Mapping actin surfaces required for functional interactions in vivo

An in vivo strategy to identify amino acids of actin required for functional interactions with actin-binding proteins was developed. This approach is based on the assumption that an actin mutation that specifically impairs the interaction with an actin-binding protein will cause a phenotype similar to a null mutation in the gene that encodes the actin-binding protein. 21 actin mutations were analyzed in budding yeast, and specific regions of actin subdomain 1 were implicated in the interaction with fimbrin, an actin filament-bundling protein. Mutations in this actin subdomain were shown to be, like a null allele of the yeast fimbrin gene (SAC6), lethal in combination with null mutations in the ABP1 and SLA2 genes, and viable in combination with a null mutation in the SLA1 gene. Biochemical experiments with act1-120 actin (E99A, E100A) verified a defect in the fimbrin-actin interaction. Genetic interactions between mutant alleles of the yeast actin gene and null alleles of the SAC6, ABP1, SLA1, and SLA2 genes also demonstrated that the effects of the 21 actin mutations are diverse and allowed four out of seven pseudo-wild-type actin alleles to be distinguished from the wild-type gene for the first time, providing evidence for functional redundancy between different surfaces of actin.     

Screens for Extragenic Mutations That Fail to Complement Act1 Alleles Identify Genes That Are Important for Actin Function in Saccharomyces Cerevisiae

Null mutations in SAC6 and ABP1, genes that encode actin-binding proteins, failed to complement the temperature-sensitive phenotype caused by a mutation in the ACT1 gene. To identify novel genes whose protein products interact with actin, mutations that fail to complement act1-1 or act1-4, two temperature-sensitive alleles of ACT1, were isolated. A total of 14 extragenic noncomplementing mutations and 12 new alleles of ACT1 were identified in two independent screens. The 14 extragenic noncomplementing mutations represent alleles of at least four different genes, ANC1, ANC2, ANC3 and ANC4 (Actin NonComplementing). Mutations in the ANC1 gene were shown to cause osmosensitivity and defects in actin organization; phenotypes that are similar to those caused by act1 mutations. We conclude that the ANC1 gene product plays an important role in actin cytoskeletal function. The 12 new alleles of ACT1 will be useful for further elucidation of the functions of actin in yeast.     

Unexpected combinations of null mutations in genes encoding the actin cytoskeleton are lethal in yeast.

To understand the role of the actin cytoskeleton in cell physiology, and how actin-binding proteins regulate the actin cytoskeleton in vivo, we and others previously identified actin-binding proteins in Saccharomyces cerevisiae and studied the effect of null mutations in the genes for these proteins. A null mutation of the actin gene (ACT1) is lethal, but null mutations in the tropomyosin (TPM1), fimbrin (SAC6), Abp1p (ABP1), and capping protein (CAP1 and CAP2) genes have relatively mild or no effects. We have now constructed double and triple mutants lacking 2 or 3 of these actin-binding proteins, and studied the effect of the combined mutations on cell growth, morphology, and organization of the actin cytoskeleton. Double mutants lacking fimbrin and either Abp1p or capping protein show negative synthetic effects on growth, in the most extreme case resulting in lethality. All other combinations of double mutations and the triple mutant lacking tropomyosin, Abp1p, and capping protein, are viable and their phenotypes are similar to or only slightly more severe than those of the single mutants. Therefore, the synthetic phenotypes are highly specific. We confirmed this specificity by overexpression of capping protein and Abp1p in strains lacking fimbrin. Thus, while overexpression of these proteins has deleterious effects on actin organization in wild-type strains, no synthetic phenotype was observed in the absence of fimbrin. We draw two important conclusions from these results. First, since mutations in pairs of actin-binding protein genes cause inviability, the actin cytoskeleton of yeast does not contain a high degree of redundancy. Second, the lack of structural and functional homology among these genetically redundant proteins (fimbrin and capping protein or Abp1p) indicates that they regulate the actin cytoskeleton by different mechanisms. Determination of the molecular basis for this surprising conclusion will provide unique insights into the essential mechanisms that regulate the actin cytoskeleton.     

