RESUMEN
The Drosophila egg chamber (EC) starts as a spherical tissue at the beginning. With maturation, the outer follicle cells of EC collectively migrate in a direction perpendicular to the anterior-posterior axis, to shape EC from spherical to ellipsoidal. Filamentous actin (F-actin) plays a significant role in shaping individual migratory cells to the overall EC shape, like in every cell migration. The primary focus of this article is to unveil the function of different Actin Binding Proteins (ABPs) in regulating mature Drosophila egg shape. We have screened 66 ABPs, and the genetic screening data revealed that individual knockdown of Arp2/3 complex genes and the "capping protein ß" (cpb) gene have severely altered the egg phenotype. Arpc1 and cpb RNAi mediated knockdown resulted in the formation of spherical eggs which are devoid of dorsal appendages. Studies also showed the role of Arpc1 and cpb on the number of laid eggs and follicle cell morphology. Furthermore, the depletion of Arpc1 and cpb resulted in a change in F-actin quantity. Together, the data indicate that Arpc1 and cpb regulate Drosophila egg shape, F-actin management, egg-laying characteristics and dorsal appendages formation.
Asunto(s)
Actinas , Proteínas de Drosophila , Morfogénesis , Animales , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Actinas/metabolismo , Actinas/genética , Femenino , Morfogénesis/genética , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Proteínas de Capping de la Actina/metabolismo , Proteínas de Capping de la Actina/genética , Óvulo/metabolismo , Óvulo/crecimiento & desarrolloRESUMEN
Cell processes require precise regulation of actin polymerization that is mediated by plus-end regulatory proteins. Detailed mechanisms that explain plus-end dynamics involve regulators with opposing roles, including factors that enhance assembly, e.g., the formin mDia1, and others that stop growth (capping protein, CP). We explore IQGAP1's roles in regulating actin filament plus-ends and the consequences of perturbing its activity in cells. We confirm that IQGAP1 pauses elongation and interacts with plus ends through two residues (C756 and C781). We directly visualize the dynamic interplay between IQGAP1 and mDia1, revealing that IQGAP1 displaces the formin to influence actin assembly. Using four-color TIRF, we show that IQGAP1's displacement activity extends to formin-CP "decision complexes," promoting end-binding protein turnover at plus-ends. Loss of IQGAP1 or its plus-end activities disrupts morphology and migration, emphasizing its essential role. These results reveal a new role for IQGAP1 in promoting protein turnover on filament ends and provide new insights into how plus-end actin assembly is regulated in cells.
Asunto(s)
Proteínas de Capping de la Actina , Citoesqueleto de Actina , Forminas , Proteínas Activadoras de ras GTPasa , Animales , Humanos , Proteínas de Capping de la Actina/metabolismo , Proteínas de Capping de la Actina/genética , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Movimiento Celular , Forminas/metabolismo , Células HeLa , Unión Proteica , Proteínas Activadoras de ras GTPasa/metabolismo , Proteínas Activadoras de ras GTPasa/genética , Ratones , Células 3T3 NIHRESUMEN
Capping protein (CP) binds to the barbed end of an actin-filament and inhibits its elongation. CARMIL binds CP and dissociates it from the barbed end of the actin-filament. The binding of CARMIL peptide alters the flexibility of CP, which is considered to facilitate the dissociation. Twinfilin also binds to CP through its C-terminal tail. The complex structures of the CP/twinfilin-tail (TW-tail) peptide indicate that the binding sites of CARMIL and TW-tail overlap. However, TW-tail binding does not facilitate the dissociation of CP from the barbed end. We extensively investigated the flexibilities of CP in the CP/TW-tail or CP/CARMIL complexes using an elastic network model and concluded that TW-tail binding does not alter the flexibility of CP. Our extensive analysis also highlighted that the strong contacts of peptides with the two domains of CP, that is, the CP-L and CP-S domains, are key to changing the flexibilities of CP. CARMIL peptides can interact strongly with both of the domains, while TW-tail peptides exclusively interact with the CP-S domain because the binding site of TW-tail on CP relatively shifts to the CP-S domain compared with that of CP/CARMIL. This result supports our hypothesis that the dissociation of CP from the barbed end is regulated by the flexibility of CP.
