Comparative study of the steady-state subcellular distribution of lysosome-associated membrane glycoprotein-2 (LAMP-2) isoforms with GYXXΦ-type tyrosine-based motifs that interact differently with four adaptor protein (AP) complexes.

Lysosome-associated membrane protein-1 and -2 (LAMP-1 and LAMP-2, respectively) are type I transmembrane proteins. LAMP-2 comprises three splice isoforms (LAMP-2A, -B and-C) with different cytoplasmic tails (CTs). These three CTs possess different tyrosine-based motifs (GYXXΦ, where Φ is a bulky hydrophobic amino acid) at their C-termini. Interactions between tyrosine-based motifs and µ-subunits of four tetrameric adaptor protein (AP) complexes are necessary for their vesicular transport to lysosomes. Little is known about how the interaction strengths of these tyrosine motifs with µ-subunits affect the localization of isoforms to lysosomes. The interactions were first investigated using a yeast two-hybrid system to address this question. LAMP-2A-CT interacted with all four µ-subunits (µ1, µ2, µ3A and µ4 of AP-1, AP-2, AP-3 and AP-4, respectively). The interaction with µ3A was more robust than that with other µ-subunits. LAMP-2B-CT interacted exclusively and moderately with µ3A. LAMP-2C-CT did not detectably interact with any of the four µ-subunits. Immunofluorescence microscopy showed that all isoforms were localized in late endosomes and lysosomes. LAMP-2C was present in the plasma membrane and early endosomes; however, LAMP-2A and -2B were barely detectable in these organelles. In cell fractionation, LAMP-2A was the most abundant in the dense lysosomes, whereas LAMP-2C was significantly present in the low-density fraction containing the plasma membrane and early endosomes, in addition to the dense lysosomes. LAMP-2B considerably existed in the low-density late endosomal fraction. These data strongly suggest that the LAMP-2 isoforms are distributed differently in endocytic organelles depending on their interaction strengths with AP-3.

Regulation of Hook1-mediated endosomal sorting of clathrin-independent cargo by γ-taxilin.

In clathrin-independent endocytosis, Hook1, a microtubule- and cargo-tethering protein, participates in sorting of cargo proteins such as CD98 (encoded by SLC3A2) and CD147 (encoded by BSG) into recycling endosomes. However, the molecular mechanism that regulates Hook1-mediated endosomal sorting is not fully understood. In the present study, we found that γ-taxilin is a novel regulator of Hook1-mediated endosomal sorting. γ-Taxilin depletion promoted both CD98-positive tubular formation and CD98 recycling. Conversely, overexpression of γ-taxilin inhibited the CD98-positive tubular formation. Depletion of Hook1, or Rab10 or Rab22a (which are both involved in Hook1-mediated endosomal sorting), attenuated the effect of γ-taxilin depletion on the CD98-positive tubular formation. γ-Taxilin depletion promoted CD147-mediated spreading of HeLa cells, suggesting that γ-taxilin might be a pivotal player in various cellular functions in which Hook1-mediated cargo proteins are involved. γ-Taxilin bound to the C-terminal region of Hook1 and inhibited its interaction with CD98; the latter interaction is necessary for sorting CD98. We suggest that γ-taxilin negatively regulates the sorting of Hook1-mediated cargo proteins into recycling endosomes by interfering with the interactions between Hook1 and the cargo proteins.

Involvement of lysosomal integral membrane protein-2 in the activation of autophagy.

Lysosomal integral membrane protein-2 (LIMP-2) is a type III transmembrane protein that is highly glycosylated and mainly localized to the lysosomal membrane. The diverse functions of LIMP-2 are currently being uncovered; however, its participation in macroautophagy, usually described as autophagy, has not yet been well-investigated. To determine the possible involvement of LIMP-2 in autophagic activity, we examined the intracellular amount of microtubule-associated protein 1 light chain 3 (LC3)-II, which is well-correlated with autophagosome levels, in exogenous rat LIMP-2-expressing COS7 and HEK293 cells. Transient or stable expression of LIMP-2-myc significantly increased the levels of LC3-II. Conversely, knockdown of LIMP-2 decreased the LC3-II levels in NIH3T3 cells. Furthermore, approaches using lysosomal protease inhibitors and mCherry-GFP-LC3 fluorescence suggested that exogenous expression of LIMP-2 increased the biogenesis of autophagosomes rather than decreased the lysosomal turnover of LC3-II. Considering the results of the biochemical assay and the quantitative fluorescence assay together, it is suggested that LIMP-2 has a possible involvement in autophagic activity, especially autophagosome biogenesis.

