RESUMEN
Transport of macromolecules from the nucleus to the cytoplasm is essential for nearly all cellular and developmental events, and when mis-regulated, is associated with diseases, tumor formation/growth, and cancer progression. Nuclear Envelope (NE)-budding is a newly appreciated nuclear export pathway for large macromolecular machineries, including those assembled to allow co-regulation of functionally related components, that bypasses canonical nuclear export through nuclear pores. In this pathway, large macromolecular complexes are enveloped by the inner nuclear membrane, transverse the perinuclear space, and then exit through the outer nuclear membrane to release its contents into the cytoplasm. NE-budding is a conserved process and shares many features with nuclear egress mechanisms used by herpesviruses. Despite its biological importance and clinical relevance, little is yet known about the regulatory and structural machineries that allow NE-budding to occur in any system. Here we summarize what is currently known or proposed for this intriguing nuclear export process.
Asunto(s)
Herpesviridae , Membrana Nuclear , Membrana Nuclear/metabolismo , Transporte Activo de Núcleo Celular/fisiología , Herpesviridae/metabolismo , Citoplasma/metabolismo , Núcleo Celular/metabolismoRESUMEN
Energy-demanding processes, such as cell growth, migration, and differentiation, are tension modulated, begging the question whether metabolism and mechanical tension are tightly linked. A recent report by Park et al. shows that stiffness in the extracellular matrix (ECM) promotes reorganization of actin, resulting in enhanced glycolysis.
Asunto(s)
Señales (Psicología) , Citoesqueleto , Actinas/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , GlucólisisRESUMEN
Understanding the role of nonequilibrium driving in self-organization is crucial for developing a predictive description of biological systems, yet it is impeded by their complexity. The actin cytoskeleton serves as a paradigm for how equilibrium and nonequilibrium forces combine to give rise to self-organization. Motivated by recent experiments that show that actin filament growth rates can tune the morphology of a growing actin bundle cross-linked by two competing types of actin-binding proteins [S. L. Freedman et al., Proc. Natl. Acad. Sci. U.S.A. 116, 16192-16197 (2019)], we construct a minimal model for such a system and show that the dynamics of a growing actin bundle are subject to a set of thermodynamic constraints that relate its nonequilibrium driving, morphology, and molecular fluxes. The thermodynamic constraints reveal the importance of correlations between these molecular fluxes and offer a route to estimating microscopic driving forces from microscopy experiments.
Asunto(s)
Biopolímeros/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Transporte de Proteínas/fisiología , TermodinámicaRESUMEN
The actin cytoskeleton, a dynamic network of actin filaments and associated F-actin-binding proteins, is fundamentally important in eukaryotes. α-Actinins are major F-actin bundlers that are inhibited by Ca2+ in nonmuscle cells. Here we report the mechanism of Ca2+-mediated regulation of Entamoeba histolytica α-actinin-2 (EhActn2) with features expected for the common ancestor of Entamoeba and higher eukaryotic α-actinins. Crystal structures of Ca2+-free and Ca2+-bound EhActn2 reveal a calmodulin-like domain (CaMD) uniquely inserted within the rod domain. Integrative studies reveal an exceptionally high affinity of the EhActn2 CaMD for Ca2+, binding of which can only be regulated in the presence of physiological concentrations of Mg2+ Ca2+ binding triggers an increase in protein multidomain rigidity, reducing conformational flexibility of F-actin-binding domains via interdomain cross-talk and consequently inhibiting F-actin bundling. In vivo studies uncover that EhActn2 plays an important role in phagocytic cup formation and might constitute a new drug target for amoebic dysentery.