Synthetic-lethal interactions identify two novel genes, SLA1 and SLA2, that control membrane cytoskeleton assembly in Saccharomyces cerevisiae

Abplp is a yeast cortical actin-binding protein that contains an SH3 domain similar to those found in signal transduction proteins that function at the membrane/cytoskeleton interface. Although no detectable phenotypes are associated with a disruption allele of ABP1, mutations that create a requirement for this protein have now been isolated in the previously identified gene SAC6 and in two new genes, SLA1 and SLA2. The SAC6 gene encodes yeast fimbrin, an actin filament-bundling protein. Null mutations in SLA1 and SLA2 cause temperature-sensitive growth defects. Sla1p contains three SH3 domains and is essential for the proper formation of the cortical actin cytoskeleton. The COOH terminus of Sla2p contains a 200 amino acid region with homology to the COOH terminus of talin, a membrane cytoskeletal protein which is a component of fibroblast focal adhesions. Sla2p is required for cellular morphogenesis and polarization of the cortical cytoskeleton. In addition, synthetic-lethal interactions were observed for double- mutants containing null alleles of SLA2 and SAC6. In total, the mutant phenotypes, sequences, and genetic interactions indicate that we have identified novel proteins that cooperate to control the dynamic cytoskeletal rearrangements that are required for the development of cell polarity in budding yeast.     

Dominant Suppressors of Yeast Actin Mutations That Are Reciprocally Suppressed

A gene whose product is likely to interact with yeast actin was identified by the isolation of pseudorevertants carrying dominant suppressors of the temperature-sensitive (Ts) act1-1 mutation. Of 30 independent revertants analyzed, 29 were found to carry extragenic suppressor mutations and of these, 24/24 tested were found to be linked to each other. This linkage group identifies a new gene SAC6, whose product, by several genetic criteria, is likely to interact intimately with actin. First, although act1-1 sac6 strains are temperature-independent (Ts+), 4/17 sac6 mutant alleles tested are Ts in an ACT1+ background. Moreover, four Ts+ pseudorevertants of these ACT1+ sac6 mutants carry suppressor mutations in ACT1; significantly, three of these are again Ts in a SAC6+ background, and are most likely new act1 mutant alleles. Thus, mutations in ACT1 and SAC6 can suppress each other's defects. Second, sac6 mutations can suppress the Ts defects of the act1-1 and act1-2, but not act1-4, mutations. This allele specificity indicates the sac6 mutations do not suppress by simply bypassing the function of actin at high temperature. Third, act1-4 sac6 strains have a growth defect greater than that due to either of the single mutations alone, again suggesting an interaction between the two proteins. The mutant sac6 gene was cloned on the basis of dominant suppression from an act1-1 sac6 mutant library, and was then mapped to chromosome IV, less than 2 cM from ARO1.     

Mutations That Enhance the Cap2 Null Mutant Phenotype in Saccharomyces Cerevisiae Affect the Actin Cytoskeleton, Morphogenesis and Pattern of Growth

Mutations conferring synthetic lethality in combination with null mutations in CAP2, the gene encoding the β subunit of capping protein of Saccharomyces cerevisiae, were obtained in a colony color assay. Monogenic inheritance was found for four mutations, which were attributed to three genetic loci. One mutation, sac6-69, is in the gene encoding fimbrin, another actin-binding protein, which was expected because null mutations in SAC6 and CAP2 are known to be synthetic-lethal. The other two loci were designated slc for synthetic lethality with cap2. These loci include the mutations slc1-66, slc1-87 and slc2-107. The slc mutations are semi-dominant, as shown by incomplete complementation in slc/SLC cap2/cap2 heterozygotes. The slc mutations and sac6-69 interact with each other, as shown by enhanced phenotypes in diheterozygotes. Moreover, the haploid slc2-107 sac6-69 double mutant is inviable. In a CAP2 background, the slc mutations lead to temperature and osmotic sensitivity. They alter the distribution of the actin cytoskeleton, including deficits in the presence of actin cables and the polarization of cortical actin patches. The slc mutations also lead to a pseudomycelial growth pattern. Together these results suggest that slc1 and slc2 encode components of the actin cytoskeleton in yeast and that the actin cytoskeleton can regulate the patterns of growth.     

Genetic Analysis to the Fimbrin-Actin Binding Interaction in Saccharomyces Cerevisiae

Yeast fimbrin is encoded by the SAC6 gene, mutations of which suppress temperature-sensitive mutations in the actin gene (ACT1). To examine the mechanism of suppression, we have sequenced 17 sac6 suppressor alleles, and found that they change nine different residues, all of which cluster in three regions of one of the two actin-binding domains of Sac6p. Two of these clusters occur in highly conserved regions (ABS1 and ABS3) that have been strongly implicated in the binding of related proteins to actin. The third cluster changes residues not previously implicated in the interaction with actin. As changes in any of nine different residues can suppress several different act1 alleles, it is likely that the suppressors restore the overall affinity, rather than specific lost interactions, between Sac6p and actin. Using mutagenesis, we have identified two mutations of the second actin-binding domain that can also suppress the act1 mutations of interest. This result suggests the two actin-binding domains of Sac6p interact with the same region of the actin molecule. However, differences in strength of suppression of temperature-sensitivity and sporulation indicate that the two actin-binding domains are distinct, and explain why second-domain mutations were not identified previously.     

