Rho1p-Bni1p-Spa2p Interactions: Implication in Localization of Bni1p at the Bud Site and Regulation of the Actin Cytoskeleton in Saccharomyces cerevisiae

Rho1p is a yeast homolog of mammalian RhoA small GTP-binding protein. Rho1p is localized at the growth sites and required for bud formation. We have recently shown that Bni1p is a potential target of Rho1p and that Bni1p regulates reorganization of the actin cytoskeleton through interactions with profilin, an actin monomer-binding protein. Using the yeast two-hybrid screening system, we cloned a gene encoding a protein that interacted with Bni1p. This protein, Spa2p, was known to be localized at the bud tip and to be implicated in the establishment of cell polarity. The C-terminal 254 amino acid region of Spa2p, Spa2p(1213–1466), directly bound to a 162-amino acid region of Bni1p, Bni1p(826–987). Genetic analyses revealed that both the bni1 and spa2 mutations showed synthetic lethal interactions with mutations in the genes encoding components of the Pkc1p-mitogen-activated protein kinase pathway, in which Pkc1p is another target of Rho1p. Immunofluorescence microscopic analysis showed that Bni1p was localized at the bud tip in wild-type cells. However, in the spa2 mutant, Bni1p was not localized at the bud tip and instead localized diffusely in the cytoplasm. A mutant Bni1p, which lacked the Rho1p-binding region, also failed to be localized at the bud tip. These results indicate that both Rho1p and Spa2p are involved in the localization of Bni1p at the growth sites where Rho1p regulates reorganization of the actin cytoskeleton through Bni1p.

Sla2p Is Associated with the Yeast Cortical Actin Cytoskeleton via Redundant Localization Signals

Sla2p, also known as End4p and Mop2p, is the founding member of a widely conserved family of actin-binding proteins, a distinguishing feature of which is a C-terminal region homologous to the C terminus of talin. These proteins may function in actin cytoskeleton-mediated plasma membrane remodeling. A human homologue of Sla2p binds to huntingtin, the protein whose mutation results in Huntington’s disease. Here we establish by immunolocalization that Sla2p is a component of the yeast cortical actin cytoskeleton. Deletion analysis showed that Sla2p contains two separable regions, which can mediate association with the cortical actin cytoskeleton, and which can provide Sla2p function. One localization signal is actin based, whereas the other signal is independent of filamentous actin. Biochemical analysis showed that Sla2p exists as a dimer in vivo. Two-hybrid analysis revealed two intramolecular interactions mediated by coiled-coil domains. One of these interactions appears to underlie dimer formation. The other appears to contribute to the regulation of Sla2p distribution between the cytoplasm and plasma membrane. The data presented are used to develop a model for Sla2p regulation and interactions.

Distinct Actions and Cooperative Roles of ROCK and mDia in Rho Small G Protein-induced Reorganization of the Actin Cytoskeleton in Madin–Darby Canine Kidney Cells

Rho, a member of the Rho small G protein family, regulates the formation of stress fibers and focal adhesions in various types of cultured cells. We investigated here the actions of ROCK and mDia, both of which have been identified to be putative downstream target molecules of Rho, in Madin–Darby canine kidney cells. The dominant active mutant of RhoA induced the formation of parallel stress fibers and focal adhesions, whereas the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, and the dominant active mutant of mDia induced the weak formation of parallel stress fibers without affecting the formation of focal adhesions. In the presence of C3 ADP-ribosyltransferase for Rho, the dominant active mutant of ROCK induced the formation of stellate stress fibers and focal adhesions, whereas the dominant active mutant of mDia induced only the diffuse localization of actin filaments. These results indicate that ROCK and mDia show distinct actions in reorganization of the actin cytoskeleton. The dominant negative mutant of either ROCK or mDia inhibited the formation of stress fibers and focal adhesions, indicating that both ROCK and mDia are necessary for the formation of stress fibers and focal adhesions. Moreover, inactivation and reactivation of both ROCK and mDia were necessary for the 12-O-tetradecanoylphorbol-13-acetate–induced disassembly and reassembly, respectively, of stress fibers and focal adhesions. The morphologies of stress fibers and focal adhesions in the cells expressing both the dominant active mutants of ROCK and mDia were not identical to those induced by the dominant active mutant of Rho. These results indicate that at least ROCK and mDia cooperatively act as downstream target molecules of Rho in the Rho-induced reorganization of the actin cytoskeleton.