The Major Lysosomal Membrane Proteins LAMP-1 and LAMP-2 Participate in Differentiation of C2C12 Myoblasts.

Lysosomes are organelles that play a crucial role in the degradation of endocytosed molecules, phagocytosed macromolecules and autophagic substrates. The membrane of lysosomes contains several highly glycosylated membrane proteins, and lysosome-associated membrane protein (LAMP)-1 and LAMP-2 account for a major portion of the lysosomal membrane glycoproteins. Although it is well known that LAMP-2 deficiency causes Danon disease, which is characterized by cardiomyopathy, myopathy and mental retardation, the roles of lysosomal membrane proteins including LAMP-1 and LAMP-2 in myogenesis are not fully understood. In this study, to understand the role of LAMP proteins in the course of differentiation of myoblasts into myotubes, we used C2C12 myoblasts and found that the protein and mRNA levels of LAMP-1 and LAMP-2 were increased in the course of differentiation of C2C12 myoblasts into myotubes. Then, we investigated the effects of LAMP-1 or LAMP-2 knockdown on C2C12 myotube formation, and found that LAMP-1 or LAMP-2 depletion impaired the differentiation of C2C12 myoblasts and reduced the diameter of C2C12 myotubes. LAMP-2 knockdown more severely impaired C2C12 myotube formation compared with LAMP-1 knockdown, and knockdown of LAMP-1 did not exacerbate the suppressive effects of LAMP-2 knockdown on C2C12 myotube formation. In addition, knockdown of LAMP-1 or LAMP-2 decreased the expression levels of myogenic regulatory factors, MyoD and myogenin. These results demonstrate that both LAMP-1 and LAMP-2 are involved in C2C12 myotube formation and LAMP-2 may contribute dominantly to it.

γ-Taxilin temporally regulates centrosome disjunction in a Nek2A-dependent manner.

Never in mitosis A-related kinase 2A (Nek2A), a centrosomal serine/threonine kinase, is involved in mitotic progression by regulating the centrosome cycle. Particularly, Nek2A is necessary for dissolution of the intercentriole linkage between the duplicated centrosomes prior to mitosis. Nek2A activity roughly parallels its cell cycle-dependent expression levels, but the precise mechanism regulating its activity remains unclear. In this study, we found that γ-taxilin co-localized with Nek2A at the centrosome during interphase and interacted with Nek2A in yeast two-hybrid and pull-down assays and that γ-taxilin regulated centrosome disjunction in a Nek2A-dependent manner. γ-Taxilin depletion increased the number of cells with striking splitting of centrosomes. The precocious splitting of centrosomes induced by γ-taxilin depletion was attenuated by Nek2A depletion, suggesting that γ-taxilin depletion induces the Nek2A-mediated dissolution of the intercentriole linkage between the duplicated centrosomes nevertheless mitosis does not yet begin. Taken together with the result that γ-taxilin protein expression levels were decreased at the onset of mitosis, we propose that γ-taxilin participates in Nek2A-mediated centrosome disjunction as a negative regulator through its interaction with Nek2A.

ß-Taxilin participates in differentiation of C2C12 myoblasts into myotubes.

Myogenesis is required for the development of skeletal muscle. Accumulating evidence indicates that the expression of several genes are upregulated during myogenesis and these genes play pivotal roles in myogenesis. However, the molecular mechanism underlying myogenesis is not fully understood. In this study, we found that ß-taxilin, which is specifically expressed in the skeletal muscle and heart tissues, was progressively expressed during differentiation of C2C12 myoblasts into myotubes, prompting us to investigate the role of ß-taxilin in myogenesis. In C2C12 cells, knockdown of ß-taxilin impaired the fusion of myoblasts into myotubes, and decreased the diameter of myotubes. We also found that ß-taxilin interacted with dysbindin, a coiled-coil-containing protein. Knockdown of dysbindin conversely promoted the fusion of myoblasts into myotubes and increased the diameter of myotubes in C2C12 cells. Furthermore, knockdown of dysbindin attenuated the inhibitory effect of ß-taxilin depletion on myotube formation of C2C12 cells. These results demonstrate that ß-taxilin participates in myogenesis through suppressing the function of dysbindin to inhibit the differentiation of C2C12 myoblasts into myotubes.