Asunto(s)
Proteínas de Capping de la Actina , Proteínas de Microfilamentos , Proteínas de Microfilamentos/metabolismo , Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/metabolismo , Unión Proteica , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Péptidos/químicaRESUMEN
The capping of barbed filament ends is a fundamental mechanism for actin regulation. Capping protein controls filament growth and actin turnover in cells by binding to the barbed ends of the filaments with high affinity and slow off-rate. The interaction between capping protein and actin is regulated by capping protein interaction (CPI) motif proteins. We identified a novel CPI motif protein, Bsp1, which is involved in cytokinesis and endocytosis in budding yeast. We demonstrate that Bsp1 is an actin binding protein with a high affinity for capping protein via its CPI motif. In cells, Bsp1 regulates capping protein at endocytic sites and is a major recruiter of capping protein to the cytokinetic actin ring. Lastly, we define Bsp1-related proteins as a distinct fungi-specific CPI protein group. Our results suggest that Bsp1 promotes actin filament capping by the capping protein. This study establishes Bsp1 as a new capping protein regulator and promising candidate to regulate actin networks in fungi.
Asunto(s)
Actinas , Citocinesis , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos/metabolismo , Endocitosis , Proteínas de Capping de la Actina/metabolismoRESUMEN
Cellular functions of actin capping protein (CP) regulators are poorly understood. Di Pietro and colleagues (https://doi.org/10.1083/jcb.202306154) shed unprecedented light on this topic using budding yeast. Two proteins with CPI (capping protein interacting) motifs recruit CP to sites of actin assembly, while a third contributes to CP turnover.
Asunto(s)
Actinas , Saccharomycetales , Actinas/genética , Actinas/metabolismo , Unión Proteica , Saccharomycetales/genética , Proteínas de Capping de la Actina/genética , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismoRESUMEN
Clathrin-mediated endocytosis depends on polymerization of a branched actin network to provide force for membrane invagination. A key regulator in branched actin network formation is actin capping protein (CP), which binds to the barbed end of actin filaments to prevent the addition or loss of actin subunits. CP was thought to stochastically bind actin filaments, but recent evidence shows CP is regulated by a group of proteins containing CP-interacting (CPI) motifs. Importantly, how CPI motif proteins function together to regulate CP is poorly understood. Here, we show Aim21 and Bsp1 work synergistically to recruit CP to the endocytic actin network in budding yeast through their CPI motifs, which also allosterically modulate capping strength. In contrast, twinfilin works downstream of CP recruitment, regulating the turnover of CP through its CPI motif and a non-allosteric mechanism. Collectively, our findings reveal how three CPI motif proteins work together to regulate CP in a stepwise fashion during endocytosis.
Asunto(s)
Proteínas de Capping de la Actina , Actinas , Endocitosis , Proteínas de Saccharomyces cerevisiae , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Clatrina/metabolismo , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Adaptadoras del Transporte Vesicular/metabolismoRESUMEN
Cytoadherence and migration are crucial for pathogens to establish colonization in the host. In contrast to a nonadherent isolate of Trichomonas vaginalis, an adherent one expresses more actin-related machinery proteins with more active flagellate-amoeboid morphogenesis, amoeba migration, and cytoadherence, activities that were abrogated by an actin assembly blocker. By immunoprecipitation coupled with label-free quantitative proteomics, an F-actin capping protein (T. vaginalis F-actin capping protein subunit α [TvFACPα]) was identified from the actin-centric interactome. His-TvFACPα was detected at the barbed end of a growing F-actin filament, which inhibited elongation and possessed atypical activity in binding G-actin in in vitro assays. TvFACPα partially colocalized with F-actin at the parasite pseudopod protrusion and formed a protein complex with α-actin through its C-terminal domain. Meanwhile, TvFACPα overexpression suppressed F-actin polymerization, amoeboid morphogenesis, and cytoadherence in this parasite. Ser2 phosphorylation of TvFACPα enriched in the amoeboid stage of adhered trophozoites was reduced by a casein kinase II (CKII) inhibitor. Site-directed mutagenesis and CKII inhibitor treatment revealed that Ser2 phosphorylation acts as a switching signal to alter TvFACPα actin-binding activity and the consequent actin cytoskeleton behaviors. Through CKII signaling, TvFACPα also controls the conversion of adherent trophozoites from amoeboid migration to the flagellate form with axonemal motility. Together, CKII-dependent Ser2 phosphorylation regulates TvFACPα binding to actin to fine-tune cytoskeleton dynamics and drive crucial behaviors underlying host colonization by T. vaginalis. IMPORTANCE Trichomoniasis is one of the most prevalent nonviral sexually transmitted diseases. T. vaginalis cytoadherence to urogenital epithelium cells is the first step in the colonization of the host. However, studies on the mechanisms of cytoadherence have focused mainly on the role of adhesion molecules, and their effects are limited when analyzed by loss- or gain-of-function assays. This study proposes an extra pathway in which the actin cytoskeleton mediated by a capping protein α-subunit may play roles in parasite morphogenesis, cytoadherence, and motility, which are crucial for colonization. Once the origin of the cytoskeleton dynamics could be manipulated, the consequent activities would be controlled as well. This mechanism may provide new potential therapeutic targets to impair this parasite infection and relieve the increasing impact of drug resistance on clinical and public health.