Regulation of the Actin Cytoskeleton by Thrombin in Human Endothelial Cells: Role of Rho Proteins in Endothelial Barrier Function

Endothelial barrier function is regulated at the cellular level by cytoskeletal-dependent anchoring and retracting forces. In the present study we have examined the signal transduction pathways underlying agonist-stimulated reorganization of the actin cytoskeleton in human umbilical vein endothelial cells. Receptor activation by thrombin, or the thrombin receptor (proteinase-activated receptor 1) agonist peptide, leads to an early increase in stress fiber formation followed by cortical actin accumulation and cell rounding. Selective inhibition of thrombin-stimulated signaling systems, including Gi/o (pertussis toxin sensitive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light chain kinase, effectively blocked thrombin-induced cell rounding and retraction. The contribution of Rho to these effects was analyzed by using bacterial toxins that either activate or inhibit the GTPase. Escherichia coli cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorganization of polymerized actin into stress fibers but failed to induce cell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C3 fused to the B fragment of diphtheria toxin caused loss of stress fibers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfection experiments using either constitutively active (V12) or dominant-interfering (N17) mutants. Expression of RacV12 mimicked the effect of thrombin on cell rounding, and RacN17 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier function and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.

Remodeling of the Actin Cytoskeleton Is Coordinately Regulated by Protein Kinase C and the ADP-Ribosylation Factor Nucleotide Exchange Factor ARNO

ARNO is a member of a family of guanine-nucleotide exchange factors with specificity for the ADP-ribosylation factor (ARF) GTPases. ARNO possesses a central catalytic domain with homology to yeast Sec7p and an adjacent C-terminal pleckstrin homology (PH) domain. We have previously shown that ARNO localizes to the plasma membrane in vivo and efficiently catalyzes ARF6 nucleotide exchange in vitro. In addition to a role in endocytosis, ARF6 has also been shown to regulate assembly of the actin cytoskeleton. To determine whether ARNO is an upstream regulator of ARF6 in vivo, we examined the distribution of actin in HeLa cells overexpressing ARNO. We found that, while expression of ARNO leads to disassembly of actin stress fibers, it does not result in obvious changes in cell morphology. However, treatment of ARNO transfectants with the PKC agonist phorbol 12-myristate 13-acetate results in the dramatic redistribution of ARNO, ARF6, and actin into membrane protrusions resembling lamellipodia. This process requires ARF activation, as actin rearrangement does not occur in cells expressing a catalytically inactive ARNO mutant. PKC phosphorylates ARNO at a site immediately C-terminal to its PH domain. However, mutation of this site had no effect on the ability of ARNO to regulate actin rearrangement, suggesting that phosphorylation of ARNO by PKC does not positively regulate its activity. Finally, we demonstrate that an ARNO mutant lacking the C-terminal PH domain no longer mediates cytoskeletal reorganization, indicating a role for this domain in appropriate membrane localization. Taken together, these data suggest that ARNO represents an important link between cell surface receptors, ARF6, and the actin cytoskeleton.

The Yeast GRD20 Gene Is Required for Protein Sorting in the trans-Golgi Network/Endosomal System and for Polarization of the Actin Cytoskeleton

The proper localization of resident membrane proteins to the trans-Golgi network (TGN) involves mechanisms for both TGN retention and retrieval from post-TGN compartments. In this study we report identification of a new gene, GRD20, involved in protein sorting in the TGN/endosomal system of Saccharomyces cerevisiae. A strain carrying a transposon insertion allele of GRD20 exhibited rapid vacuolar degradation of the resident TGN endoprotease Kex2p and aberrantly secreted ∼50% of the soluble vacuolar hydrolase carboxypeptidase Y. The Kex2p mislocalization and carboxypeptidase Y missorting phenotypes were exhibited rapidly after loss of Grd20p function in grd20 temperature-sensitive mutant strains, indicating that Grd20p plays a direct role in these processes. Surprisingly, little if any vacuolar degradation was observed for the TGN membrane proteins A-ALP and Vps10p, underscoring a difference in trafficking patterns for these proteins compared with that of Kex2p. A grd20 null mutant strain exhibited extremely slow growth and a defect in polarization of the actin cytoskeleton, and these two phenotypes were invariably linked in a collection of randomly mutagenized grd20 alleles. GRD20 encodes a hydrophilic protein that partially associates with the TGN. The discovery of GRD20 suggests a link between the cytoskeleton and function of the yeast TGN.