TSG101, a tumor susceptibility gene, bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression.

BACKGROUND: Tumor susceptibility gene 101 (TSG101) was initially identified in fibroblasts as a tumor suppressor gene but subsequent studies show that TSG101 also functions as a tumor-enhancing gene in some epithelial tumor cells. Although previous studies have unraveled diverse biological functions of TSG101, the precise mechanism by which TSG101 is involved in carcinogenesis and tumor progression in a bidirectional and multifaceted manner remains unclear. METHODS: To reveal the mechanism underlying bidirectional modulation of cell invasion by TSG101, we used RNA interference to examine whether TSG101 depletion bidirectionally modulated matrix metalloproteinase (MMP)-9 expression in different cell types. RESULTS: TSG101 depletion promoted cell invasion of HT1080 cells but contrarily reduced cell invasion of HeLaS3 cells. In HT1080 cells, TSG101 depletion increased both baseline and phorbol 12-myristate 13-acetate (PMA)-induced MMP-9 secretion through enhancing MMP-9 mRNA expression, but did not affect the expression or activation of MMP-2. In contrast, TSG101 depletion decreased PMA-induced MMP-9 secretion through reducing MMP-9 mRNA expression in HeLaS3 cells. TSG101 depletion had little impact on the signaling pathways required for the activation of transcription of MMP-9 or MMP-9 mRNA stability in either cell line. CONCLUSION: TSG101 bidirectionally modulates cell invasion through regulating MMP-9 mRNA expression in different cell types. Our results provide a mechanistic context for the role of TSG101 in cell invasion as a multifaceted gene.

α-Taxilin interacts with sorting nexin 4 and participates in the recycling pathway of transferrin receptor.

Membrane traffic plays a crucial role in delivering proteins and lipids to their intracellular destinations. We previously identified α-taxilin as a binding partner of the syntaxin family, which is involved in intracellular vesicle traffic. α-Taxilin is overexpressed in tumor tissues and interacts with polymerized tubulin, but the precise function of α-taxilin remains unclear. Receptor proteins on the plasma membrane are internalized, delivered to early endosomes and then either sorted to the lysosome for degradation or recycled back to the plasma membrane. In this study, we found that knockdown of α-taxilin induced the lysosomal degradation of transferrin receptor (TfnR), a well-known receptor which is generally recycled back to the plasma membrane after internalization, and impeded the recycling of transferrin. α-Taxilin was immunoprecipitated with sorting nexin 4 (SNX4), which is involved in the recycling of TfnR. Furthermore, knockdown of α-taxilin decreased the number and length of SNX4-positive tubular structures. We report for the first time that α-taxilin interacts with SNX4 and plays a role in the recycling pathway of TfnR.

Expression of α-taxilin in the murine gastrointestinal tract: potential implication in cell proliferation.

α-Taxilin, a binding partner of the syntaxin family, is a candidate tumor marker. To gain insight into the physiological role of α-taxilin in normal tissues, we examined α-taxilin expression by Western blot and performed immunochemical analysis in the murine gastrointestinal tract where cell renewal vigorously occurs. α-Taxilin was expressed in the majority of the gastrointestinal tract and was prominently expressed in epithelial cells positive for Ki-67, a marker of actively proliferating cells. In the small intestine, α-taxilin was expressed in transient-amplifying cells and crypt base columnar cells intercalated among Paneth cells. In the corpus and antrum of the stomach, α-taxilin was expressed in cells localized in the lower pit and at the gland, respectively, but not in parietal or zymogenic cells. During development of the small intestine, α-taxilin was expressed in Ki-67-positive regions. Inhibition of cell proliferation by suppression of the Notch cascade using a γ-secretase inhibitor led to a decrease in α-taxilin- and Ki-67-positive cells in the stomach. These results suggest that expression of α-taxilin is regulated in parallel with cell proliferation in the murine gastrointestinal tract.

The apical and basolateral secretion of Wnt11 and Wnt3a in polarized epithelial cells is regulated by different mechanisms.

