Histology,histochemistry and ultrastructure of pre-embryogenic cells determined for direct somatic embryogenesis in the palm tree Syagrus oleracea

Somatic embryogenesis in palm trees is, in general, a slow and highly complex process, with a predominance of the indirect route and, consequently, a lack of knowledge about the direct route. We present new knowledge related to the morphological, histochemical and ultrastructural aspects of the transition from somatic to embryogenic cells and direct formation of somatic embryos from mature zygotic embryos of Syagrus oleracea, a palm tree. The results support the general concept that 2,4-dichlorophenoxyacetic acid plays a critical role for the formation of somatic embryos of direct and multicellular origin. Seven days in medium with auxin were enough for the identification of embryogenic cells. These cells had a set of characteristics corresponding to totipotent stem cells. At 14 days on induction medium, nodular formations were observed in the distal region of inoculated embryos, which evolved into globular somatic embryos. At 120 days on induction medium, the quality of the somatic embryos was compromised. The dynamics of the mobilization of reserve compounds was also demonstrated, with emphasis on starch and protein as energy sources required for the embryogenic process. This study shows for the first time the anatomical and ultrastructural events involved in direct somatic embryogenesis in a palm tree and incites the scientific community to return to the discussion of classical concepts related to direct somatic embryogenesis, especially regarding the characteristics and location of determined pre-embryogenic cells.     

Dynamic actin remodeling during epithelial-mesenchymal transition depends on increased moesin expression

Remodeling of actin filaments is necessary for epithelial-mesenchymal transition (EMT); however, understanding of how this is regulated in real time is limited. We used an actin filament reporter and high-resolution live-cell imaging to analyze the regulated dynamics of actin filaments during transforming growth factor-β-induced EMT of mammary epithelial cells. Progressive changes in cell morphology were accompanied by reorganization of actin filaments from thin cortical bundles in epithelial cells to thick, parallel, contractile bundles that disassembled more slowly but remained dynamic in transdifferentiated cells. We show that efficient actin filament remodeling during EMT depends on increased expression of the ezrin/radixin/moesin (ERM) protein moesin. Cells suppressed for moesin expression by short hairpin RNA had fewer, thinner, and less stable actin bundles, incomplete morphological transition, and decreased invasive capacity. These cells also had less α-smooth muscle actin and phosphorylated myosin light chain in cortical patches, decreased abundance of the adhesion receptor CD44 at membrane protrusions, and attenuated autophosphorylation of focal adhesion kinase. Our findings suggest that increased moesin expression promotes EMT by regulating adhesion and contractile elements for changes in actin filament organization. We propose that the transciptional program driving EMT controls progressive remodeling of actin filament architectures.     

Reorganisation of peripheral actin filaments as a prelude to exocytosis

Evidence is presented, from studies on the adrenal chromaffin cell, that reorganisation of the cortical actin network is necessary to allow granules to reach exocytotic sites in stimulated cells. This reorganisation may involve changes in actin filament cross-linking, assembly and interactions with secretory granule and plasma membranes. The possibility is discussed that cytoskeletal elements including the membrane-binding proteins caldesmon, p70 and p36 may be involved in granule-plasmalemmal interactions immediately prior to exocytosis.     

Adenosine diphosphate ribosylation factor-GTPase-activating protein stimulates the transport of AUX1 endosome, which relies on actin cytoskeletal organization in rice root development

Polar auxin transport, which depends on polarized subcellular distribution of AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) influx carriers and PIN-FORMED (PIN) efflux carriers, mediates various processes of plant growth and development. Endosomal recycling of PIN1 is mediated by an adenosine diphosphate (ADP)ribosylation factor (ARF)-GTPase exchange factor protein, GNOM. However, the mediation of auxin influx carrier recycling is poorly understood. Here, we report that overexpression of OsAGAP, an ARF-GTPase-activating protein in rice, stimulates vesicle transport from the plasma membrane to the Golgi apparatus in protoplasts and transgenic plants and induces the accumulation of early endosomes and AUX1. AUX1 endosomes could partially colocalize with FM4-64 labeled early endosome after actin disruption. Furthermore, OsAGAP is involved in actin cytoskeletal organization, and its overexpression tends to reduce the thickness and bundling of actin filaments. Fluorescence recovery after photobleaching analysis revealed exocytosis of the AUX1 recycling endosome was not affected in the OsAGAP overexpression cells, and was only slightly promoted when the actin filaments were completely disrupted by Lat B. Thus, we propose that AUX1 accumulation in the OsAGAP overexpression and actin disrupted cells may be due to the fact that endocytosis of the auxin influx carrier AUX1 early endosome was greatly promoted by actin cytoskeleton disruption.     