Phosphorylation of protein kinase N by phosphoinositide-dependent protein kinase-1 mediates insulin signals to the actin cytoskeleton

Growth factors such as insulin regulate phosphatidylinositol 3-kinase-dependent actin cytoskeleton rearrangement in many types of cells. However, the mechanism by which the insulin signal is transmitted to the actin cytoskeleton remains largely unknown. Yeast two-hybrid screening revealed that the phosphatidylinositol 3-kinase downstream effector phosphoinositide-dependent protein kinase-1 (PDK1) interacted with protein kinase N (PKN), a Rho-binding Ser/Thr protein kinase potentially implicated in a variety of cellular events, including phosphorylation of cytoskeletal components. PDK1 and PKN interacted in vitro and in intact cells, and this interaction was mediated by the kinase domain of PDK1 and the carboxyl terminus of PKN. In addition to a direct interaction, PDK1 also phosphorylated Thr774 in the activation loop and activated PKN. Insulin treatment or ectopic expression of the wild-type PDK1 or PKN, but not protein kinase Cζ, induced actin cytoskeleton reorganization and membrane ruffling in 3T3-L1 fibroblasts and Rat1 cells that stably express the insulin receptor (Rat1-IR). However, the insulin-stimulated actin cytoskeleton reorganization in Rat1-IR cells was prevented by expression of kinase-defective PDK1 or PDK1-phosphorylation site-mutated PKN. Thus, phosphorylation by PDK1 appears to be necessary for PKN to transduce signals from the insulin receptor to the actin cytoskeleton.

Role of Rac in controlling the actin cytoskeleton and chemotaxis in motile cells

We have used the chemotactic ability of Dictyostelium cells to examine the roles of Rho family members, known regulators of the assembly of F-actin, in cell movement. Wild-type cells polarize with a leading edge enriched in F-actin toward a chemoattractant. Overexpression of constitutively active Dictyostelium Rac1B61L or disruption of DdRacGAP1, which encodes a Dictyostelium Rac1 GAP, induces membrane ruffles enriched with actin filaments around the perimeter of the cell and increased levels of F-actin in resting cells. Whereas wild-type cells move linearly toward the cAMP source, Rac1B61L and Ddracgap1 null cells make many wrong turns and chemotaxis is inefficient, which presumably results from the unregulated activation of F-actin assembly and pseudopod extension. Cells expressing dominant-negative DdRac1B17N do not have a well-defined F-actin-rich leading edge and do not protrude pseudopodia, resulting in very poor cell motility. From these studies and assays examining chemoattractant-mediated F-actin assembly, we suggest DdRac1 regulates the basal levels of F-actin assembly, its dynamic reorganization in response to chemoattractants, and cellular polarity during chemotaxis.

Dopamine D2 and D3 receptors are linked to the actin cytoskeleton via interaction with filamin A

We have used a yeast two-hybrid approach to uncover protein interactions involving the D2-like subfamily of dopamine receptors. Using the third intracellular loop of the D2S and D3 dopamine receptors as bait to screen a human brain cDNA library, we identified filamin A (FLN-A) as a protein that interacts with both the D2 and D3 subtypes. The interaction with FLN-A was specific for the D2 and D3 receptors and was independently confirmed in pull-down and coimmunoprecipitation experiments. Deletion mapping localized the dopamine receptor–FLN-A interaction to the N-terminal segment of the D2 and D3 dopamine receptors and to repeat 19 of FLN-A. In cultures of dissociated rat striatum, FLN-A and D2 receptors colocalized throughout neuronal somata and processes as well as in astrocytes. Expression of D2 dopamine receptors in FLN-A-deficient M2 melanoma cells resulted in predominant intracellular localization of the D2 receptors, whereas in FLN-A-reconstituted cells, the D2 receptor was predominantly localized at the plasma membrane. These results suggest that FLN-A may be required for proper cell surface expression of the D2 dopamine receptors. Association of D2 and D3 dopamine receptors with FLN-A provides a mechanism whereby specific dopamine receptor subtypes may be functionally linked to downstream signaling components via the actin cytoskeleton.

Striking a balance: Modulation of the actin cytoskeleton by Salmonella

Salmonella spp. have evolved the ability to enter into cells that are normally nonphagocytic. The internalization process is the result of a remarkable interaction between the bacteria and the host cells. Immediately on contact, Salmonella delivers a number of bacterial effector proteins into the host cell cytosol through the function of a specialized organelle termed the type III secretion system. Initially, two of the delivered proteins, SopE and SopB, stimulate the small GTP-binding proteins Cdc42 and Rac. SopE is an exchange factor for these GTPases, and SopB is an inositol polyphosphate phosphatase. Stimulation of Cdc42 and Rac leads to marked actin cytoskeleton rearrangements, which are further enhanced by SipA, a Salmonella protein also delivered into the host cell by the type III secretion system. SipA lowers the critical concentration of G-actin, stabilizes F-actin at the site of bacterial entry, and increases the bundling activity of the host-cell protein T-plastin (fimbrin). The cellular responses stimulated by Salmonella are short-lived; therefore, immediately after bacterial entry, the cell regains its normal architecture. Remarkably, this process is mediated by SptP, another target of the type III secretion system. SptP exert its function by serving as a GTPase-activating protein for Cdc42 and Rac, turning these G proteins off after their stimulation by the bacterial effectors SopE and SopB. The balanced interaction of Salmonella with host cells constitutes a remarkable example of the sophisticated nature of a pathogen/host relationship shaped by evolution through a longstanding coexistence.