Asunto(s)
Trichomonas vaginalis , Trichomonas vaginalis/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas de Capping de la Actina/metabolismoRESUMEN
Actin dynamics play an important role in tissue morphogenesis, yet the control of actin filament growth takes place at the molecular level. A challenge in the field is to link the molecular function of actin regulators with their physiological function. Here, we report an in vivo role of the actin-capping protein CAP-1 in the Caenorhabditis elegans germline. We show that CAP-1 is associated with actomyosin structures in the cortex and rachis, and its depletion or overexpression led to severe structural defects in the syncytial germline and oocytes. A 60% reduction in the level of CAP-1 caused a twofold increase in F-actin and non-muscle myosin II activity, and laser incision experiments revealed an increase in rachis contractility. Cytosim simulations pointed to increased myosin as the main driver of increased contractility following loss of actin-capping protein. Double depletion of CAP-1 and myosin or Rho kinase demonstrated that the rachis architecture defects associated with CAP-1 depletion require contractility of the rachis actomyosin corset. Thus, we uncovered a physiological role for actin-capping protein in regulating actomyosin contractility to maintain reproductive tissue architecture.
Asunto(s)
Actomiosina , Caenorhabditis elegans , Animales , Actomiosina/metabolismo , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Actinas/metabolismo , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Miosinas/metabolismo , Células Germinativas/metabolismoRESUMEN
How cells simultaneously assemble actin structures of distinct sizes, shapes, and filamentous architectures is still not well understood. Here, we used budding yeast as a model to investigate how competition for the barbed ends of actin filaments might influence this process. We found that while vertebrate capping protein (CapZ) and formins can simultaneously associate with barbed ends and catalyze each other's displacement, yeast capping protein (Cap1/2) poorly displaces both yeast and vertebrate formins. Consistent with these biochemical differences, in vivo formin-mediated actin cable assembly was strongly attenuated by the overexpression of CapZ but not Cap1/2. Multiwavelength live cell imaging further revealed that actin patches in cap2∆ cells acquire cable-like features over time, including recruitment of formins and tropomyosin. Together, our results suggest that the activities of S. cerevisiae Cap1/2 have been tuned across evolution to allow robust cable assembly by formins in the presence of high cytosolic levels of Cap1/2, which conversely limit patch growth and shield patches from formins.