The Yeast Gene, MDM20, Is Necessary for Mitochondrial Inheritance and Organization of the Actin Cytoskeleton

In Saccharomyces cerevisiae, the growing bud inherits a portion of the mitochondrial network from the mother cell soon after it emerges. Although this polarized transport of mitochondria is thought to require functions of the cytoskeleton, there are conflicting reports concerning the nature of the cytoskeletal element involved. Here we report the isolation of a yeast mutant, mdm20, in which both mitochondrial inheritance and actin cables (bundles of actin filaments) are disrupted. The MDM20 gene encodes a 93-kD polypeptide with no homology to other characterized proteins. Extra copies of TPM1, a gene encoding the actin filament–binding protein tropomyosin, suppress mitochondrial inheritance defects and partially restore actin cables in mdm20Δ cells. Synthetic lethality is also observed between mdm20 and tpm1 mutant strains. Overexpression of a second yeast tropomyosin, Tpm2p, rescues mutant phenotypes in the mdm20 strain to a lesser extent. Together, these results provide compelling evidence that mitochondrial inheritance in yeast is an actin-mediated process. MDM20 and TPM1 also exhibit the same pattern of genetic interactions; mutations in MDM20 are synthetically lethal with mutations in BEM2 and MYO2 but not SAC6. Although MDM20 and TPM1 are both required for the formation and/or stabilization of actin cables, mutations in these genes disrupt mitochondrial inheritance and nuclear segregation to different extents. Thus, Mdm20p and Tpm1p may act in vivo to establish molecular and functional heterogeneity of the actin cytoskeleton.     

Actin mutations that show suppression with fimbrin mutations identify a likely fimbrin-binding site on actin

Actin interacts with a large number of different proteins that modulate its assembly and mediate its functions. One such protein is the yeast actin-binding protein Sac6p, which is homologous to vertebrate fimbrin (Adams, A. E. M., D. Botstein, and D. G. Drubin. 1991. Nature (Lond.). 354:404-408.). Sac6p was originally identified both genetically (Adams, A. E. M., and D. Botstein. 1989. Genetics. 121:675-683.) by dominant, reciprocal suppression of a temperature-sensitive yeast actin mutation (act1-1), as well as biochemically (Drubin, D. G., K. G. Miller, and D. Botstein. 1988. J. Cell Biol. 107: 2551-2561.). To identify the region on actin that interacts with Sac6p, we have analyzed eight different act1 mutations that show suppression with sac6 mutant alleles, and have asked whether (a) these mutations occur in a small defined region on the crystal structure of actin; and (b) the mutant actins are defective in their interaction with Sac6p in vitro. Sequence analysis indicates that all of these mutations change residues that cluster in the small domain of the actin crystal structure, suggesting that this region is an important part of the Sac6p-binding domain. Biochemical analysis reveals defects in the ability of several of the mutant actins to bind Sac6p, and a reduction in Sac6p-induced cross-linking of mutant actin filaments. Together, these observations identify a likely site of interaction of fimbrin on actin.     

Suppressors of Yeast Actin Mutations

Suppressors of a temperature-sensitive mutation (act1-1) in the single actin gene of Saccharomyces cerevisiae were selected that had simultaneously acquired a cold-sensitive growth phenotype. Five genes, called SAC (suppressor of actin) were defined by complementation tests; both suppression and cold-sensitive phenotypes were recessive. Three of the genes (SAC1, SAC2 and SAC3) were subjected to extensive genetic and phenotypic analysis, including molecular cloning. Suppression was found to be allele-specific with respect to actin alleles. The sac mutants, even in ACT1+ genetic backgrounds, displayed phenotypes similar to those of actin mutants, notably aberrant organization of intracellular actin and deposition of chitin at the cell surface. These results are interpreted as being consistent with the idea that the SAC genes encode proteins that interact with actin, presumably as components or controllers of the assembly or stability of the yeast actin cytoskeleton. Two unexpected properties of the SAC1 gene were noted. Disruptions of the gene indicated that its function is essential only at temperatures below about 17 degrees and all sac1 alleles are inviable when combined with act1-2. These properties are interpreted in the context of the evolution of the actin cytoskeleton of yeast.     