Asunto(s)
Actinina/metabolismo , Calcio/farmacología , Entamoeba histolytica/metabolismo , Actinina/química , Actinina/genética , Dominio Catalítico , Entamoeba histolytica/genética , Regulación de la Expresión Génica , Modelos Moleculares , Conformación Proteica , Dominios ProteicosRESUMEN
We have previously shown that angiotensin-converting enzyme 2 (ACE2), an enzyme counterbalancing the deleterious effects of angiotensin type 1 receptor activation by production of vasodilatory peptides Angiotensin (Ang)-(1-9) and Ang-(1-7), is internalized and degraded in lysosomes following chronic Ang-II treatment. However, the molecular mechanisms involved in this effect remain unknown. In an attempt to identify the accessory proteins involved in this effect, we conducted a proteomic analysis in ACE2-transfected HEK293T cells. A single protein, fascin-1, was found to differentially interact with ACE2 after Ang-II treatment for 4 h. The interactions between fascin-1 and ACE2 were confirmed by confocal microscopy and co-immunoprecipitation. Overexpression of fascin-1 attenuates the effects of Ang-II on ACE2 activity. In contrast, downregulation of fascin-1 severely decreased ACE2 enzymatic activity. Interestingly, in brain homogenates from hypertensive mice, we observed a significant reduction of fascin-1, suggesting that the levels of this protein may change in cardiovascular diseases. In conclusion, we identified fascin-1 as an ACE2-accessory protein, interacting with the enzyme in an Ang-II dependent manner and contributing to the regulation of enzyme activity.
Asunto(s)
Actinas , Enzima Convertidora de Angiotensina 2 , Proteínas Portadoras , Proteínas de Microfilamentos , Actinas/metabolismo , Angiotensina I/metabolismo , Angiotensina II/metabolismo , Animales , Proteínas Portadoras/metabolismo , Células HEK293 , Humanos , Ratones , Proteínas de Microfilamentos/metabolismo , Fragmentos de Péptidos/metabolismo , ProteómicaRESUMEN
The post-translational modifications (PTMs), which are crucial in the regulation of protein functions, have great potential as biomarkers of cancer status. Fascin (Fascin actin-bundling protein 1, FSCN1), a key protein in the formation of filopodia that is structurally based on actin filaments (F-actin), is significantly associated with tumor invasion and metastasis. Studies have revealed various regulatory mechanisms of human Fascin, including PTMs. Although a number of Fascin PTM sites have been identified, their exact functions and clinical significance are much less explored. This review explores studies on the functions of Fascin and briefly discusses the regulatory mechanisms of Fascin. Next, to review the role of Fascin PTMs in cell biology and their associations with metastatic disease, we discuss the advances in the characterization of Fascin PTMs, including phosphorylation, ubiquitination, sumoylation, and acetylation, and the main regulatory mechanisms are discussed. Fascin PTMs may be potential targets for therapy for metastatic disease.
Asunto(s)
Citoesqueleto de Actina , Seudópodos , Humanos , Línea Celular Tumoral , Seudópodos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismoRESUMEN
The actin cytoskeleton operates in a multitude of cellular processes including cell shape and migration, mechanoregulation, and membrane or organelle dynamics. However, its filamentous properties and functions inside the mammalian cell nucleus are less well explored. We previously described transient actin assembly at mitotic exit that promotes nuclear expansion during chromatin decondensation. Here, we identify non-muscle α-actinin 4 (ACTN4) as a critical regulator to facilitate F-actin reorganization and bundling during postmitotic nuclear expansion. ACTN4 binds to nuclear actin filament structures, and ACTN4 clusters associate with nuclear F-actin in a highly dynamic fashion. ACTN4 but not ACTN1 is required for proper postmitotic nuclear volume expansion, mediated by its actin-binding domain. Using super-resolution imaging to quantify actin filament numbers and widths in individual nuclei, we find that ACTN4 is necessary for postmitotic nuclear actin reorganization and actin filament bundling. Our findings uncover a nuclear cytoskeletal function for ACTN4 to control nuclear size and chromatin organization during mitotic cell division.