Asunto(s)
Proteínas de Capping de la Actina , Actinas , Proteínas de Saccharomyces cerevisiae , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citosol/metabolismo , Forminas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteína CapZ/metabolismoRESUMEN
The infection of a mammalian host by the pathogenic fungus Candida albicans involves fungal resistance to reactive oxygen species (ROS)-induced DNA damage stress generated by the defending macrophages or neutrophils. Thus, the DNA damage response in C. albicans may contribute to its pathogenicity. Uncovering the transcriptional changes triggered by the DNA damage-inducing agent MMS in many model organisms has enhanced the understanding of their DNA damage response processes. However, the transcriptional regulation triggered by MMS remains unclear in C. albicans. Here, we explored the global transcription profile in response to MMS in C. albicans and identified 306 defined genes whose transcription was significantly affected by MMS. Only a few MMS-responsive genes, such as MGT1, DDR48, MAG1, and RAD7, showed potential roles in DNA repair. GO term analysis revealed that a large number of induced genes were involved in antioxidation responses, and some downregulated genes were involved in nucleosome packing and IMP biosynthesis. Nevertheless, phenotypic assays revealed that MMS-induced antioxidation gene CAP1 and glutathione metabolism genes GST2 and GST3 showed no direct roles in MMS resistance. Furthermore, the altered transcription of several MMS-responsive genes exhibited RAD53-related regulation. Intriguingly, the transcription profile in response to MMS in C. albicans shared a limited similarity with the pattern in S. cerevisiae, including COX17, PRI2, and MGT1. Overall, C. albicans cells exhibit global transcriptional changes to the DNA damage agent MMS; these findings improve our understanding of this pathogen's DNA damage response pathways.
Asunto(s)
Candida albicans , Metilmetanosulfonato , Proteínas de Capping de la Actina/genética , Proteínas de Capping de la Actina/metabolismo , Animales , Candida albicans/efectos de los fármacos , Candida albicans/genética , Candida albicans/metabolismo , Daño del ADN/efectos de los fármacos , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Mamíferos/metabolismo , Metilmetanosulfonato/farmacología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Capping protein Arp2/3 myosin I linker (CARMIL) proteins are multi-domain scaffold proteins that regulate actin dynamics by regulating the activity of capping protein (CP). Here, we characterize CARMIL-GAP (GAP for GTPase-activating protein), a Dictyostelium CARMIL isoform that contains a â¼130 residue insert that, by homology, confers GTPase-activating properties for Rho-related GTPases. Consistent with this idea, this GAP domain binds Dictyostelium Rac1a and accelerates its rate of GTP hydrolysis. CARMIL-GAP concentrates with F-actin in phagocytic cups and at the leading edge of chemotaxing cells, and CARMIL-GAP-null cells exhibit pronounced defects in phagocytosis and chemotactic streaming. Importantly, these defects are fully rescued by expressing GFP-tagged CARMIL-GAP in CARMIL-GAP-null cells. Finally, rescue with versions of CARMIL-GAP that lack either GAP activity or the ability to regulate CP show that, although both activities contribute significantly to CARMIL-GAP function, the GAP activity plays the bigger role. Together, our results add to the growing evidence that CARMIL proteins influence actin dynamics by regulating signaling molecules as well as CP, and that the continuous cycling of the nucleotide state of Rho GTPases is often required to drive Rho-dependent biological processes.
Asunto(s)
Proteínas de Capping de la Actina , Dictyostelium , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas Portadoras/metabolismo , Dictyostelium/genética , Dictyostelium/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismoRESUMEN
Cyclase-associated protein (CAP) is an actin binding protein that has been initially described as partner of the adenylyl cyclase in yeast. In all vertebrates and some invertebrate species, two orthologs, named CAP1 and CAP2, have been described. CAP1 and CAP2 are characterized by a similar multidomain structure, but different expression patterns. Several molecular studies clarified the biological function of the different CAP domains, and they shed light onto the mechanisms underlying CAP-dependent regulation of actin treadmilling. However, CAPs are crucial elements not only for the regulation of actin dynamics, but also for signal transduction pathways. During recent years, human genetic studies and the analysis of gene-targeted mice provided important novel insights into the physiological roles of CAPs and their involvement in the pathogenesis of several diseases. In the present review, we summarize and discuss recent progress in our understanding of CAPs' physiological functions, focusing on heart, skeletal muscle and central nervous system as well as their involvement in the mechanisms controlling metabolism. Remarkably, loss of CAPs or impairment of CAPs-dependent pathways can contribute to the pathogenesis of different diseases. Overall, these studies unraveled CAPs complexity highlighting their capability to orchestrate structural and signaling pathways in the cells.
