Microfilaments and membranes.

Microfilaments are intimately involved in many plasma and internal membrane functions. Recent studies of microfilament-membrane linking proteins and non-filamentous myosins implicate microfilaments in diverse functions, including transmembrane signaling and vesicular transport. Evidence from animal and yeast cells suggests that microfilaments are regulated by protein phosphosphorylation, small GTP-binding proteins and associations involving SH3 domains.

Regulation of cortical structure by the ezrin-radixin-moesin protein family.

Molecules involved in ERM (ezrin-radixin-moesin) based attachment of membrane proteins to the cortical cytoskeleton in cell surface structures have been identified. In lymphocytes, a direct interaction is seen with extracellular matrix receptors and intercellular adhesion molecules. In polarized epithelial cells, an adaptor molecule named EBP50 provides a bridge between the amino-terminal domain of ezrin and the cytoplasmic regions of plasma membrane proteins, including the cystic fibrosis transmembrane conductance regulator (CFTR) and the beta2 adrenergic receptor. ERM proteins are conformationally regulated - binding sites for EBP50 and F actin are masked in the dormant molecules and activation leads to exposure of these sites. The mechanism of activation, however, remains to be fully elucidated. ERM proteins also play a role in the Rho and Rac signaling pathways: activated ERM proteins can dissociate Rho-GDI (GDP dissociation inhibitor) from Rho and thereby activate Rho-dependent pathways.

Rapid phosphorylation and reorganization of ezrin and spectrin accompany morphological changes induced in A-431 cells by epidermal growth factor.

Addition of EGF to human carcinoma A-431 cells is known to induce membrane ruffling after approximately 2 min (Chinkers, M., J. A. McKanna, and S. Cohen. 1979. J. Cell Biol. 83:260-265) and the phosphorylation of a protein referred to as p81, a known substrate for various protein-tyrosine kinases (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273). Ezrin, a Mr approximately 80,000 cytoskeletal protein of the isolated chicken microvillar core, is present in actin-containing cell surface structures of a wide variety of cells (Bretscher, A. 1983. J. Cell Biol. 97:425-432). Ezrin was then found to be homologous to p81 and to be phosphorylated on tyrosine in response to EGF (Gould, K. L., J. A. Cooper, A. Bretscher, and T. Hunter. 1986. J. Cell Biol. 102:660-669). Here, the purification of ezrin from human placenta is described. Antibodies to human ezrin, together with antibodies to other microfilament-associated proteins, were used to follow the distribution and phosphorylation of these proteins in A-431 cells after EGF treatment. EGF induces the formation of microvillar-like surface structures on these cells within 30 s and these give way to membrane ruffles at approximately 2-5 min after EGF addition; the cells then round up after approximately 10-20 min. Ezrin is recruited into the microvillar-like structures and the membrane ruffles, and is phosphorylated on tyrosine and serine in a time course that parallels the formation and disappearance of these surface structures. Spectrin is recruited into the membrane ruffles and shows a similar rapid kinetics of phosphorylation, but only on serine residues, and remains phosphorylated through the rounding up of the cells. The microvillar-like structures and membrane ruffles are also enriched in fimbrin and alpha-actinin. Myosin becomes rapidly reorganized into a striated pattern that is consistent with it playing a role in cell rounding. These results show that two cortical proteins, ezrin and spectrin, become phosphorylated in a time course coincident with remodeling of the cell surface. The results are consistent with the notion that ezrin phosphorylation may play a role in the formation of cell surface projections whereas spectrin phosphorylation may be involved in remodelling of more planar areas of the cell surface.

Purification of an 80,000-dalton protein that is a component of the isolated microvillus cytoskeleton, and its localization in nonmuscle cells.

The microvillus cytoskeleton, isolated from chicken intestinal epithelial cell brush borders, is known to contain five major protein components, the 110,000-dalton polypeptide, villin (95,000 daltons), fimbrin (68,000 daltons), actin (43,000 daltons), and calmodulin (17,000 daltons). In this paper we describe our first step in studying the minor components of the isolated core. We have so far identified and purified an 80,000-dalton polypeptide that was present in the isolated structure in approximately 0.7% the molar abundance of actin. Antibodies to the 80,000-dalton component did not react with other microvillus core proteins, and, when used in indirect immunofluorescence microscopy, they stained the microvilli of intestinal epithelial cells fixed in situ. The 80,000-dalton component therefore appears to be a newly-identified, authentic component of intestinal microvilli in vivo and of isolated microvillus cores. Immunological studies demonstrate that the 80,000-dalton component is widely distributed in nonmuscle cells. Indirect immunofluorescence microscopy reveals that it is particularly enriched in surface structures, such as blebs, microvilli, and retraction fibers of cultured cells.