The receptor tyrosine phosphatase Dlar and integrins organize actin filaments in the Drosophila follicular epithelium.

BACKGROUND: Regulation of actin structures is instrumental in maintaining proper cytoarchitecture in many tissues. In the follicular epithelium of Drosophila ovaries, a system of actin filaments is coordinated across the basal surface of cells encircling the oocyte. These filaments have been postulated to regulate oocyte elongation; however, the molecular components that control this cytoskeletal array are not yet understood. RESULTS: We find that the receptor tyrosine phosphatase (RPTP) Dlar and integrins are involved in organizing basal actin filaments in follicle cells. Mutations in Dlar and the common beta-integrin subunit mys cause a failure in oocyte elongation, which is correlated with a loss of proper actin filament organization. Immunolocalization shows that early in oogenesis Dlar is polarized to membranes where filaments terminate but becomes generally distributed late in development, at which time beta-integrin and Enabled specifically associate with actin filament terminals. Rescue experiments point to the early period of polar Dlar localization as critical for its function. Furthermore, clonal analysis shows that loss of Dlar or mys influences actin filament polarity in wild-type cells that surround mutant tissues, suggesting that communication between neighboring cells regulates cytoskeletal organization. Finally, we find that two integrin alpha subunits encoded by mew and if are required for proper oocyte elongation, implying that multiple components of the ECM are instructive in coordinating actin fiber polarity. CONCLUSIONS: Dlar cooperates with integrins to coordinate actin filaments at the basal surface of the follicular epithelium. To our knowledge, this is the first direct demonstration of an RPTP's influence on the actin cytoskeleton.     

Do phytotropins inhibit auxin efflux by impairing vesicle traffic?

Phytotropins such as 1-N-naphthylphthalamic acid (NPA) strongly inhibit auxin efflux, but the mechanism of this inhibition remains unknown. Auxin efflux is also strongly decreased by the vesicle trafficking inhibitor brefeldin A (BFA). Using suspension-cultured interphase cells of the BY-2 tobacco (Nicotiana tabacum L. cv Bright-Yellow 2) cell line, we compared the effects of NPA and BFA on auxin accumulation and on the arrangement of the cytoskeleton and endoplasmic reticulum (ER). The inhibition of auxin efflux (stimulation of net accumulation) by both NPA and BFA occurred rapidly with no measurable lag. NPA had no observable effect on the arrangement of microtubules, actin filaments, or ER. Thus, its inhibitory effect on auxin efflux was not mediated by perturbation of the cytoskeletal system and ER. BFA, however, caused substantial alterations to the arrangement of actin filaments and ER, including a characteristic accumulation of actin in the perinuclear cytoplasm. Even at saturating concentrations, NPA inhibited net auxin efflux far more effectively than did BFA. Therefore, a proportion of the NPA-sensitive auxin efflux carriers may be protected from the action of BFA. Maximum inhibition of auxin efflux occurred at concentrations of NPA substantially below those previously reported to be necessary to perturb vesicle trafficking. We found no evidence to support recent suggestions that the action of auxin transport inhibitors is mediated by a general inhibition of vesicle-mediated protein traffic to the plasma membrane.     

Arabidopsis actin depolymerizing factor4 modulates the stochastic dynamic behavior of actin filaments in the cortical array of epidermal cells

Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells.     