Asunto(s)
Actinina , Actinas , Citoesqueleto de Actina , Actinina/genética , Actinas/genética , Animales , Núcleo Celular , CitoesqueletoRESUMEN
Pseudomonas aeruginosa uses a type III secretion system (T3SS) to inject cytotoxic effector proteins into host cells. The promiscuous nucleotidyl cyclase, exoenzyme Y (ExoY), is one of the most common effectors found in clinical P. aeruginosa isolates. Recent studies have revealed that the nucleotidyl cyclase activity of ExoY is stimulated by actin filaments (F-actin) and that ExoY alters actin cytoskeleton dynamics in vitro, via an unknown mechanism. The actin cytoskeleton plays an important role in numerous key biological processes and is targeted by many pathogens to gain competitive advantages. We utilized total internal reflection fluorescence microscopy, bulk actin assays, and EM to investigate how ExoY impacts actin dynamics. We found that ExoY can directly bundle actin filaments with high affinity, comparable with eukaryotic F-actin-bundling proteins, such as fimbrin. Of note, ExoY enzymatic activity was not required for F-actin bundling. Bundling is known to require multiple actin-binding sites, yet small-angle X-ray scattering experiments revealed that ExoY is a monomer in solution, and previous data suggested that ExoY possesses only one actin-binding site. We therefore hypothesized that ExoY oligomerizes in response to F-actin binding and have used the ExoY structure to construct a dimer-based structural model for the ExoY-F-actin complex. Subsequent mutational analyses suggested that the ExoY oligomerization interface plays a crucial role in mediating F-actin bundling. Our results indicate that ExoY represents a new class of actin-binding proteins that modulate the actin cytoskeleton both directly, via F-actin bundling, and indirectly, via actin-activated nucleotidyl cyclase activity.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Bacterianas/metabolismo , Glucosiltransferasas/metabolismo , Pseudomonas aeruginosa/enzimología , Citoesqueleto de Actina/ultraestructura , Factores Despolimerizantes de la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Actinas/ultraestructura , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/ultraestructura , Glucosiltransferasas/química , Glucosiltransferasas/genética , Glucosiltransferasas/ultraestructura , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Modelos Moleculares , Mutación/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
BACKGROUND: Metastasis is the predominant cause for cancer morbidity and mortality accounting for approximatively 90% of cancer deaths. The actin-bundling protein L-plastin has been proposed as a metastatic marker and phosphorylation on its residue Ser5 is known to increase its actin-bundling activity. We recently showed that activation of the ERK/MAPK signalling pathway leads to L-plastin Ser5 phosphorylation and that the downstream kinases RSK1 and RSK2 are able to directly phosphorylate Ser5. Here we investigate the involvement of the PI3K pathway in L-plastin Ser5 phosphorylation and the functional effect of this phosphorylation event in breast cancer cells. METHODS: To unravel the signal transduction network upstream of L-plastin Ser5 phosphorylation, we performed computational modelling based on immunoblot analysis data, followed by experimental validation through inhibition/overexpression studies and in vitro kinase assays. To assess the functional impact of L-plastin expression/Ser5 phosphorylation in breast cancer cells, we either silenced L-plastin in cell lines initially expressing endogenous L-plastin or neoexpressed L-plastin wild type and phosphovariants in cell lines devoid of endogenous L-plastin. The established cell lines were used for cell biology experiments and confocal microscopy analysis. RESULTS: Our modelling approach revealed that, in addition to the ERK/MAPK pathway and depending on the cellular context, the PI3K pathway contributes to L-plastin Ser5 phosphorylation through its downstream kinase SGK3. The results of the transwell invasion/migration assays showed that shRNA-mediated knockdown of L-plastin in BT-20 or HCC38 cells significantly reduced cell invasion, whereas stable expression of the phosphomimetic L-plastin Ser5Glu variant led to increased migration and invasion of BT-549 and MDA-MB-231 cells. Finally, confocal image analysis combined with zymography experiments and gelatin degradation assays provided evidence that L-plastin Ser5 phosphorylation promotes L-plastin recruitment to invadopodia, MMP-9 activity and concomitant extracellular matrix degradation. CONCLUSION: Altogether, our results demonstrate that L-plastin Ser5 phosphorylation increases breast cancer cell invasiveness. Being a downstream molecule of both ERK/MAPK and PI3K/SGK pathways, L-plastin is proposed here as a potential target for therapeutic approaches that are aimed at blocking dysregulated signalling outcome of both pathways and, thus, at impairing cancer cell invasion and metastasis formation. Video abstract.