Asunto(s)
Actinas , Proteínas de Saccharomyces cerevisiae , Proteínas de Capping de la Actina/metabolismo , Actinas/metabolismo , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas del Citoesqueleto/metabolismo , Genética Humana , Humanos , Ratones , Proteínas de Microfilamentos/metabolismo , Unión Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Apicomplexan parasites, such as Plasmodium spp., rely on an unusual actomyosin motor, termed glideosome, for motility and host cell invasion. The actin filaments are maintained by a small set of essential regulators, which provide control over actin dynamics in the different stages of the parasite life cycle. Actin filament capping proteins (CPs) are indispensable heterodimeric regulators of actin dynamics. CPs have been extensively characterized in higher eukaryotes, but their role and functional mechanism in Apicomplexa remain enigmatic. Here, we present the first crystal structure of a homodimeric CP from the malaria parasite and compare the homo- and heterodimeric CP structures in detail. Despite retaining several characteristics of a canonical CP, the homodimeric Plasmodium berghei (Pb)CP exhibits crucial differences to the canonical heterodimers. Both homo- and heterodimeric PbCPs regulate actin dynamics in an atypical manner, facilitating rapid turnover of parasite actin, without affecting its critical concentration. Homo- and heterodimeric PbCPs show partially redundant activities, possibly to rescue actin filament capping in life cycle stages where the ß-subunit is downregulated. Our data suggest that the homodimeric PbCP also influences actin kinetics by recruiting lateral actin dimers. This unusual function could arise from the absence of a ß-subunit, as the asymmetric PbCP homodimer lacks structural elements essential for canonical barbed end interactions suggesting a novel CP binding mode. These findings will facilitate further studies aimed at elucidating the precise actin filament capping mechanism in Plasmodium.
Asunto(s)
Proteínas de Capping de la Actina , Antígenos de Protozoos , Malaria/parasitología , Plasmodium/metabolismo , Proteínas Protozoarias , Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/metabolismo , Antígenos de Protozoos/química , Antígenos de Protozoos/metabolismo , Cinética , Modelos Moleculares , Unión Proteica , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismoRESUMEN
Heterodimeric capping protein (CP/CapZ) is an essential factor for the assembly of branched actin networks, which push against cellular membranes to drive a large variety of cellular processes. Aside from terminating filament growth, CP potentiates the nucleation of actin filaments by the Arp2/3 complex in branched actin networks through an unclear mechanism. Here, we combine structural biology with in vitro reconstitution to demonstrate that CP not only terminates filament elongation, but indirectly stimulates the activity of Arp2/3 activating nucleation promoting factors (NPFs) by preventing their association to filament barbed ends. Key to this function is one of CP's C-terminal "tentacle" extensions, which sterically masks the main interaction site of the terminal actin protomer. Deletion of the ß tentacle only modestly impairs capping. However, in the context of a growing branched actin network, its removal potently inhibits nucleation promoting factors by tethering them to capped filament ends. End tethering of NPFs prevents their loading with actin monomers required for activation of the Arp2/3 complex and thus strongly inhibits branched network assembly both in cells and reconstituted motility assays. Our results mechanistically explain how CP couples two opposed processes-capping and nucleation-in branched actin network assembly.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Melanocitos/metabolismo , Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/genética , Citoesqueleto de Actina/ultraestructura , Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/genética , Actinas/química , Actinas/genética , Animales , Sitios de Unión , Bovinos , Citoesqueleto/ultraestructura , Gelsolina/química , Gelsolina/genética , Gelsolina/metabolismo , Regulación de la Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Cinética , Melanocitos/citología , Melanoma Experimental/genética , Melanoma Experimental/metabolismo , Melanoma Experimental/patología , Ratones , Modelos Moleculares , Profilinas/química , Profilinas/genética , Profilinas/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Timo/citología , Timo/metabolismo , Proteína Neuronal del Síndrome de Wiskott-Aldrich/química , Proteína Neuronal del Síndrome de Wiskott-Aldrich/genética , Proteína Neuronal del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Clathrin- and actin-mediated endocytosis is a fundamental process in eukaryotic cells. Previously, we discovered