Depletion of intracellular potassium disrupts coated pits and reversibly inhibits cell polarization during fibroblast spreading

To learn more about the possible role of the coated pits endocytic pathway in cell adhesion, we studied attachment and spreading of fibroblasts whose coated pits were disrupted by depletion of intercellular potassium. Fibroblasts incubated in suspension in potassium-free medium lost 80% of their intracellular potassium within 10 min and showed disrupted coated pits based on fluorescence staining of clathrin. Potassium-depleted cells attached and spread on fibronectin-coated substrata over the same time course (15 min-2 h) as control cells. Unlike controls, however, potassium-depleted fibroblasts attained a radial morphology with circumferentially organized actin filament bundles and were unable to make the transition to a polarized morphology with stress fibers. In the radially spread fibroblasts, fibronectin receptors and vinculin colocalized in focal adhesion sites and appeared to be membrane insertion points for circumferentially arranged actin filament bundles, but these sites were much smaller than the focal adhesion plaques in polarized cells. The effects of potassium depletion on cell adhesion were reversible. Within 1 h after switching K(+)-depleted fibroblasts to medium containing KCl, cells developed a polarized morphology with actin stress fibers inserting into focal adhesion plaques. Coated pits also reformed on the cell surface during this time. Because formation of focal adhesion plaques preceded reappearance of clathrin-coated pits at the cell margins, it seems unlikely that coated pits play a direct role in adhesion plaque assembly. Polarization of fibroblasts upon addition of KCl was inhibited by ouabain showing that intracellular potassium was required for activity. Polarization also was inhibited when potassium-depleted cells were switched to potassium-containing medium under hypertonic or acidified conditions, both of which have been shown to inhibit receptor- mediated endocytosis. Our results suggest that the coated pit endocytic pathway is not required for initial attachment, spreading, and formation of focal adhesions by fibroblasts, but may play a role in cell polarization.     

Endothelial actin cytoskeleton remodeling during mechanostimulation with fluid shear stress

Fluid shear stress stimulation induces endothelial cells to elongate and align in the direction of applied flow. Using the complementary techniques of photoactivation of fluorescence and fluorescence recovery after photobleaching, we have characterized endothelial actin cytoskeleton dynamics during the alignment process in response to steady laminar fluid flow and have correlated these results to motility. Alignment requires 24 h of exposure to fluid flow, but the cells respond within minutes to flow and diminish their movement by 50%. Although movement slows, the actin filament turnover rate increases threefold and the percentage of total actin in the polymerized state decreases by 34%, accelerating actin filament remodeling in individual cells within a confluent endothelial monolayer subjected to flow to levels used by dispersed nonconfluent cells under static conditions for rapid movement. Temporally, the rapid decrease in filamentous actin shortly after flow stimulation is preceded by an increase in actin filament turnover, revealing that the earliest phase of the actin cytoskeletal response to shear stress is net cytoskeletal depolymerization. However, unlike static cells, in which cell motility correlates positively with the rate of filament turnover and negatively with the amount polymerized actin, the decoupling of enhanced motility from enhanced actin dynamics after shear stress stimulation supports the notion that actin remodeling under these conditions favors cytoskeletal remodeling for shape change over locomotion. Hours later, motility returned to pre-shear stress levels but actin remodeling remained highly dynamic in many cells after alignment, suggesting continual cell shape optimization. We conclude that shear stress initiates a cytoplasmic actin-remodeling response that is used for endothelial cell shape change instead of bulk cell translocation. atherosclerosis; cytoskeletal dynamics; endothelial cells; mechanotransduction     

Effects of myosin ATPase inhibitor 2,3-butanedione 2-monoxime on distributions of myosins, F-actin, microtubules, and cortical endoplasmic reticulum in maize root apices

2,3-Butanedione 2-monoxime (BDM) is a general inhibitor of myosin ATPases of eukaryotic cells, and its effects on animal and yeast cells are well described. Using immunofluorescence and electron microscopy, we have analyzed the impacts of BDM on distributions of plant myosins, actin filaments (AFs), microtubules (MTs), and cortical endoplasmic reticulum (ER) elements in various cell types of maize root apices. Treatment of growing maize roots with BDM altered the typical distribution patterns of unconventional plant myosin VIII and of putative maize homologue(s) of myosin II. This pharmacological agent also induced a broad range of impacts on AFs and on cortical ER elements associated with plasmodesmata and pit fields. BDM-mediated effects on the actomyosin cytoskeleton were especially pronounced in cells of the root transition zone. Additionally, BDM elicited distinct reactions in the MT cytoskeleton; endoplasmic MTs vanished in all cells of the transition zone and cortical MTs assembled in increased amounts preferentially at plasmodesmata and pit-fields. Our data indicate that AFs and MTs interact together via BDM-sensitive plant myosins, which can be considered as putative integrators of the plant cytoskeleton. Morphometric analysis revealed that cell growth was prominently inhibited in the transition zone and the apical part, but not the central part, of the elongation region. Obviously, myosin-based contractility of the actin cytoskeleton is essential for the developmental progression of root cells through the transition zone.     