Fimbrin, a new microfilament-associated protein present in microvilli and other cell surface structures.

A 68,000 mol wt polypeptide has been identified as one of the few major proteins in the microfilament bundles of the microvilli present on intestinal epithelial cells. Antibodies against the purified protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein have been used in indirect immunofluorescence microscopy on several cultured cells. The protein is found particularly prominent in membrane ruffles, microspikes, and microvilli.

Mapping of the microvillar 110K-calmodulin complex: calmodulin-associated or -free fragments of the 110-kD polypeptide bind F-actin and retain ATPase activity.

The 110K-calmodulin complex isolated from intestinal microvilli is an ATPase consisting of one polypeptide chain of 110 kD in association with three to four calmodulin molecules. This complex is presumably the link between the actin filaments in the microvillar core and the surrounding cell membrane. To study its structural regions, we have partially cleaved the 110K-calmodulin complex with alpha-chymotrypsin; calmodulin remains essentially intact under the conditions used. As determined by 125I-calmodulin overlays, ion exchange chromatography, and actin-binding assays, a 90-kD digest fragment generated in EGTA remains associated with calmodulin. The 90K-calmodulin complex binds actin in an ATP-reversible manner and decorates actin filaments with an arrow-head appearance similar to that found after incubation of F-actin with the parent complex; binding occurs in either calcium- or EGTA-containing buffers. ATPase activity of the 90-kD digest closely resembles the parent complex. In calcium a digest mixture containing fragments of 78 kD, a group of three at approximately 40 kD, and a 32-kD fragment (78-kD digest mixture) is generated with alpha-chymotrypsin at a longer incubation time; no association of these fragments with calmodulin is observed. Time courses of digestions and cyanogen bromide cleavage indicate that the 78-kD fragment derives from the 90-kD peptide. The 78-kD mixture can also hydrolyze ATP. Furthermore, removal of the calmodulin by ion exchange chromatography from this 78-kD mixture had no effect on the ATPase activity of the digest, indicating that the ATPase activity resides on the 110-kD polypeptide. The 78 kD, two of the three fragments at approximately 40 kD, and the 32-kD fragments associate with F-actin in an ATP-reversible manner. Electron microscopy of actin filaments after incubation with the 78-kD digest mixture reveals coated filaments, although the prominent arrowhead appearance characteristic of the parent complex is not observed. These data indicate that calmodulin is not required either for the ATPase activity or the ATP-reversible binding of the 110K-calmodulin complex to F-actin. In addition, since all the fragments that bind F-actin do so in an ATP-reversible manner, the sites required for F-actin binding and ATP reversibility likely reside nearby.

Identification and localization of immunoreactive forms of caldesmon in smooth and nonmuscle cells: a comparison with the distributions of tropomyosin and alpha-actinin.

Caldesmon is an F-actin cross-linking protein of chicken gizzard smooth muscle whose F-actin binding activity can be regulated in vitro by Ca2+-calmodulin (Sobue, K., Y. Muramoto, M. Fujita, and S. Kakiuchi, 1981, Proc. Natl. Acad. Sci. USA, 78:5652-5655). It is a rod-shaped, heat-stable, F-actin bundling protein and is the most abundant F-actin cross-linking protein of chicken gizzard smooth muscle presently known (Bretscher, A., 1984, J. Biol. Chem., 259:12873-12880). We report the use of polyclonal antibodies to caldesmon to investigate its distribution and localization in other cells. Using immune blotting procedures, we have detected immunoreactive, heat-stable forms of caldesmon in cultured cells having either approximately the same apparent polypeptide molecular weight as gizzard caldesmon (120,000-140,000) or a substantially lower molecular weight (71,000-77,000). Through use of affinity-purified antibodies in indirect immunofluorescence microscopy, we have localized the immunoreactive forms to the terminal web of the brush border of intestinal epithelial cells and to the stress fibers and ruffling membranes of cultured cells. At the light microscope level caldesmon is distributed in a periodic fashion along stress fibers that is coincident with the distribution of tropomyosin and complementary to the distribution of alpha-actinin.