Asunto(s)
Neoplasias de la Mama/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Quinasas/metabolismo , Neoplasias de la Mama/patología , Línea Celular , Movimiento Celular , Femenino , Humanos , Glicoproteínas de Membrana/genética , Proteínas de Microfilamentos/genética , Modelos Biológicos , Invasividad Neoplásica , Fosforilación , Serina/metabolismoRESUMEN
Many eukaryotes accomplish cell division by building and constricting a medial actomyosin-based cytokinetic ring (CR). In Schizosaccharomyces pombe, a Hippo-related signaling pathway termed the septation initiation network (SIN) controls CR formation, maintenance, and constriction. However, how the SIN regulates integral CR components was unknown. Here, we identify the essential cytokinetic formin Cdc12 as a key CR substrate of SIN kinase Sid2. Eliminating Sid2-mediated Cdc12 phosphorylation leads to persistent Cdc12 clustering, which prevents CR assembly in the absence of anillin-like Mid1 and causes CRs to collapse when cytokinesis is delayed. Molecularly, Sid2 phosphorylation of Cdc12 abrogates multimerization of a previously unrecognized Cdc12 domain that confers F-actin bundling activity. Taken together, our findings identify a SIN-triggered oligomeric switch that modulates cytokinetic formin function, revealing a novel mechanism of actin cytoskeleton regulation during cell division.
Asunto(s)
Citocinesis/fisiología , Proteínas del Citoesqueleto/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/citología , Schizosaccharomyces/metabolismo , Citoesqueleto de Actina/metabolismo , Citocinesis/genética , Proteínas del Citoesqueleto/genética , Fosforilación , Proteínas Quinasas/metabolismo , Multimerización de Proteína , Estructura Terciaria de Proteína , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Eliminación de SecuenciaRESUMEN
Phagocytosis, macropinocytosis and antigen presentation by dendritic cells (DC) requires reorganization of the actin cytoskeleton. Drebrin (Dbn1) is an actin binding and stabilizing protein with roles in endocytosis, formation of dendrite spines in neurons and coordinating cell-cell synapses in immune cells. However, its role in DC phagocytosis and antigen presentation is unknown. These studies now report that silencing of Dbn1 in DC resulted in restrained cell surface display of receptors, most notably MHC class I and II and co-stimulatory molecules. This, as expected, resulted in impaired antigen-specific T-cell activation and proliferation. Studies additionally revealed that knockdown of Dbn1 in DC impaired macropinocytosis and phagocytosis. However, there was a concomitant increase in fluid-phase uptake, suggesting that Dbn1 is responsible for the differential control of macropinocytosis versus micropinocytosis activities. Taken together, these findings now reveal that Dbn1 plays a major role in coordinating the actin cytoskeletal activities responsible for antigen presentation in DC.
