus nonpolarized epithelial cells.

Cell-cell contact changes the dynamics of lamellar activity in nontransformed epitheliocytes but not in their ras-transformed descendants.

We investigated the structural and functional alterations of active lamellae during initial cell-cell collision and establishment of cell-cell contacts in wounded cultures of nontransformed rat epitheliocytes (IAR-2 line) and their ras-transformed descendants (C4 line). Typically, the leading edges of nontransformed cells formed multiple transient contacts followed by establishment of small, stable contacts that would undergo lateral expansion. Formation and expansion of the contact area was accompanied by accumulation of the cell-cell adhesion molecules E-cadherin, beta-catenin, and plakoglobin. During lateral expansion, the circumferential bundles of actin filaments, characteristic of IAR-2 cells, disassembled at the site of stable contact forming a concave arc-like actin bundle between adjacent cells at the expanding edge. Pseudopodial activity was completely inhibited in the contact zone and partially inhibited at the free lamellar edges adjacent to the zone of contact. Con A-coated beads on the plasma membrane at the zone of contact stopped undergoing centripetal transport but now moved along the cell-cell boundary. On the other hand, ras-transformed cells developed overlapping lamellae and exhibited no detectable change in activity of lamellae, localization of adhesion molecules, and organization of the actin cytoskeleton. We propose that contact-induced reorganization of cell surface adhesion molecules and the underlying cortical cytoskeleton leads to development of lateral traction that may be an essential element in inducing expansion of the contact and in inhibiting local pseudopodial activity.

Dynamics of active lamellae in cultured epithelial cells: effects of expression of exogenous N-ras oncogene.

We examined the functional consequences of cellular transformation of rat IAR-2 epithelial cells, by a mutant N-ras oncogene, on the dynamics of active lamellae, structures that play an important role in cell motility, adhesion, and surface-receptor capping. Lamellar activity was assessed by measuring the rate of outer-edge pseudopodial activity and by analyzing the motility of Con A-coated beads placed on lamellar surfaces with optical tweezers. Although transformation dramatically affected the shape and size of active cellular lamellae, there was little detectable effect on either pseudopodial activity or bead movement. To investigate the potential relationship between functional lamellar activity and the microtubule cytoskeleton, lamellar activity was examined in nontransformed and transformed cells treated with the microtubule-disrupting drug nocodazole. In the absence of microtubules, transformed cells were less polarized and possessed decreased rates of pseudopodial and bead motility. On the basis of these observations, it is suggested that ras-induced transformation of epithelial cells consists of two cytoskeletal modifications: overall diminished actin cytoskeletal dynamics in lamellae and reorganization of the microtubule cytoskeleton that directs pseudopodial activity to smaller polarized lamellae.

Identification of coelomocyte unconventional myosin and its association with in vivo particle/vesicle motility.

Sea urchin coelomocytes undergo an inducible structural transformation from petalloid to filopodial form during the 'clotting' response in sea urchins. Using a petalloid coelomocyte model, stimulated coelomocytes exhibited bidirectional particle/vesicle motility with a broad distribution of velocities, ranging from 0.02 to 0.12 microns s-1 in the outward bound direction. Coelomocytes treated with the microtubule-disrupting drug, nocodazole, continued to exhibit outward particle/vesicle movements along linear paths with an average velocity of 0.028 +/- 0.006 microns s-1. We partially purified a 110 kDa polypeptide possessing K+EDTA-, Ca2(+)-, Mg2(+)- and F-actin-activated Mg(2+)-ATPase activities characteristic of myosin-like motor proteins. The 110 kDa protein immuno-crossreacted with both affinity-purified, anti-brush border unconventional myosin-I polyclonal antibodies and anti-Acanthamoeba myosin head monoclonal antibodies. By indirect immunofluorescence, the 110 kDa unconventional myosin was localized to clusters of particles/vesicles within the perinuclear region of unstimulated coelomocytes, an area containing numerous mitochondria, acidic, lysosomal and Golgi organelles. Indirect immunofluorescence of partially transformed and filopodial coelomocytes detected a diminution of perinuclear staining with a concomitant appearance of stained linear arrays of particles/vesicles, enhanced staining of peripheral lamellae, and staining of the entire length of the filopodia. Subfractionation of unstimulated coelomocyte homogenates on linear sucrose gradients identified distinct peaks of ATPase activity associated with fractions containing conventional and 110 kDa unconventional myosin. Unconventional myosin-containing fractions were found to have numerous particles that stained with anti-brush border unconventional myosin-I antibodies and the lipophilic dye, DiOC6. Thus, coelomocytes demonstrate activatable movements of particles/vesicles in cells devoid of microtubules and possess an unconventional myosin, which may be the motor protein driving particle/vesicle translocation.