Characterization of TPM1 disrupted yeast cells indicates an involvement of tropomyosin in directed vesicular transport.

Disruption of the yeast tropomyosin gene TPM1 results in the apparent loss of actin cables from the cytoskeleton (Liu, H., and A. Bretscher. 1989. Cell. 57:233-242). Here we show that TPM1 disrupted cells grow slowly, show heterogeneity in cell size, have delocalized deposition of chitin, and mate poorly because of defects in both shmooing and cell fusion. The transit time of alpha-factor induced a-agglutinin secretion to the cell surface is longer than in isogenic wild-type strains, and some of the protein is mislocalized. Many of the TPM1-deleted cells contain abundant vesicles, similar in morphology to late secretory vesicles, but without an abnormal accumulation of intermediates in the delivery of either carboxypeptidase Y to the vacuole or invertase to the cell surface. Combinations of the TPM1 disruption with sec13 or sec18 mutations, which affect early steps in the secretory pathway, block vesicle accumulation, while combinations with sec1, sec4 or sec6 mutations, which affect a late step in the secretory pathway, have no effect on the vesicle accumulation. The phenotype of the TPM1 disrupted cells is very similar to that of a conditional mutation in the MYO2 gene, which encodes a myosin-like protein (Johnston, G. C., J. A. Prendergast, and R. A. Singer. 1991. J. Cell Biol. 113:539-551). The myo2-66 conditional mutation shows synthetic lethality with the TPM1 disruption, indicating that the MYO2 and TPM1 gene products may be involved in the same, or parallel function. We conclude that tropomyosin, and by inference actin cables, may facilitate directed vesicular transport of components to the correct location on the cell surface.

Identification of EPI64, a TBC/rabGAP domain-containing microvillar protein that binds to the first PDZ domain of EBP50 and E3KARP.

The cortical scaffolding proteins EBP50 (ERM-binding phosphoprotein-50) and E3KARP (NHE3 kinase A regulatory protein) contain two PDZ (PSD-95/DlgA/ZO-1-like) domains followed by a COOH-terminal sequence that binds to active ERM family members. Using affinity chromatography, we identified polypeptides from placental microvilli that bind the PDZ domains of EBP50. Among these are 64- and/or 65-kD differentially phosphorylated polypeptides that bind preferentially to the first PDZ domain of EBP50, as well as to E3KARP, and that we call EPI64 (EBP50-PDZ interactor of 64 kD). The gene for human EPI64 lies on chromosome 22 where nine exons specify a protein of 508 residues that contains a Tre/Bub2/Cdc16 (TBC)/rab GTPase-activating protein (GAP) domain. EPI64 terminates in DTYL, which is necessary for binding to the PDZ domains of EBP50, as a mutant ending in DTYLA no longer interacts. EPI64 colocalizes with EBP50 and ezrin in syncytiotrophoblast and cultured cell microvilli, and this localization in cultured cells is abolished by introduction of the DTYLA mutation. In addition to EPI64, immobilized EBP50 PDZ domains retain several polypeptides from placental microvilli, including an isoform of nadrin, a rhoGAP domain-containing protein implicated in regulating vesicular transport. Nadrin binds EBP50 directly, probably through its COOH-terminal STAL sequence. Thus, EBP50 appears to bind membrane proteins as well as factors potentially involved in regulating membrane traffic.

Calcium-regulated cooperative binding of the microvillar 110K-calmodulin complex to F-actin: formation of decorated filaments.