Wnts are glycan- and lipid-modified morphogens that are important for cellular responses, but how Wnts are secreted in polarized epithelial cells remains unclear. Although Wntless (Wls) has been shown to interact with Wnts and support their secretion, the role of Wls in the sorting of Wnts to the final destination in polarized epithelial cells have not been clarified. Glycosylation was shown to be important for the sorting of some transmembrane and secreted proteins, but glycan profiles and their roles in the polarized secretion of Wnts has not yet been demonstrated. Here we show the apical and basolateral secretion of Wnts is regulated by different mechanisms. Wnt11 and Wnt3a were secreted apically and basolaterally, respectively, in polarized epithelial cells. Wls was localized to the basolateral membrane. Mass-spectrometric analyses revealed that Wnt11 is modified with complex/hybrid(Asn40)-, high-mannose(Asn90)- and high-mannose/hybrid(Asn300)-type glycans and that Wnt3a is modified with two high-mannose-type glycans (Asn87 and Asn298). Glycosylation processing at Asn40 and galectin-3 were required for the apical secretion of Wnt11, whereas clathrin and adaptor protein-1 were required for the basolateral secretion of Wnt3a. By the fusion of the Asn40 glycosylation site of Wnt11, Wnt3a was secreted apically. The recycling of Wls by AP-2 was necessary for the basolateral secretion of Wnt3a but not for the apical secretion of Wnt11. These results suggest that Wls has different roles in the polarized secretion of Wnt11 and Wnt3a and that glycosylation processing of Wnts decides their secretory routes.

Identification of a second amphipathic lipid-packing sensor-like motif that contributes to Gcs1p function in the early endosome-to-TGN pathway.

ArfGAPs, GTPase-activating proteins for Arf small GTPases, are involved in multiple steps of vesicle formation of various transport pathways. Amphipathic lipid-packing sensor (ALPS) motif was first identified in the C-terminal regions of ArfGAP1 and its yeast homologue Gcs1p as a region that adsorbs preferentially onto highly curved membranes by folding into an amphipathic α-helix (AH). We previously showed that Gcs1p functionally interacted with the phospholipid flippase Cdc50p-Drs2p in the early endosome-to-TGN retrieval pathway. In this study, we performed functional analyses of the C-terminal region of Gcs1p containing ALPS. Hydrophobic cluster analysis suggested that there is another potential AH-forming region downstream of ALPS in Gcs1p. Mutational analysis suggested that the ALPS motif is important for the Gcs1p function in the Golgi-to-ER retrograde pathway, whereas ALPS and the predicted AH region redundantly function in the post-Golgi pathways including the early endosome-to-TGN pathway. Liposome flotation assay indicated that this downstream region preferentially interacted with liposomes of smaller size. The region containing the ALPS motif was also required for the interaction with SNARE proteins including Snc1p and Tlg1p. These results suggest that ALPS and the predicted AH region are involved in the regulation and function of Gcs1p by interacting with membrane phospholipids and vesicle proteins.

Localization of glypican-4 in different membrane microdomains is involved in the regulation of Wnt signaling.

Glypicans are members of the heparan sulfate proteoglycans (HSPGs) and are involved in various growth factor signaling mechanisms. Although HSPGs affect the ß-catenin-dependent and -independent pathways of Wnt signaling, how they regulate distinct Wnt pathways is not clear. It has been suggested that the ß-catenin-dependent pathway is initiated through receptor endocytosis in lipid raft microdomains and the independent pathway is activated through receptor endocytosis in non-lipid raft microdomains. Here, evidence is presented that glypican-4 (GPC4) is localized to both membrane microdomains and that the localization affects its ability to regulate distinct Wnt pathways. GPC4 bound to Wnt3a and Wnt5a, which activate the ß-catenin-dependent and -independent pathways, respectively, and colocalized with Wnts on the cell surface. LRP6, one of Wnt3a coreceptors, was present in lipid raft microdomains, whereas Ror2, one of Wnt5a coreceptors, was localized to non-lipid raft microdomains. Expression of GPC4 enhanced the Wnt3a-dependent ß-catenin pathway and the Wnt5a-dependent ß-catenin-independent pathway, and knockdown of GPC4 suppressed both pathways. A GPC4 mutant that was localized to only non-lipid raft microdomains inhibited the ß-catenin-dependent pathway but enhanced the ß-catenin-independent pathway. These results suggest that GPC4 concentrates Wnt3a and Wnt5a to the vicinity of their specific receptors in different membrane microdomains, thereby regulating distinct Wnt signaling.