Tda2 as a new yeast dynein light chain (DLC) that works with Aim21 to regulate actin assembly during endocytosis. Here we show Tda2 functions as a dimerization engine bringing two Aim21 molecules together using a novel binding surface different than the canonical DLC ligand binding groove. Point mutations on either protein that diminish the Tda2-Aim21 interaction in vitro cause the same in vivo phenotype as TDA2 deletion showing reduced actin capping protein (CP) recruitment and increased filamentous actin at endocytic sites. Remarkably, chemically induced dimerization of Aim21 rescues the endocytic phenotype of TDA2 deletion. We also uncovered a CP interacting motif in Aim21, expanding its function to a fundamental cellular pathway and showing such motif exists outside mammalian cells. Furthermore, specific disruption of this motif causes the same deficit of actin CP recruitment and increased filamentous actin at endocytic sites as AIM21 deletion. Thus, the data indicate the Tda2-Aim21 complex functions in actin assembly primarily through CP regulation. Collectively, our results provide a mechanistic view of the Tda2-Aim21 complex and its function in actin network regulation at endocytic sites.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Proteínas del Citoesqueleto/metabolismo , Endocitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Citoesqueleto de Actina/metabolismo , Multimerización de ProteínaRESUMEN
Twinfilin is a conserved actin regulator that interacts with actin capping protein (CP) via C terminus residues (TWtail) that exhibits sequence similarity with the CP interaction (CPI) motif of CARMIL. Here we report the crystal structure of TWtail in complex with CP. Our structure showed that although TWtail and CARMIL CPI bind CP to an overlapping surface via their middle regions, they exhibit different CP-binding modes at both termini. Consequently, TWtail and CARMIL CPI restrict the CP in distinct conformations of open and closed forms, respectively. Interestingly, V-1, which targets CP away from the TWtail binding site, also favors the open-form CP. Consistently, TWtail forms a stable ternary complex with CP and V-1, a striking contrast to CARMIL CPI, which rapidly dissociates V-1 from CP. Our results demonstrate that TWtail is a unique CP-binding motif that regulates CP in a manner distinct from CARMIL CPI.
Asunto(s)
Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Pollos , Cristalografía por Rayos X , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Modelos Moleculares , Unión Proteica , Estructura Cuaternaria de ProteínaRESUMEN
Heterodimeric capping protein (CP) binds the rapidly growing barbed ends of actin filaments and prevents the addition (or loss) of subunits. Capping activity is generally considered to be essential for actin-based motility induced by Arp2/3 complex nucleation. By stopping barbed end growth, CP favors nucleation of daughter filaments at the functionalized surface where the Arp2/3 complex is activated, thus creating polarized network growth, which is necessary for movement. However, here using an in vitro assay where Arp2/3 complex-based actin polymerization is induced on bead surfaces in the absence of CP, we produce robust polarized actin growth and motility. This is achieved either by adding the actin polymerase Ena/VASP or by boosting Arp2/3 complex activity at the surface. Another actin polymerase, the formin FMNL2, cannot substitute for CP, showing that polymerase activity alone is not enough to override the need for CP. Interfering with the polymerase activity of Ena/VASP, its surface recruitment or its bundling activity all reduce Ena/VASP's ability to maintain polarized network growth in the absence of CP. Taken together, our findings show that CP is dispensable for polarized actin growth and motility in situations where surface-directed polymerization is favored by whatever means over the growth of barbed ends in the network.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Proteínas de Unión al ADN/metabolismo , Forminas/metabolismo , Animales , Ratones , Polimerizacion , Conejos , PorcinosRESUMEN
Improper lengths of actin-thin filaments are associated with altered contractile activity and lethal myopathies. Leiomodin, a member of the tropomodulin family of proteins, is critical in thin filament assembly and maintenance; however, its role is under dispute. Using nuclear magnetic resonance data and molecular dynamics simulations, we generated the first atomic structural model of the binding interface between the tropomyosin-binding site of cardiac leiomodin and the N-terminus of striated muscle tropomyosin. Our structural data indicate that the leiomodin/tropomyosin complex only forms at the pointed end of thin filaments, where the tropomyosin N-terminus is not blocked by an adjacent tropomyosin protomer. This discovery