The rho-GAP encoded by BEM2 regulates cytoskeletal structure in budding yeast.

Microfilaments are required for polarized growth and morphogenesis in Saccharomyces cerevisiae. To accomplish this, actin cables and patches are redistributed during the cell cycle to direct secretory components to appropriate sites for cell growth. A major component of actin cables is tropomyosin I, encoded by TPM1, that determines or stabilizes these structures. Disruption of TPM1 is not lethal but results in the loss of actin cables and confers a partial defect in polarized secretion. Using a synthetic lethal screen, we have identified seven mutations residing in six genes whose products are required in the absence of Tpm1p. Each mutant exhibited a morphological defect, suggesting a functional link to the actin cytoskeleton. Complementation cloning of one mutation revealed that it lies in BEM2, which encodes a GTPase-activating protein for the RHO1 product. bem2 mutations also show synthetic lethality with rho1 and mutations in certain other cytoskeletal genes (ACT1, MYO1, MYO2, and SAC6) but not with mutations in several noncytoskeletal genes. These data therefore provide a genetic link between the GAP encoded by BEM2 and the functional organization of microfilaments. In addition, we show that bem2 mutations confer benomyl sensitivity and have abnormal microtubule arrays, suggesting that the BEM2 product may also be involved directly or indirectly in regulating microtubule function.     

Mutations Synthetically Lethal with Tpm1δ Lie in Genes Involved in Morphogenesis

Yeast contains two genes, TPM1 and TPM2, encoding tropomyosins, either of which can provide an essential function in the yeast cytoskeleton. To elucidate more clearly the function of the major tropomyosin, encoded by TPM1, we have isolated mutations that confer synthetic lethality with the null mutant of TPM1. Here we describe a phenotypic and genetic analysis of mutations in TSL1/BEM2, TSL2, TSL3, TSL5, and TSL6 (tropomyosin synthetic lethal). All the mutants exhibit clear morphological and some actin cytoskeletal defects, but are not noticeably defective in secretion, endocytosis, or organelle segregation. The lethality conferred by tsl tpm1δ mutations could be specifically suppressed by either TPM1 or an additional copy of TPM2. This implies that the essential function compromised in the tsl tpm1δ constructs is the same essential function for which Tpm1p or Tpm2p is necessary. Synthetic interactions and unlinked noncomplementation were observed between the tsl mutants, suggesting that they participate in related functions involving morphogenesis. In support of this, tsl6-1 was identified as an allele of the nonessential gene SLT2 or MPK1 whose product is a MAP kinase regulating cell wall synthesis. These results indicate that this synthetic lethality approach provides a sensitive screen for the isolation of mutations affecting morphogenesis, many of which are likely to be in nonessential genes, like BEM2 and SLT2.     

A nuclear protein with sequence similarity to proteins implicated in human acute leukemias is important for cellular morphogenesis and actin cytoskeletal function in Saccharomyces cerevisiae.

The cellular functions of the product of the Saccharomyces cerevisiae ANC1 (actin non-complementing) gene were investigated. ANC1 was previously identified in a screen for mutations that enhance the defect caused by a mutation in the actin gene. Here, we show that anc1-1 and anc1 delta 1::HIS3 (gene deletion) mutants exhibit a novel combination of defects in the organization of the actin cytoskeleton and the localization of Spa2p, a protein implicated in polarity development and cytokinesis. Morphological abnormalities exhibited by anc1 mutants include failure to form a mating projection in response to alpha-factor and development of swollen or elongated cell shapes during proliferation. These morphological aberrations correlate with cytoskeletal defects that were also observed. These phenotypes demonstrate that Anc1p is important for actin function and for the functions of other proteins involved in morphogenesis. In further support of these roles for Anc1p, the anc1 delta 1::HIS3 mutation was found to be synthetically lethal in combination with a null mutation in SLA1, a gene that is important for membrane cytoskeleton function. Surprisingly, Anc1p was found to be a nuclear protein and to have sequence similarity to the human proteins ENL and AF-9. These human proteins are implicated in the development of a subset of acute lymphoblastic leukemias, acute myeloid leukemias, and lymphomas. Our findings suggest that changes in the functions or organization of actin filaments might contribute to the establishment of the neoplastic state for these leukemias and lymphomas.     