Sea urchin egg 100-kDa dynamin-related protein: identification of and localization to intracellular vesicles.

We have initiated a study to identify possible tubulin dimer binding proteins from unfertilized sea urchin eggs utilizing tubulin dimer coupled to Sepharose 4B. A 100-kDa polypeptide has been isolated and its interaction with tubulin/microtubules examined. Solution assembly studies indicated the 100-kDa polypeptide potentiated microtubule assembly. The 100-kDa protein also bundled and cosedimented with polymerized microtubules. Rabbit polyclonal anti-egg 100-kDa antibodies cross-reacted with a 100-kDa polypeptide in the eggs of several different sea urchin species and with mammalian brain dynamin, a 100-kDa microtubule binding protein. These biochemical characterizations and immunological results suggest that the sea urchin 100-kDa microtubule binding protein appears to be related to the dynamin protein family. To gain insight into the functional role of sea urchin egg 100-kDa dynamin-related protein, we immunolocalized egg 100-kDa protein in cryosections and isolated cortices of unfertilized eggs. Egg 100-kDa protein was localized to the monolayer of cortical granules that underlie the plasma membrane and to the inner cytoplasm. Immunolocalizations of egg 100-kDa protein in cortices isolated from fertilized eggs detailed the redistribution of the egg 100-kDa onto cortically positioned vesicles. Egg 100-kDa dynamin-related protein was expressed throughout early development with approximately equivalent amounts present in the unfertilized egg and embryo through early gastrulation, followed by an abrupt decrease in the level of expression by the pluteus larval stage.

A novel vesicle-associated protein (VAP-1) in sea urchin eggs containing multiple RNA-binding consensus sequences.

We have identified a novel high molecular weight, vesicle-associated protein (VAP-1) in the eggs of the sea urchin Strongylocentrotus purpuratus. Biochemical fractionation and immunofluorescence analysis of unfertilized eggs indicate that VAP-1 is a peripheral membrane protein associated with microsomal membrane fractions. Sequence analysis of partial VAP-1 cDNA clones reveals that the protein contains at least four RNA-binding consensus sequences. The RNA-binding sequences are separated by several glycine rich domains and this organization, RNA-binding domains separated by glycine rich sequences, is common to several RNA-binding proteins including the heterogeneous ribonuclear protein A1 and nucleolin. The characteristics of VAP-1 suggest that the protein may function as a multidomain RNA-binding protein. The possibility that VAP-1 may play a role in nuclear RNA processing is also discussed.

Reversible structural alterations of undifferentiated and differentiated human neuroblastoma cells induced by phorbol ester.

Morphological alterations in the structure of undifferentiated and morphologically differentiated human neuroblastoma cells induced by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, were examined by video microscopy and immunomorphology. In undifferentiated cells, PMA induced the formation of motile actin-rich lamellas and of stable cylindrical processes rich in microtubules. Formation of stable processes resulted either from the collapse of lamellas or the movement of the cell body away from the base of a process. In differentiated cells, PMA induced the rapid extension of small lamellas and subsequent formation of short-lived elongated processes from the lateral edges of neurites. Additionally, growth cones exhibited enhanced modulation in shape after PMA treatment. These reversible reorganizations were similar to the actinoplast-tubuloplast transformations exhibited by PMA-treated fibroblasts. We suggest that actinoplast-tubuloplast reorganizations play essential roles in morphogenesis where stable cytoplasmic extensions are induced by external stimuli. In particular, PMA-induced reorganizations of neural cells in culture may be a model for morphological modulations that occur in nerve tissue.