Detection of actin assembly by fluorescence energy transfer

Fluorescence energy transfer was used to measure the assembly and disassembly of actin filaments. Actin was labeled at cysteine 373 with an energy donor (5-iodoacetamidofluorescein) or an energy acceptor (tetramethylrhodamine iodoacetamide or eosin iodoacetamide). Donor- labeled actin and acceptor-labeled actin were coassembled. The dependence of the transfer efficiency on the mole fraction of acceptor- labeled actin showed that the radial coordinate of the label at cysteine 373 is approximately 35 A, which means that this site is located near the outer surface of the filament. The distance between a donor and the closest acceptor in such a filament is 58 A. The increase in fluorescence after the mixing of actin filaments containing both donor and acceptor with unlabeled filaments showed that there is a slow continuous exchange of actin units. The rate of exchange was markedly accelerated when the filaments were sonicated. The rapid loss of energy transfer caused by mechanical shear probably resulted from an increase in the number of filament ends, which in turn accelerated the exchange of monomeric actin units. Energy transfer promises to be a valuable tool in characterizing the assembly and dynamics of actin and other cytoskeletal and contractile proteins in vitro and in intact cells.     

Cytoskeletal organization affects cellular responses to cytochalasins: comparison of a normal line and its transformant

The relationships between cytoskeletal network organization and cellular response to cytochalasin D (CD) in a normal rat fibroblast cell line (Hmf-n) and its spontaneous transformant (tHmf-e), with markedly different cytoskeletal phenotypes, were compared (using immunofluorescence, electron microscopy, and DNAse I assay for actin content). Hmf-n have prominent, polar stress fiber (SF) arrays terminating in vinculin adhesion plaques whereas tHmf-e, which are apolar, epithelioid cells with dense plasma membrane-associated actin networks, lack SF and adhesion plaques. Hmf-n exposed to CD become markedly retracted and dendritic, SF-derived actin aggregates form large endoplasmic masses, and discrete tabular aggregates at the distal ends of retraction processes. Prolonged exposure leads to recession of process, cellular rounding, and development of large cystic vacuoles. tHmf-e cells exposed to similar doses of CD display a diagnostically different response; retraction is less drastic, cells retain broad processes containing scattered actin aggregates in discrete foci often associated with plasma membrane, large tabular aggregates are never found and processes persist throughout long exposure, vacuolation is uncommon. The CD-induced microfilamentous aggregates in Hmf-n are composed of short, kinky filament fragments forming a felt-like skein, often aggregates contain a more ordered array of roughly parallel fragments, while those of tHmf-e are very short, kinky, randomly orientated filaments imparting a distinctly granular nature to the mass. Total actin content and the amount of actin associated with detergent-resistant cytoskeletons increase following CD exposure in both cell types. Throughout exposure to CD, the actin-associated contractile proteins tropomyosin, myosin, and alpha-actinin co-localize within the actin aggregates in both cell types. Fodrin, the protein linking cortical actin to membrane, co-localizes with actin aggregates in tHmf-e cells and most, but not all, such aggregates in Hmf-n cells, consistent with their stress fiber derivation. Vinculin is lost from the tabular aggregates at the distal ends of retraction processes in Hmf-n cells concomitant with the fragmentation and contraction of SF. The aborized processes in both cells types contain strikingly similar axial cores of bundled vimentin filaments associated with passively compressed microtubules. The characteristic CD-induced distribution of actin filament aggregates and redistribution of vimentin in these cell types also occur when cells are allowed to respread from the rounded state in the presence of CD.     