Ras Regulates the Polarity of the Yeast Actin Cytoskeleton through the Stress Response Pathway

Polarized growth in yeast requires cooperation between the polarized actin cytoskeleton and delivery of post-Golgi secretory vesicles. We have previously reported that loss of the major tropomyosin isoform, Tpm1p, results in cells sensitive to perturbations in cell polarity. To identify components that bridge these processes, we sought mutations with both a conditional defect in secretion and a partial defect in polarity. Thus, we set up a genetic screen for mutations that conferred a conditional growth defect, showed synthetic lethality with tpm1Δ, and simultaneously became denser at the restrictive temperature, a hallmark of secretion-defective cells. Of the 10 complementation groups recovered, the group with the largest number of independent isolates was functionally null alleles of RAS2. Consistent with this, ras2Δ and tpm1Δ are synthetically lethal at 35°C. We show that ras2Δ confers temperature-sensitive growth and temperature-dependent depolarization of the actin cytoskeleton. Furthermore, we show that at elevated temperatures ras2Δ cells are partially defective in endocytosis and show a delocalization of two key polarity markers, Myo2p and Cdc42p. However, the conditional enhanced density phenotype of ras2Δ cells is not a defect in secretion. All the phenotypes of ras2Δ cells can be fully suppressed by expression of yeast RAS1 or RAS2 genes, human Ha-ras, or the double disruption of the stress response genes msn2Δmsn4Δ. Although the best characterized pathway of Ras function in yeast involves activation of the cAMP-dependent protein kinase A pathway, activation of the protein kinase A pathway does not fully suppress the actin polarity defects, suggesting that there is an additional pathway from Ras2p to Msn2/4p. Thus, Ras2p regulates cytoskeletal polarity in yeast under conditions of mild temperature stress through the stress response pathway.

Movement of yeast cortical actin cytoskeleton visualized in vivo.

Fusion proteins between the green fluorescent protein (GFP) and the cytoskeleton proteins Act1p (actin), Sac6p (yeast fimbrin homolog), and Abp1p in budding yeast (Saccharomyces cerevisiae) localize to the cortical actin patches. The actin fusions could not function as the sole actin source in yeast, but fusions between the actin-binding proteins Abp1p and Sac6p complement fully the phenotypes associated with their gene deletions. Direct observation in vivo reveals that the actin cortical patches move. Movement of actin patches is constrained to the asymmetric distribution of the patches in growing cells, and this movement is greatly reduced when metabolic inhibitors such as sodium azide are added. Fusion protein-labeled patches are normally distributed during the yeast cell cycle and during mating. In vivo observation made possible the visualization of actin patches during sporulation as well.

H-ras, K-ras, and inner plasma membrane raft proteins operate in nanoclusters with differential dependence on the actin cytoskeleton

Plasma membrane compartmentalization imposes lateral segregation on membrane proteins that is important for regulating signal transduction. We use computational modeling of immunogold spatial point patterns on intact plasma membrane sheets to test different models of inner plasma membrane organization. We find compartmentalization at the nanoscale level but show that a classical raft model of preexisting stable domains into which lipid raft proteins partition is incompatible with the spatial point patterns generated by the immunogold labeling of a palmitoylated raft marker protein. Rather, approximate to 30% of the raft protein exists in cholesterol-dependent nanoclusters, with approximate to 70% distributed as monomers. The cluster/monomer ratio (number of proteins in clusters/number of proteins outside clusters) is independent of expression level. H-rasG12V and K-rasG12V proteins also operate in nanoclusters with fixed cluster/monomer ratios that are independent of expression level. Detailed calibration of the immunogold imaging protocol suggests that radii of raft and RasG12V protein nanoclusters may be as small as 11 and 6 nm, respectively, and shows that the nanoclusters contain small numbers (6.0-7.7) of proteins. Raft nanoclusters do not form if the actin cytoskeleton is disassembled. The formation of K-rasG12V but not H-rasG12V nanoclusters also is actin-dependent. K-rasG12V but not H-rasG12V signaling is abrogated by actin cytoskeleton disassembly, which shows that nanoclustering is critical for Ras function. These findings argue against stable preexisting domains on the inner plasma membrane in favor of dynamic actively regulated nanoclusters similar to those proposed for the outer plasma membrane. RasG12V nanoclusters may facilitate the assembly of essential signal transduction complexes.