An anti-Wnt5a antibody suppresses metastasis of gastric cancer cells in vivo by inhibiting receptor-mediated endocytosis.

Wnt5a is a representative ligand that activates the ß-catenin-independent pathway in Wnt signaling. It was reported that the expression of Wnt5a in human gastric cancer is associated with aggressiveness and poor prognosis and that knockdown of Wnt5a reduces the ability of gastric cancer cells to metastasize in nude mice. Therefore, Wnt5a and its signaling pathway might be important targets for the therapy of gastric cancer. The aim of this study was to examine whether an anti-Wnt5a antibody affects metastasis of gastric cancer cells. One anti-Wnt5a polyclonal antibody (pAb5a-5) inhibited migration and invasion activities in vitro of gastric cancer cells with a high expression level of Wnt5a. Previously, it was shown that Wnt5a induces the internalization of receptors, which is required for Wnt5a-dependent activation of Rac1. pAb5a-5 inhibited Wnt5a-dependent internalization of receptors, thereby suppressed Wnt5a-dependent activation of Rac1. Laminin γ2 is one of target genes of Wnt5a signaling and Rac1 was involved in its expression. pAb5a-5 also inhibited Wnt5a-dependent expression of laminin γ2. In an experimental liver metastasis assay, gastric cancer cells were introduced into the spleens of nude mice. Laminin γ2 was required for liver metastatic ability of gastric cancer cells in vivo. Furthermore, intraperitoneal injection of pAb5a-5 inhibited the metastatic ability of gastric cancer cells. These results suggest that an anti-Wnt5a antibody was capable of suppressing Wnt5a-dependent internalization of receptors, resulting in the prevention of metastasis of gastric cancer cells by inhibiting the activation of Rac1 and the expression of laminin γ2.

LRP6 is internalized by Dkk1 to suppress its phosphorylation in the lipid raft and is recycled for reuse.

Beta-catenin-mediated Wnt signaling is crucial in animal development and tumor progression. The phosphorylation of low-density lipoprotein receptor-related protein 6 (LRP6), a single-span transmembrane Wnt receptor, plays a vital role in this signaling. Dickkopf1 (Dkk1) has been shown to inhibit the Wnt-beta-catenin pathway, but the mechanism is not yet clear. Here, evidence is presented that Wnt3a-dependent phosphorylation of LRP6 occurs in the lipid raft and that Dkk1 inhibits the formation of a complex between LRP6 and casein kinase 1gamma (CK1gamma) by removing LRP6 from the lipid raft. Dkk1 internalized LRP6 in a Rab5-dependent mechanism to prevent phosphorylation mediated by CK1gamma. The internalized LRP6 was recycled back in a Rab11-dependent mechanism to the cell-surface membrane, and the recycled LRP6 again responded to Wnt3a and Dkk1. Internalized Dkk1 was trafficked in a Rab7-mediated route and degraded in the lysosome. These results suggest that Dkk1 induces the internalization of LRP6 to suppress its phosphorylation in the lipid raft and allows subsequent recycling of LRP6 so that it can be reused for signaling.

Wnt5a regulates distinct signalling pathways by binding to Frizzled2.

Wnt5a regulates multiple intracellular signalling cascades, but how Wnt5a determines the specificity of these pathways is not well understood. This study examined whether the internalization of Wnt receptors affects the ability of Wnt5a to regulate its signalling pathways. Wnt5a activated Rac in the beta-catenin-independent pathway, and Frizzled2 (Fz2) and Ror1 or Ror2 were required for this action. Fz2 was internalized through a clathrin-mediated route in response to Wnt5a, and inhibition of clathrin-dependent internalization suppressed the ability of Wnt5a to activate Rac. As another action of Wnt5a, it inhibited Wnt3a-dependent lipoprotein receptor-related protein 6 (LRP6) phosphorylation and beta-catenin accumulation. Wnt3a-dependent phosphorylation of LRP6 was enhanced in Wnt5a knockout embryonic fibroblasts. Fz2 was also required for the Wnt3a-dependent accumulation of beta-catenin, and Wnt5a competed with Wnt3a for binding to Fz2 in vitro and in intact cells, thereby inhibiting the beta-catenin pathway. This inhibitory action of Wnt5a was not affected by the impairment of clathrin-dependent internalization. These results suggest that Wnt5a regulates distinct pathways through receptor internalization-dependent and -independent mechanisms.