Analysis of dynamin isoforms in mammalian brain: dynamin-1 expression is spatially and temporally regulated during postnatal development.

In adult rat brain, the microtubule-associated protein dynamin is composed of a closely spaced polypeptide doublet of approximately 100 kDa. Using an antibody preparation that is monospecific for dynamin-1 (the higher molecular mass isoform) we examined the temporal and regional expression of dynamin-1 in developing rat brain. Analysis of whole rat brain homogenates established that prior to postnatal day 9, dynamin-1 was present only at very low levels and thereafter its expression steadily increased with adult levels being attained by postnatal day 23. In individual regions of the brain, dynamin-1 levels were highest in cortex, amygdala, and striatum, significantly lower in olfactory bulb, cerebellum, and midbrain, and lowest in brainstem. During postnatal development, each of the regions exhibited approximately the same time course of protein expression except for a slight lag in expression in olfactory bulb. The spatial and temporal patterns of expression of dynamin-1 correlate with the establishment and/or maintenance of mature neuronal structure and function rather than dendritic or axonal outgrowth.

Controlled micromanipulation of human sperm in three dimensions with an infrared laser optical trap: effect on sperm velocity.

Individual human sperm can be micromanipulated in three dimensions using a 1.06 microns Nd:YAG laser trap. Single sperm swimming with velocities in the range of 65 to 85 microns/sec can be trapped with 40 mW of power through 120 seconds without a deleterious effect on velocity. Even though it will be necessary to further evaluate the effects of laser light on specific functions of sperm, our data suggest that decreasing the time of manipulation to a minimum will increase the safety of the micromanipulation procedures. Laser traps may play a role in assisted reproductive technology by facilitating the selective transport of individual sperm.

Sea urchin egg villin: identification of villin in a non-epithelial cell from an invertebrate species.

Fertilization of sea urchin eggs results in the rapid polymerization of actin filaments and subsequent formation of a brush border-like cortical cytoskeleton. A 110 x 10(3) Mr (110K) actin binding protein has been purified from extracts of unfertilized Strongylocentrotus purpuratus eggs. Analysis of polymerization kinetics using fluorescence and viscometry assays demonstrated that 110K accelerated the nucleation phase of actin assembly only in the presence of elevated Ca2+. The Ca(2+)-mediated effects were correlated with a decrease in sedimentable polymer and a decrease in average filament length. Addition of Ca2+ to solutions of 110K and F-actin, polymerized in the presence of EGTA, resulted in a precipitous drop in viscosity and the decreased viscosity was fully reversible upon chelation of Ca2+. The Ca2+ threshold for 110K activation was in the 10(-6) to 10(-7) M range. Nucleated assembly experiments using Limulus sperm acrosomal processes demonstrated that egg 110K capped the barbed ends of actin filaments. In the absence of Ca2+, 110K organized actin filaments into bundles at pH values less than 7.4. Anti-egg 110K antibody crossreacted with chicken intestinal epithelial cell villin and anti-porcine villin headpiece monoclonal antibody crossreacted with 110K. Further, 110K possesses an approximately 10 x 10(3) Mr terminal polypeptide segment that is immunologically related to villin headpiece. These studies demonstrate that sea urchin egg 110K is functionally, immunologically and structurally related to villin, an actin binding protein expressed in specific epithelial tissues in vertebrates. Consequently, this finding provides insight into the potential mechanisms that might determine the genesis of the cortical brush border cytoarchitecture in sea urchin eggs and further sheds light on the evolution of the villin protein family.