Action of cytochalasin D on cytoskeletal networks

Extraction of SC-1 cells (African green monkey kidney) with the detergent Triton X-100 in combination with stereo high-voltage electron microscopy of whole mount preparations has been used as an approach to determine the mode of action of cytochalasin D on cells. The cytoskeleton of extracted BSC-1 cells consists of substrate-associated filament bundles (stress fibers) and a highly cross-linked network of four major filament types extending throughout the cell body; 10-nm filaments, actin microfilaments, microtubules, and 2- to 3-nm filaments. Actin filaments and 2- to 3-nm filaments form numerous end- to-side contacts with other cytoskeletal filaments. Cytochalasin D treatment severely disrupts network organization, increases the number of actin filament ends, and leads to the formation of filamentous aggregates or foci composed mainly of actin filaments. Metabolic inhibitors prevent filament redistribution, foci formation, and cell arborization, but not disorganization of the three-dimensional filament network. In cells first extracted and then treated with cytochalasin D, network organization is disrupted, and the number of free filament ends is increased. Supernates of preparations treated in this way contain both short actin filaments and network fragments (i.e., actin filaments in end-to-side contact with other actin filaments). It is proposed that the dramatic effects of cytochalasin D on cells result from both a direct interaction of the drug with the actin filament component of cytoskeletal networks and a secondary cellular response. The former leads to an immediate disruption of the ordered cytoskeletal network that appears to involve breaking of actin filaments, rather than inhibition of actin filament-filament interactions (i.e., disruption of end-to-side contacts). The latter engages network fragments in an energy-dependent (contractile) event that leads to the formation of filament foci.     

Astrocyte process growth induction by actin breakdown

cAMP analogues such as dibutyryl cAMP (dBcAMP) have been shown to induce the formation of processes in cultured primary astrocytes. We observe that the processes form by elongation as well as the previously reported retraction of cytoplasm around cytoskeletal elements. The most prominent cytoskeletal change that occurs in response to dBcAMP is a rearrangement of actin filaments characterized by a loss of cortical F-actin staining and the appearance of actin filament staining at the tips of the processes. If cortical actin filaments are disrupted with dihydrocytochalasin B, processes form that are similar to those induced by dBcAMP suggesting that the disruption of the cortical actin network is the pivotal step in process formation. Reorganization of the actin filament network in response to cAMP is accompanied by a decrease in phosphate incorporation into the regulatory light chain of myosin (MLC). Two selective inhibitors of MLC kinase (MLCK), ML-9 and KT5926, as well as a calmodulin antagonist (W7), which would also inhibit MLCK activation, all induce astrocytic process growth implicating MLCK as a control point in process initiation. We also found that dBcAMP and ML-9 both cause a decrease in the phosphate content of actin depolymerizing factor, suggesting that this protein and myosin light chain are the effectors of actin cytoskeleton reorganization and process growth.     

Smooth muscle length adaptation and actin filament length: a network model of the cytoskeletal dysregulation

Length adaptation of the airway smooth muscle cell is attributable to cytoskeletal remodeling. It has been proposed that dysregulated actin filaments may become longer in asthma, and that such elongation would prevent a parallel-to-series transition of contractile units, thus precluding the well-known beneficial effects of deep inspirations and tidal breathing. To test the potential effect that actin filament elongation could have in overall muscle mechanics, we present an extremely simple model. The cytoskeleton is represented as a 2-D network of links (contractile filaments) connecting nodes (adhesion plaques). Such a network evolves in discrete time steps by forming and dissolving links in a stochastic fashion. Links are formed by idealized contractile units whose properties are either those from normal or elongated actin filaments. Oscillations were then imposed on the network to evaluate both the effects of breathing and length adaptation. In response to length oscillation, a network with longer actin filaments showed smaller decreases of force, smaller increases in compliance, and higher shortening velocities. Taken together, these changes correspond to a network that is refractory to the effects of breathing and therefore approximates an asthmatic scenario. Thus, an extremely simple model seems to capture some relatively complex mechanics of airway smooth muscle, supporting the idea that dysregulation of actin filament length may contribute to excessive airway narrowing.     