The 110K-calmodulin complex of intestinal microvilli is believed to be the link between the actin filaments comprising the core bundle and the surrounding cell membrane. Although not the first study describing a purification scheme for the 110K-calmodulin complex, a procedure for the isolation of stable 110K-calmodulin complex both pure and in high yield is presented; moreover, isolation is without loss of the associated calmodulin molecules since a previously determined ratio in isolated microvillar cytoskeletons of calmodulin to 110-kD polypeptide of 3.3:1 is preserved. We have found that removal of calmodulin from the complex by the calmodulin antagonists W7 or W13 results in precipitation of the 110-kD polypeptide with calmodulin remaining in solution. The interaction of 110K-calmodulin with beef skeletal muscle F-actin has been examined. Cosedimentation assays of 110K-calmodulin samples incubated with F-actin show the amount of 110K-calmodulin associating with F-actin to be ATP, calcium, and protein concentration dependent; however, relatively salt independent. In calcium, approximately 30% of the calmodulin remains in the supernatant rather than cosedimenting with the 110-kD polypeptide and actin. Electron microscopy of actin filaments after incubation with 110K-calmodulin in either calcium- or EGTA-containing buffers show polarized filaments often laterally associated. Each individual actin filament is seen to exhibit an arrowhead appearance characteristic of actin filaments after their incubation with myosin fragments, heavy meromyosin and subfragment 1. In some cases projections having a 33-nm periodicity are observed. This formation of periodically spaced projections on actin filaments provides further compelling evidence that the 110K-calmodulin complex is the bridge between actin and the microvillar membrane.

Reassociation of microvillar core proteins: making a microvillar core in vitro.

Intestinal epithelia have a brush border membrane of numerous microvilli each comprised of a cross-linked core bundle of 15-20 actin filaments attached to the surrounding membrane by lateral cross-bridges; the cross-bridges are tilted with respect to the core bundle. Isolated microvillar cores contain actin (42 kD) and three other major proteins: fimbrin (68 kD), villin (95 kD), and the 110K-calmodulin complex. The addition of ATP to detergent-treated isolated microvillar cores has previously been shown to result in loss of the lateral cross-bridges and a corresponding decrease in the amount of the 110-kD polypeptide and calmodulin associated with the core bundle. This provided the first evidence to suggest that these lateral cross-bridges to the membrane are comprised at least in part by a 110-kD polypeptide complexed with calmodulin. We now demonstrate that purified 110K-calmodulin complex can be readded to ATP-treated, stripped microvillar cores. The resulting bundles display the same helical and periodic arrangement of lateral bridges as is found in vivo. In reconstitution experiments, actin filaments incubated in EGTA with purified fimbrin and villin form smooth-sided bundles containing an apparently random number of filaments. Upon addition of 110K-calmodulin complex, the bundles, as viewed by electron microscopy of negatively stained images, display along their entire length helically arranged projections with the same 33-nm repeat of the lateral cross-bridges found on microvilli in vivo; these bridges likewise tilt relative to the bundle. Thus, reconstitution of actin filaments with fimbrin, villin, and the 110K-calmodulin complex results in structures remarkably similar to native microvillar cores. These data provide direct proof that the 110K-calmodulin is the cross-bridge protein and indicate that actin filaments bundled by fimbrin and villin are of uniform polarity and lie in register. The arrangement of the cross-bridge arms on the bundle is determined by the structure of the core filaments as fixed by fimbrin and villin; a contribution from the membrane is not required.

Parallel secretory pathways to the cell surface in yeast.

Saccharomyces cerevisiae mutants that have a post-Golgi block in the exocytic pathway accumulate 100-nm vesicles carrying secretory enzymes as well as plasma membrane and cell-wall components. We have separated the vesicle markers into two groups by equilibrium isodensity centrifugation. The major population of vesicles contains Bg12p, an endoglucanase destined to be a cell-wall component, as well as Pma1p, the major plasma membrane ATPase. In addition, Snc1p, a synaptobrevin homologue, copurifies with these vesicles. Another vesicle population contains the periplasmic enzymes invertase and acid phosphatase. Both vesicle populations also contain exoglucanase activity; the major exoglucanase normally secreted from the cell, encoded by EXG1, is carried in the population containing periplasmic enzymes. Electron microscopy shows that both vesicle groups have an average diameter of 100 nm. The late secretory mutants sec1, sec4, and sec6 accumulate both vesicle populations, while neither is detected in wild-type cells, early sec mutants, or a sec13 sec6 double mutant. Moreover, a block in endocytosis does not prevent the accumulation of either vesicle species in an end4 sec6 double mutant, further indicating that both populations are of exocytic origin. The accumulation of two populations of late secretory vesicles indicates the existence of two parallel routes from the Golgi to the plasma membrane.