Asunto(s)
Presentación de Antígeno , Células Dendríticas/inmunología , Regulación de la Expresión Génica/inmunología , Antígenos de Histocompatibilidad Clase II/inmunología , Antígenos de Histocompatibilidad Clase I/inmunología , Neuropéptidos/inmunología , Fagocitosis , Animales , Citoesqueleto/genética , Citoesqueleto/inmunología , Células Dendríticas/citología , Técnicas de Inactivación de Genes , Antígenos de Histocompatibilidad Clase I/genética , Antígenos de Histocompatibilidad Clase II/genética , Sinapsis Inmunológicas/genética , Sinapsis Inmunológicas/inmunología , Activación de Linfocitos/genética , Ratones , Ratones Transgénicos , Neuropéptidos/genética , Linfocitos T/citología , Linfocitos T/inmunologíaRESUMEN
Auditory reception relies on the perception of mechanical stimuli by stereocilia and its conversion to electrochemical signal. Mechanosensory stereocilia are abundant in actin, which provides them with structural conformity necessary for perception of auditory stimuli. Out of three major classes of actin-bundling proteins, plastin 1 encoded by PLS1, is highly expressed in stereocilia and is necessary for their regular maintenance. A missense PLS1 variant associated with autosomal dominant hearing loss (HL) in a small family has recently been reported. Here, we present another PLS1 missense variant, c.805G > A (p.E269K), in a Turkish family with autosomal dominant non-syndromic HL confirming the causative role of PLS1 mutations in HL. We propose that HL due to the p.E269K variant is from the loss of a stable PLS1-ACTB interaction.
Asunto(s)
Genes Dominantes , Pérdida Auditiva/genética , Glicoproteínas de Membrana/genética , Proteínas de Microfilamentos/genética , Mutación/genética , Secuencia de Aminoácidos , Secuencia de Bases , Familia , Femenino , Humanos , Masculino , Glicoproteínas de Membrana/química , Proteínas de Microfilamentos/química , Proteínas Mutantes/química , Linaje , TurquíaRESUMEN
Actin bundling protein 34 (ABP34) is the one of 11 actin-crosslinking proteins identified in Dictyostelium discoideum, a novel model organism for the study of actin-associated neurodegenerative disorders such as Alzheimer's disease and Huntington's disease. ABP34 localizes at the leading and trailing edges of locomotory cells, i.e., at the cell cortex, filopodia, and pseudopodia. Functionally, it serves to stabilize membrane-associated actin at sites of cell-cell contact. In addition, this small crosslinking protein is involved in actin bundle formation, and its bundling activity is regulated by the concentration of calcium ion. Several studies have sought to determine the mechanism underlying the calcium-regulated actin bundling activity of ABP34, but it remains unclear. Using several mutational and structural analyses, we revealed that calcium binding to the EF2 motif disrupts the inter-domain interaction between the N- and C-domains, thereby inhibiting the actin bundling activity of ABP34. This finding provides clues about the pathogenesis of neurodegenerative disorders related to actin bundling.
Asunto(s)
Actinas/metabolismo , Calcio/metabolismo , Proteínas de Microfilamentos/antagonistas & inhibidores , Factor 2 de Elongación Peptídica/metabolismo , Proteínas Protozoarias/antagonistas & inhibidores , Sitios de Unión , Cromatografía en Gel , Dictyostelium/química , Dictyostelium/metabolismo , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Factor 2 de Elongación Peptídica/química , Proteínas Protozoarias/genética , Proteínas Protozoarias/metabolismoRESUMEN
Fascin is an actin-bundling protein that, among immune cells, is restricted to expression in dendritic cells (DCs). Previous reports have suggested that fascin plays an important role in governing DC antigen presentation to CD4+ T cells. However, no report has clearly linked the receptor-ligand engagement that can direct downstream regulation of fascin expression. In this study, bone marrow-derived DCs from wild-type versus CD40-knockout C57BL/6 mice were used to elucidate the mechanisms of fascin expression and activity upon CD40-CD40 ligand (CD40L) engagement. These investigations now show that CD40 engagement governs fascin expression in DCs to promote CD4+ T-cell cytokine production. Absence of CD40 signaling resulted in diminished fascin expression in DCs and was associated with impaired CD4+ T-cell responses. Furthermore, the study found that loss of CD40-CD40L engagement resulted in reduced DC-T-cell contacts. Rescue by ectopic fascin expression in CD40-deficient DCs was able to re-establish sustained contacts with T cells and restore cytokine production. Taken together, these results show that cross-talk through CD40-CD40L signaling drives elevated fascin expression in DCs to support acquisition of full T-cell responses.