ORIGIN AND DEVELOPMENT OF POLLEN EMBRYOIDS AND POLLEN CALLUSES IN CULTURED ANTHER SEGMENTS OF HYOSCYAMUS NIGER (HENBANE)

The dedifferentiation of pollen grains of Hyoscyamus niger (henbane) into embryoids and calluses was examined by culturing identical segments of the same anther in a mineral salt-sucrose basal medium and in the basal medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid, respectively. Addition of auxin enhanced anther efficiency but did not affect the number of embryogenic pollen grains of an anther segment transformed into calluses. In anther segments cultured in the basal medium, the organogenetic part of the pollen embryoid was formed by the division of the generative cell alone, or by the division of both generative and vegetative cells. More or less similar pathways were followed by pollen grains of anther segments cultured in a medium containing auxin to form calluses. Culture of anther segments in a medium containing a high concentration of auxin (50.0 mg/l) led to a significant reduction in the yield of calluses which were formed almost entirely by the division of both generative and vegetative cells. The bearing of these observations on the role of auxin in determining the pathway of differentiation of embryogenic pollen grains in cultured anther segments is considered. The appearance of embryogenic pollen grains in close proximity to the tapetum as seen in longitudinal sections of cultured anther segments has suggested a role for a gradient of tapetal factors in embryogenic induction.     

In Vitro Reconstitution of Cortical Actin Assembly Sites in Budding Yeast

We have developed a biochemical approach for identifying the components of cortical actin assembly sites in polarized yeast cells, based on a permeabilized cell assay that we established for actin assembly in vitro. Previous analysis indicated that an activity associated with the cell cortex promotes actin polymerization in the bud. After inactivation by a chemical treatment, this activity can be reconstituted back to the permeabilized cells from a cytoplasmic extract. Fractionation of the extract revealed that the reconstitution depends on two sequentially acting protein factors. Bee1, a cortical actin cytoskeletal protein with sequence homology to Wiskott-Aldrich syndrome protein, is required for the first step of the reconstitution. This finding, together with the severe defects in actin organization associated with the bee1 null mutation, indicates that Bee1 protein plays a direct role in controlling actin polymerization at the cell cortex. The factor that acts in the second step of the reconstitution has been identified by conventional chromatography. It is composed of a novel protein, Pca1. Sequence analysis suggests that Pca1 has the potential to interact with SH3 domain-containing proteins and phospholipids.     

Identification of a potential structural marker for embryogenic competency in the Brassica napus spp. oleifera embryogenic tissue

The Brassica napus secondary embryogenesis system requires no exogenous growth regulator to stimulate embryo development. It is stable embryogenically over a long period of culture and has a distinct pre-embryogenic stage. This system was used to investigate the morphological and cellular changes occurring in the embryogenic tissue compared to non-embryogenic tissue using various microscopy techniques. A unique ultrastructural feature designated the extracellular matrix (ECM) was observed on the surface of pre-embryogenic embryoids but not on the non-embryogenic individuals. The ECM layer was found to be dominant in the pre-embryogenic stage and reduced to fragments during embryo growth and development in mature embryogenic tissue. This is a novel aspect of the phenotype previously unreported in the Brassica system. This structure might be linked to acquisition of embryogenic competence.     

The human WASP-interacting protein, WIP, activates the cell polarity pathway in yeast.

WIP, the Wiskott-Aldrich syndrome protein-interacting protein, is a human protein involved in actin polymerization and redistribution in lymphoid cells. The mechanism by which WIP reorganizes actin cytoskeleton is unknown. WIP is similar to yeast verprolin, an actin- and myosin-interacting protein required for polarized morphogenesis. To determine whether WIP and verprolin are functional homologues, we analyzed the function of WIP in yeast. WIP suppresses the growth defects of VRP1 missense and null mutations as well as the defects in cytoskeletal organization and endocytosis observed in vrp1-1 cells. The ability of WIP to replace verprolin is dependent on its WH2 actin binding domain and a putative profilin binding domain. Immunofluorescence localization of WIP in yeast cells reveals a pattern consistent with its function at the cortical sites of growth. Thus, like verprolin, WIP functions in yeast to link the polarity development pathway and the actin cytoskeleton to generate cytoskeletal asymmetry. A role for WIP in cell polarity provides a framework for unifying, under a common paradigm, distinct molecular defects associated with immunodeficiencies like Wiskott-Aldrich syndrome.