Microvillus 110K-calmodulin: effects of nucleotides on isolated cytoskeletons and the interaction of the purified complex with F-actin.

Microvilli isolated from intestinal epithelial cells contain a cytoskeletal Mr 110,000 polypeptide complexed with calmodulin (110K-CM) that is believed to link the microfilament core bundle laterally to the plasma membrane. Previous work has shown that physiological levels of ATP can partially solubilize the 110K-CM complex from isolated microvillus cytoskeletons or isolated microvilli. However, once extracted, the 110K-CM complex has been found to be difficult to maintain stably soluble in aqueous buffers. This is due to the presence of an endogenous ATPase (approximately 100 nmol Pi/min per mg at 37 degrees C) in microvillus cytoskeletal preparations that depletes the ATP with subsequent precipitation of 110K-CM. Addition of ATP to such precipitates resolubilizes 110K-CM. Inclusion of an ATP regenerating system in the solubilization of 110K-CM from cytoskeletons, or membrane-bound brush borders, increases the amount of 110K-CM solubilized. Solubilization of 110K-CM from microvillus cytoskeletons was found to require a divalent cation (Mg2+, Mn2+, or Co2+, but not Zn2+) and a nucleoside triphosphate (ATP, GTP, CTP, or ITP). ADP did not solubilize 110K-CM, but could partially inhibit ATP-dependent solubilization. Solubilized 110K was phosphorylated during extraction of microvillus cores with [gamma-32P]ATP, but this was unrelated to the solubilization of 110K-CM as the endogenous kinase was specific for ATP, whereas the solubilization was not. The 110K-CM was purified using ATP extraction of brush border cytoskeletons in the presence of an ATP regenerating system, gel filtration of the solubilized extract, an ATP depletion step to specifically precipitate 110K-CM with F-actin, and resolubilization followed by phosphocellulose chromatography. The purified complex was stably soluble in aqueous buffers both in the presence and absence of ATP. It bound almost quantitatively to F-actin in the absence of ATP, and showed nucleotide solubilization characteristics from F-actin similar to that found for solubilization of 110K-CM from microvillus cores. At low ATP levels, the binding to F-actin was increased in the presence of ADP. These results suggest that the purified complex has been isolated in a native form. The data confirm and extend the studies of Howe and Mooseker (1983, J. Cell Biol., 97:974-985) using a partially purified preparation of 110K-CM and further emphasize that 110K-CM is a stably water soluble complex and not an integral membrane protein.

Localization of actin and microfilament-associated proteins in the microvilli and terminal web of the intestinal brush border by immunofluorescence microscopy.

Indirect immunofluorescence microscopy was used to localize microfilament-associated proteins in the brush border of mouse intestinal epithelial cells. As expected, antibodies to actin decorated the microfilaments of the microvilli, giving rise to a very intense fluorescence. By contrast, antibodies to myosin, tropomyosin, filamin, and alpha-actinin did not decorate the microvilli. All these antibodies, however, decorated the terminal web region of the brush border. Myosin, tropomyosin, and alpha-actinin, although present throughout the terminal web, were found to be preferentially located around the periphery of the organelle. Therefore, two classes of microfilamentous structures can be documented in the brush border. First, the highly ordered microfilaments which make up the cores of the microvilli apparently lack the associated proteins. Second, seemingly less-ordered microfilaments are found in the terminal web, in which region the myosin, tropomyosin, filamin and alpha-actinin are located.

Microinjection of villin into cultured cells induces rapid and long-lasting changes in cell morphology but does not inhibit cytokinesis, cell motility, or membrane ruffling.