The novel adaptor protein, Mti1p, and Vrp1p, a homolog of Wiskott-Aldrich syndrome protein-interacting protein (WIP), may antagonistically regulate type I myosins in Saccharomyces cerevisiae

Type I myosins in yeast, Myo3p and Myo5p (Myo3/5p), are involved in the reorganization of the actin cytoskeleton. The SH3 domain of Myo5p regulates the polymerization of actin through interactions with both Las17p, a homolog of mammalian Wiskott-Aldrich syndrome protein (WASP), and Vrp1p, a homolog of WASP-interacting protein (WIP). Vrp1p is required for both the localization of Myo5p to cortical patch-like structures and the ATP-independent interaction between the Myo5p tail region and actin filaments. We have identified and characterized a new adaptor protein, Mti1p (Myosin tail region-interacting protein), which interacts with the SH3 domains of Myo3/5p. Mti1p co-immunoprecipitated with Myo5p and Mti1p-GFP co-localized with cortical actin patches. A null mutation of MTI1 exhibited synthetic lethal phenotypes with mutations in SAC6 and SLA2, which encode actin-bundling and cortical actin-binding proteins, respectively. Although the mti1 null mutation alone did not display any obvious phenotype, it suppressed vrp1 mutation phenotypes, including temperature-sensitive growth, abnormally large cell morphology, defects in endocytosis and salt-sensitive growth. These results suggest that Mti1p and Vrp1p antagonistically regulate type I myosin functions.     

The Yeast V159N Actin Mutant Reveals Roles for Actin Dynamics In Vivo

Actin with a Val 159 to Asn mutation (V159N) forms actin filaments that depolymerize slowly because of a failure to undergo a conformational change after inorganic phosphate release. Here we demonstrate that expression of this actin results in reduced actin dynamics in vivo, and we make use of this property to study the roles of rapid actin filament turnover. Yeast strains expressing the V159N mutant (act1-159) as their only source of actin have larger cortical actin patches and more actin cables than wild-type yeast. Rapid actin dynamics are not essential for cortical actin patch motility or establishment of cell polarity. However, fluid phase endocytosis is defective in act1-159 strains. act1-159 is synthetically lethal with cofilin and profilin mutants, supporting the conclusion that mutations in all of these genes impair the polymerization/ depolymerization cycle. In contrast, act1-159 partially suppresses the temperature sensitivity of a tropomyosin mutant, and the loss of cytoplasmic cables seen in fimbrin, Mdm20p, and tropomyosin null mutants, suggesting filament stabilizing functions for these actin-binding proteins. Analysis of the cables in these double-mutant cells supports a role for fimbrin in organizing cytoplasmic cables and for Mdm20p and tropomyosin in excluding cofilin from the cables.     

Null alleles of SAC7 suppress temperature-sensitive actin mutations in Saccharomyces cerevisiae.

Extragenic suppressors of a new temperature-sensitive mutation (act1-4) in the actin gene of Saccharomyces cerevisiae were isolated in an attempt to identify genes whose products interact directly with actin. One suppressor with a cold-sensitive growth phenotype defined the new gene, SAC7, which was mapped, cloned, sequenced, and disrupted. Genetic analysis of strains that are disrupted for SAC7 demonstrated that the protein is required for normal growth and actin assembly at low temperatures. Surprisingly, null mutations in SAC7 also suppressed the temperature-sensitive growth defect caused by the act1-1 and act1-4 mutations, whereas they were lethal in combination with the temperature-sensitive allele act1-2. These results support the notion that the SAC7 gene product is involved in the normal assembly or function or both of actin.     

Suppressors of mdm20 in yeast identify new alleles of ACT1 and TPM1 predicted to enhance actin-tropomyosin interactions

The actin cytoskeleton is required for many aspects of cell division in yeast, including mitochondrial partitioning into growing buds (mitochondrial inheritance). Yeast cells lacking MDM20 function display defects in both mitochondrial inheritance and actin organization, specifically, a lack of visible actin cables and enhanced sensitivity to Latrunculin A. mdm20 mutants also exhibit a temperature-sensitive growth phenotype, which we exploited to isolate second-site suppressor mutations. Nine dominant suppressors selected in an mdm20/mdm20 background rescue temperature-sensitive growth defects and mitochondrial inheritance defects and partially restore actin cables in haploid and diploid mdm20 strains. The suppressor mutations define new alleles of ACT1 and TPM1, which encode actin and the major form of tropomyosin in yeast, respectively. The ACT1 mutations cluster in a region of the actin protein predicted to contact tropomyosin, suggesting that they stabilize actin cables by enhancing actin-tropomyosin interactions. The characteristics of the mutant ACT1 and TPM1 alleles and their potential effects on protein structure and binding are discussed.     