Wnt3a and Dkk1 regulate distinct internalization pathways of LRP6 to tune the activation of beta-catenin signaling.

Wnt and Dickkopf (Dkk) regulate the stabilization of beta-catenin antagonistically in the Wnt signaling pathway; however, the molecular mechanism is not clear. In this study, we found that Wnt3a acts in parallel to induce the caveolin-dependent internalization of low-density-lipoprotein receptor-related protein 6 (LRP6), as well as the phosphorylation of LRP6 and the recruitment of Axin to LRP6 on the cell surface membrane. The phosphorylation and internalization of LRP6 occurred independently of one another, and both were necessary for the accumulation of beta-catenin. In contrast, Dkk1, which inhibits Wnt3a-dependent stabilization of beta-catenin, induced the internalization of LRP6 with clathrin. Knockdown of clathrin suppressed the Dkk1-dependent inhibition of the Wnt3a response. Furthermore, Dkk1 reduced the distribution of LRP6 in the lipid raft fraction where caveolin is associated. These results indicate that Wnt3a and Dkk1 shunt LRP6 to distinct internalization pathways in order to activate and inhibit the beta-catenin signaling, respectively.

The functional relationship between the Cdc50p-Drs2p putative aminophospholipid translocase and the Arf GAP Gcs1p in vesicle formation in the retrieval pathway from yeast early endosomes to the TGN.

Drs2p, the catalytic subunit of the Cdc50p-Drs2p putative aminophospholipid translocase, has been implicated in conjunction with the Arf1 signaling pathway in the formation of clathrin-coated vesicles (CCVs) from the TGN. Herein, we searched for Arf regulator genes whose mutations were synthetically lethal with cdc50Delta, and identified the Arf GAP gene GCS1. Most of the examined transport pathways in the Cdc50p-depleted gcs1Delta mutant were nearly normal, including endocytic transport to vacuoles, carboxypeptidase Y sorting, and the processing and secretion of invertase. In contrast, this mutant exhibited severe defects in the early endosome-to-TGN transport pathway; proteins that are transported via this pathway, such as the v-SNARE Snc1p, the t-SNARE Tlg1p, and the chitin synthase III subunit Chs3p, accumulated in TGN-independent aberrant membrane structures. We extended our analyses to clathrin adaptors, and found that Gga1p/Gga2p and AP-1 were also involved in this pathway. The Cdc50p-depleted gga1Delta gga2Delta mutant and the gcs1Delta apl2Delta (the beta1 subunit of AP-1) mutant exhibited growth defects and intracellular Snc1p-containing membranes accumulated in these cells. These results suggest that Cdc50p-Drs2p plays an important role in the Arf1p-mediated formation of CCVs for the retrieval pathway from early endosomes to the TGN.

Identification of novel mutations in ACT1 and SLA2 that suppress the actin-cable-overproducing phenotype caused by overexpression of a dominant active form of Bni1p in Saccharomyces cerevisiae.

A formin Bni1p nucleates actin to assemble actin cables, which guide the polarized transport of secretory vesicles in budding yeast. We identified mutations that suppressed both the lethality and the excessive actin cable formation caused by overexpression of a truncated Bni1p (BNI1DeltaN). Two recessive mutations, act1-301 in the actin gene and sla2-82 in a gene involved in cortical actin patch assembly, were identified. The isolation of sla2-82 was unexpected, because cortical actin patches are required for the internalization step of endocytosis. Both act1-301 and sla2-82 exhibited synthetic growth defects with bni1Delta. act1-301, which resulted in an E117K substitution, interacted genetically with mutations in profilin (PFY1) and BUD6, suggesting that Act1-301p was not fully functional in formin-mediated polymerization. sla2-82 also interacted genetically with genes involved in actin cable assembly. Some experiments, however, suggested that the effects of sla2-82 were caused by depletion of actin monomers, because the temperature-sensitive growth phenotype of the bni1Delta sla2-82 mutant was suppressed by increased expression of ACT1. The isolation of suppressors of the BNI1DeltaN phenotype may provide a useful system for identification of actin amino-acid residues that are important for formin-mediated actin polymerization and mutations that affect the availability of actin monomers.