Villin, a Ca2(+)-regulated F-actin bundling, severing, capping, and nucleating protein, is a major component of the core of microvilli of the intestinal brush border. Its actin binding properties, tissue specificity, and expression during cell differentiation suggest that it might be involved in the organization of the microfilaments in intestinal epithelial cells to form a brush border. Recently, Friederich et al., (Friederich, E., C. Huet, M. Arpin, and D. Louvard. 1989. Cell. 59:461-475) showed that villin expression in transiently transfected fibroblasts resulted in the loss of stress fibers and the appearance of large cell surface microvilli on some cells. Here, we describe the effect of villin microinjection into cells that normally lack this protein, which has allowed us to examine the immediate and long-term effects of introducing different concentrations of villin on microfilament organization and function. Microinjected cells rapidly lost their stress fibers and the actin was reorganized into abundant villin containing cortical structures, including microspikes and, in about half the cells, large surface microvilli. This change in actin organization persisted in cells for at least 24 h, during which time they had gone through two or three cell divisions. Microinjection of villin core, that lacks the bundling activity of villin but retains all the Ca2(+)-dependent properties, disrupted the stress fiber system and had no effect on cell surface morphology. Thus, the Ca2(+)-dependent activities of villin are responsible for stress fiber disruption, and the generation of cell surface structures is a consequence of its bundling activity. Microinjection of villin led to the reorganization of myosin, tropomyosin, and alpha-actinin, proteins normally associated with stress fibers, whereas both fimbrin and ezrin, which are also components of microvillar core filaments, were readily recruited into the induced surface structures. Vinculin was also redistributed from its normal location in focal adhesions. Despite these changes in the actin cytoskeleton, cells were able to divide and undergo cytokinesis, move, spread on a substratum, and ruffle. Thus, we show that a single microfilament-associated protein can reorganize the entire microfilament structure of a cell, without interfering with general microfilament-based functions like cytokinesis, cell locomotion, and membrane ruffling.

Identification of EBP50: A PDZ-containing phosphoprotein that associates with members of the ezrin-radixin-moesin family.

Members of the ezrin-radixin-moesin (ERM) family of membrane-cytoskeletal linking proteins have NH2- and COOH-terminal domains that associate with the plasma membrane and the actin cytoskeleton, respectively. To search for ERM binding partners potentially involved in membrane association, tissue lysates were subjected to affinity chromatography on the immobilized NH2-terminal domains of ezrin and moesin, which comprise the ezrin-radixin-moesin-association domain (N-ERMAD). A collection of polypeptides at 50-53 kD from human placenta and at 58-59 kD from bovine brain bound directly to both N-ERMADs. The 50-53-kD placental proteins migrated as a major 50-kD species after phosphatase treatment, indicating that the heterogeneity is due to different phosphorylation states. We refer to these polypeptides as ERM-binding phosphoprotein 50 (EBP50). Sequence analysis of human EBP50 was used to identify an approximately 2-kb human cDNA that encodes a 357-residue polypeptide. Recombinant EBP50 binds tightly to the N-ERMADs of ezrin and moesin. Peptide sequences from the brain candidate indicated that it is closely related to EBP50. EBP50 has two PSD-95/DlgA/ZO-1-like (PDZ) domains and is most likely a homologue of rabbit protein cofactor, which is involved in the protein kinase A regulation of the renal brush border Na+/H+ exchanger. EBP50 is widely distributed in tissues, and is particularly enriched in those containing polarized epithelia. Immunofluorescence microscopy of cultured cells and tissues revealed that EBP50 colocalizes with actin and ezrin in the apical microvilli of epithelial cells, and immunoelectron microscopy demonstrated that it is specifically associated with the microvilli of the placental syncytiotrophoblast. Moreover, EBP50 and ezrin can be coimmunoprecipitated as a complex from isolated human placental microvilli. These findings show that EBP50 is a physiologically relevant ezrin binding protein. Since PDZ domains are known to mediate associations with integral membrane proteins, one mode of membrane attachment of ezrin is likely to be mediated through EBP50.

Ezrin oligomers are major cytoskeletal components of placental microvilli: a proposal for their involvement in cortical morphogenesis.

Ezrin is a component of the microvillus cytoskeleton of a variety of polarized epithelial cells and is believed to function as a membrane-cytoskeletal linker. In this study, we isolated microvilli from human placental syncytiotrophoblast as a model system for biochemical analysis of ezrin function. In contrast to intestinal microvilli, ezrin is a major protein component of placental microvilli, comprising approximately 5% of the total protein mass and present at about one quarter of the molar abundance of actin. Gel filtration and chemical cross-linking studies demonstrated that ezrin exists mainly in the form of noncovalent dimers and higher order oligomers in extracts of placental microvilli. A novel form of ezrin, apparently representing covalently cross-linked adducts, was present as a relatively minor constituent of placental microvilli. Both oligomers and adducts remained associated with the detergent-insoluble cytoskeleton, indicating a tight interaction with actin filaments. Moreover, stimulation of human A431 carcinoma cells with EGF induces the rapid formation of ezrin oligomers in vivo, thus identifying a signal transduction pathway involving ezrin oligomerization coincident with microvillus assembly. In addition to time course studies, experiments with tyrosine kinase and tyrosine phosphatase inhibitors revealed a correlation between the phosphorylation of ezrin on tyrosine and the onset of oligomer formation, consistent with the possibility that phosphorylation of ezrin might be required for the generation of stable oligomers. Based on these observations, a model for the assembly of cell surface structures is proposed.