provides evidence supporting the debated mechanism where leiomodin and tropomodulin regulate thin filament lengths by competing for thin filament binding. Data from experiments performed in cardiomyocytes provide additional support for the competition model; specifically, expression of a leiomodin mutant that is unable to interact with tropomyosin fails to displace tropomodulin at thin filament pointed ends and fails to elongate thin filaments. Together with previous structural and biochemical data, we now propose a molecular mechanism of actin polymerization at the pointed end in the presence of bound leiomodin. In the proposed model, the N-terminal actin-binding site of leiomodin can act as a "swinging gate" allowing limited actin polymerization, thus making leiomodin a leaky pointed-end cap. Results presented in this work answer long-standing questions about the role of leiomodin in thin filament length regulation and maintenance.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Proteínas de Capping de la Actina/química , Proteínas de Capping de la Actina/metabolismo , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animales , Animales Recién Nacidos , Sitios de Unión , Células Cultivadas , Proteínas del Citoesqueleto/química , Proteínas del Citoesqueleto/metabolismo , Humanos , Ratones , Modelos Moleculares , Simulación de Dinámica Molecular , Miocardio/metabolismo , Resonancia Magnética Nuclear Biomolecular , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Ratas , Sarcómeros/metabolismoRESUMEN
BACKGROUND: The PIM family kinases promote cancer cell survival and motility as well as metastatic growth in various types of cancer. We have previously identified several PIM substrates, which support cancer cell migration and invasiveness. However, none of them are known to regulate cellular movements by directly interacting with the actin cytoskeleton. Here we have studied the phosphorylation-dependent effects of PIM1 on actin capping proteins, which bind as heterodimers to the fast-growing actin filament ends and stabilize them. METHODS: Based on a phosphoproteomics screen for novel PIM substrates, we have used kinase assays and fluorescence-based imaging techniques to validate actin capping proteins as PIM1 substrates and interaction partners. We have analysed the functional consequences of capping protein phosphorylation on cell migration and adhesion by using wound healing and real-time impedance-based assays. We have also investigated phosphorylation-dependent effects on actin polymerization by analysing the protective role of capping protein phosphomutants in actin disassembly assays. RESULTS: We have identified capping proteins CAPZA1 and CAPZB2 as PIM1 substrates, and shown that phosphorylation of either of them leads to increased adhesion and migration of human prostate cancer cells. Phosphorylation also reduces the ability of the capping proteins to protect polymerized actin from disassembly. CONCLUSIONS: Our data suggest that PIM kinases are able to induce changes in actin dynamics to support cell adhesion and movement. Thus, we have identified a novel mechanism through which PIM kinases enhance motility and metastatic behaviour of cancer cells. Video abstract.
Asunto(s)
Proteínas de Capping de la Actina/metabolismo , Movimiento Celular , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteínas Proto-Oncogénicas c-pim-1/metabolismo , Actinas/metabolismo , Animales , Adhesión Celular , Línea Celular Tumoral , Extensiones de la Superficie Celular/metabolismo , Citoplasma/metabolismo , Humanos , Masculino , Ratones , Fosforilación , Multimerización de Proteína , Subunidades de Proteína/metabolismo , Proteínas Proto-Oncogénicas c-pim-1/antagonistas & inhibidoresRESUMEN
Cell migration entails networks and bundles of actin filaments termed lamellipodia and microspikes or filopodia, respectively, as well as focal adhesions, all of which recruit Ena/VASP family members hitherto thought to antagonize efficient cell motility. However, we find these proteins to act as positive regulators of migration in different murine cell lines. CRISPR/Cas9-mediated loss of Ena/VASP proteins reduced lamellipodial actin assembly and perturbed lamellipodial architecture, as evidenced by changed network geometry as well as reduction of filament length and number that was accompanied by abnormal Arp2/3 complex and heterodimeric capping protein accumulation. Loss of Ena/VASP function also abolished the formation of microspikes normally embedded in lamellipodia, but not of filopodia capable of emanating without lamellipodia. Ena/VASP-deficiency also impaired integrin-mediated adhesion accompanied by reduced traction forces exerted through these structures. Our data thus uncover novel Ena/VASP functions of these actin polymerases that are fully consistent with their promotion of cell migration.