Z-disc-associated,Alternatively Spliced,PDZ Motif-containing Protein (ZASP) Mutations in the Actin-binding Domain Cause Disruption of Skeletal Muscle Actin Filaments in Myofibrillar Myopathy

The core of skeletal muscle Z-discs consists of actin filaments from adjacent sarcomeres that are cross-linked by α-actinin homodimers. Z-disc-associated, alternatively spliced, PDZ motif-containing protein (ZASP)/Cypher interacts with α-actinin, myotilin, and other Z-disc proteins via the PDZ domain. However, these interactions are not sufficient to maintain the Z-disc structure. We show that ZASP directly interacts with skeletal actin filaments. The actin-binding domain is between the modular PDZ and LIM domains. This ZASP region is alternatively spliced so that each isoform has unique actin-binding domains. All ZASP isoforms contain the exon 6-encoded ZASP-like motif that is mutated in zaspopathy, a myofibrillar myopathy (MFM), whereas the exon 8–11 junction-encoded peptide is exclusive to the postnatal long ZASP isoform (ZASP-LΔex10). MFM is characterized by disruption of skeletal muscle Z-discs and accumulation of myofibrillar degradation products. Wild-type and mutant ZASP interact with α-actin, α-actinin, and myotilin. Expression of mutant, but not wild-type, ZASP leads to Z-disc disruption and F-actin accumulation in mouse skeletal muscle, as in MFM. Mutations in the actin-binding domain of ZASP-LΔex10, but not other isoforms, cause disruption of the actin cytoskeleton in muscle cells. These isoform-specific mutation effects highlight the essential role of the ZASP-LΔex10 isoform in F-actin organization. Our results show that MFM-associated ZASP mutations in the actin-binding domain have deleterious effects on the core structure of the Z-discs in skeletal muscle.     

Multiple Functions for Actin during Filamentous Growth of Saccharomyces cerevisiae

Saccharomyces cerevisiae is dimorphic and switches from a yeast form to a pseudohyphal (PH) form when starved for nitrogen. PH cells are elongated, bud in a unipolar manner, and invade the agar substrate. We assessed the requirements for actin in mediating the dramatic morphogenetic events that accompany the transition to PH growth. Twelve “alanine scan” alleles of the single yeast actin gene (ACT1) were tested for effects on filamentation, unipolar budding, agar invasion, and cell elongation. Some act1 mutations affect all phenotypes, whereas others affect only one or two aspects of PH growth. Tests of intragenic complementation among specific act1 mutations support the phenotypic evidence for multiple actin functions in filamentous growth. We present evidence that interaction between actin and the actin-binding protein fimbrin is important for PH growth and suggest that association of different actin-binding proteins with actin mediates the multiple functions of actin in filamentous growth. Furthermore, characterization of cytoskeletal structure in wild type and act1/act1 mutants indicates that PH cell morphogenesis requires the maintenance of a highly polarized actin cytoskeleton. Collectively, this work demonstrates that actin plays a central role in fungal dimorphism.     

Actin organization, bristle morphology, and viability are affected by actin capping protein mutations in Drosophila

Regulation of actin filament length and orientation is important in many actin-based cellular processes. This regulation is postulated to occur through the action of actin-binding proteins. Many actin-binding proteins that modify actin in vitro have been identified, but in many cases, it is not known if this activity is physiologically relevant. Capping protein (CP) is an actin-binding protein that has been demonstrated to control filament length in vitro by binding to the barbed ends and preventing the addition or loss of actin monomers. To examine the in vivo role of CP, we have performed a molecular and genetic characterization of the beta subunit of capping protein from Drosophila melanogaster. We have identified mutations in the Drosophila beta subunit-these are the first CP mutations in a multicellular organism, and unlike CP mutations in yeast, they are lethal, causing death during the early larval stage. Adult files that are heterozygous for a pair of weak alleles have a defect in bristle morphology that is correlated to disorganized actin bundles in developing bristles. Our data demonstrate that CP has an essential function during development, and further suggest that CP is required to regulate actin assembly during the development of specialized structures that depend on actin for their morphology.