The COOH-terminal domain of Myo2p, a yeast myosin V, has a direct role in secretory vesicle targeting.

MYO2 encodes a type V myosin heavy chain needed for the targeting of vacuoles and secretory vesicles to the growing bud of yeast. Here we describe new myo2 alleles containing conditional lethal mutations in the COOH-terminal tail domain. Within 5 min of shifting to the restrictive temperature, the polarized distribution of secretory vesicles is abolished without affecting the distribution of actin or the mutant Myo2p, showing that the tail has a direct role in vesicle targeting. We also show that the actin cable-dependent translocation of Myo2p to growth sites does not require secretory vesicle cargo. Although a fusion protein containing the Myo2p tail also concentrates at growth sites, this accumulation depends on the polarized delivery of secretory vesicles, implying that the Myo2p tail binds to secretory vesicles. Most of the new mutations alter a region of the Myo2p tail conserved with vertebrate myosin Vs but divergent from Myo4p, the myosin V involved in mRNA transport, and genetic data suggest that the tail interacts with Smy1p, a kinesin homologue, and Sec4p, a vesicle-associated Rab protein. The data support a model in which the Myo2p tail tethers secretory vesicles, and the motor transports them down polarized actin cables to the site of exocytosis.

Tropomyosin-containing actin cables direct the Myo2p-dependent polarized delivery of secretory vesicles in budding yeast.

The actin cytoskeleton in budding yeast consists of cortical patches and cables, both of which polarize toward regions of cell growth. Tropomyosin localizes specifically to actin cables and not cortical patches. Upon shifting cells with conditionally defective tropomyosin to restrictive temperatures, actin cables disappear within 1 min and both the unconventional class V myosin Myo2p and the secretory vesicle-associated Rab GTPase Sec4p depolarize rapidly. Bud growth ceases and the mother cell grows isotropically. When returned to permissive temperatures, tropomyosin-containing cables reform within 1 min in polarized arrays. Cable reassembly permits rapid enrichment of Myo2p at the focus of nascent cables as well as the Myo2p- dependent recruitment of Sec4p and the exocyst protein Sec8p, and the initiation of bud emergence. With the loss of actin cables, cortical patches slowly assume an isotropic distribution within the cell and will repolarize only after restoration of cables. Therefore, actin cables respond to polarity cues independently of the overall distribution of cortical patches and are able to directly target the Myo2p-dependent delivery of secretory vesicles and polarization of growth.

The protein-tyrosine kinase substrate, p81, is homologous to a chicken microvillar core protein.

p81, a protein-tyrosine kinase substrate previously identified in epidermal growth factor-treated A431 cells, is demonstrated to be homologous to ezrin, an 80-kD component of microvillar core proteins. p81 has been characterized using antiserum raised against purified chicken intestinal ezrin. p81, located by indirect immunofluorescent staining, is concentrated in surface projections of A431 cells such as microvilli and retraction fibers. None of the conditions of biochemical cell fractionation tested completely solubilizes p81; the insoluble p81 partitions as if associated with the cytoskeleton. The soluble form of p81 behaves as a monomer in all extraction procedures studied. EGF-stimulated phosphorylation of p81 does not appear to change its intracellular location. p81 exhibits a wide tissue distribution with highest levels of expression in small intestine, kidney, thymus, and lung. Intermediate levels are found in spleen, thymus, lymph nodes, and bone marrow, with low levels in brain, heart, and testes. p81 is undetectable in muscle and liver. In A431 cells, p81 is phosphorylated on serine and threonine residues. Upon EGF treatment, approximately 10% of p81 becomes phosphorylated on tyrosine, and the phosphorylation of threonine residues increases.