Asunto(s)
Presentación de Antígeno , Linfocitos T CD4-Positivos/inmunología , Antígenos CD40/inmunología , Ligando de CD40/inmunología , Proteínas Portadoras/biosíntesis , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Proteínas de Microfilamentos/biosíntesis , Animales , Antígenos CD40/deficiencia , Proteínas Portadoras/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas de Microfilamentos/inmunologíaRESUMEN
EF-hand domain-containing protein D2/Swiprosin-1 (EFhd2) is an actin-binding protein mainly expressed in the central nervous and the immune systems of mammals. Intracellular events linked to EFhd2, such as membrane protrusion formation, cell adhesion, and BCR signaling, are triggered by the association of EFhd2 and F-actin. We previously reported that Ca2+ enhances the F-actin-bundling ability of EFhd2 through maintaining a rigid parallel EFhd2-homodimer structure. It was also reported that the F-actin-bundling ability of EFhd2 is regulated by a phosphorylation-dependent mechanism. EGF-induced phosphorylation at Ser183 of EFhd2 has been shown to inhibit F-actin-bundling, leading to irregular actin dynamics at the leading edges of cells. However, the underlying mechanism of this inhibition has remained elusive. Here, we report the crystal structure of a phospho-mimicking mutant (S183E) of the EFhd2 core domain, where the actin-binding sites are located. Although the overall structure of the phospho-mimicking mutant is similar to the one of the unphosphorylated form, we observed a conformational transition from ordered to disordered structure in the linker region at the C-terminus of the mutant. Based on our structural and biochemical analyses, we suggest that phosphorylation at Ser183 of EFhd2 causes changes in the local conformational dynamics and the surface charge distribution of the actin-binding site, resulting in a re-coordination of the actin-binding sites in the dimer structure and a reduction of F-actin-bundling activity without affecting the F-actin-binding capacity.
Asunto(s)
Proteínas de Unión al Calcio/química , Proteínas de Unión al Calcio/metabolismo , Actinas/metabolismo , Sitios de Unión , Proteínas de Unión al Calcio/genética , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Mutación , Fosforilación , Conformación Proteica , Multimerización de Proteína , Serina/metabolismoRESUMEN
Evidence is accumulating for molecular microcompartments formed when proteins interact in localized domains with the cytoskeleton, organelle surfaces, and intracellular membranes. To understand the potential functional significance of protein microcompartmentation in plants, we studied the interaction of the glycolytic enzyme fructose bisphosphate aldolase with actin in Arabidopsis thaliana. Homology modelling of a major cytosolic isozyme of aldolase, FBA8, suggested that the tetrameric holoenzyme has two actin binding sites and could therefore act as an actin-bundling protein, as was reported for animal aldolases. This was confirmed by in vitro measurements of an increase in viscosity of F-actin polymerized in the presence of recombinant FBA8. Simultaneously, interaction with F-actin caused non-competitive inhibition of aldolase activity. We did not detect co-localization of an FBA8-RFP fusion protein, expressed in an fba8-knockout background, with the actin cytoskeleton using confocal laser-scanning microscopy. However, we did find evidence for a low level of interaction using FRET-FLIM analysis of FBA8-RFP co-expressed with the actin-binding protein GFP-Lifeact. Furthermore, knockout of FBA8 caused minor alterations of guard cell actin cytoskeleton morphology and resulted in a reduced rate of stomatal closure in response to decreased humidity. We conclude that cytosolic aldolase can be microcompartmented in vivo by interaction with the actin cytoskeleton and may subtly modulate guard cell behaviour as a result.
Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Arabidopsis/genética , Fructosa-Bifosfato Aldolasa/genética , Proteínas de Plantas/genética , Arabidopsis/enzimología , Arabidopsis/metabolismo , Citosol/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Microscopía Confocal , Proteínas de Plantas/metabolismoRESUMEN
The transport of germ cells across the seminiferous epithelium is composed of a series of cellular events during the epithelial cycle essential to the completion of spermatogenesis. Without the timely transport of spermatids during spermiogenesis, spermatozoa that are transformed from step 19 spermatids in the rat testis fail to reach the luminal edge of the apical compartment and enter the tubule lumen at spermiation, thereby arriving the epididymis for further maturation. Step 19 spermatids and/or sperms that remain in the epithelium beyond stage VIII of the epithelial cycle will be removed by the Sertoli cell via phagocytosis to form phagosomes and be degraded by lysosomes, leading to subfertility and/or infertility. However, the biology of spermatid transport, in particular the final events that lead to spermiation remain elusive. Based on recent data in the field, we critically evaluate the biology of spermiation herein by focusing on the actin binding proteins (ABPs) that regulate the organization of actin microfilaments at the Sertoli-spermatid interface, which is crucial for spermatid transport during this event. The hypothesis we put forth herein also highlights some specific areas of research that can be pursued by investigators in the years to come.
Asunto(s)
Proteínas de Microfilamentos/fisiología , Espermátides/fisiología , Actinas/fisiología , Animales , Humanos , Masculino , Multimerización de Proteína , Transducción de Señal , Transporte EspermáticoRESUMEN
Arp2/3 complex is an important actin filament nucleator that creates branched actin filament networks required for formation of lamellipodia and endocytic actin structures. Cellular assembly of branched actin networks frequently requires multiple Arp2/3 complex activators, called nucleation promoting factors (NPFs). We recently presented a mechanism by which cortactin, a weak NPF, can displace a more potent NPF, N-WASP, from nascent branch junctions to synergistically accelerate nucleation. The distinct roles of these NPFs in branching nucleation are surprising given their similarities. We biochemically dissected these two classes of NPFs to determine how their Arp2/3 complex and actin interacting segments modulate their influences on branched actin networks. We find that the Arp2/3 complex-interacting N-terminal acidic sequence (NtA) of cortactin has structural features distinct from WASP acidic regions (A) that are required for synergy between the two NPFs. Our mutational analysis shows that differences between NtA and A do not explain the weak intrinsic NPF activity of cortactin, but instead that cortactin is a weak NPF because it cannot recruit actin monomers to Arp2/3 complex. We use TIRF microscopy to show that cortactin bundles branched actin filaments using actin filament binding repeats within a single cortactin molecule, but that N-WASP antagonizes cortactin-mediated bundling. Finally, we demonstrate that multiple WASP family proteins synergistically activate Arp2/3 complex and determine the biochemical requirements in WASP proteins for synergy. Our data indicate that synergy between WASP proteins and cortactin may play a general role in assembling diverse actin-based structures, including lamellipodia, podosomes, and endocytic actin networks.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Cortactina/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Secuencia de Aminoácidos , Animales , Unión Competitiva , Bovinos , Análisis Mutacional de ADN , Microscopía Fluorescente , Datos de Secuencia Molecular , Músculo Esquelético/metabolismo , Unión Proteica , Seudópodos/metabolismo , Pirenos/química , Conejos , Homología de Secuencia de AminoácidoRESUMEN
The crystal structure of the 34â kDa F-actin-bundling protein ABP34 from Dictyostelium discoideum was solved by Ca(2+)/S-SAD phasing and refined at 1.89â Å resolution. ABP34 is a calcium-regulated actin-binding protein that cross-links actin filaments into bundles. Its in vitro F-actin-binding and F-actin-bundling activities were confirmed by a co-sedimentation assay and transmission electron microscopy. The co-localization of ABP34 with actin in cells was also verified. ABP34 adopts a two-domain structure with an EF-hand-containing N-domain and an actin-binding C-domain, but has no reported overall structural homologues. The EF-hand is occupied by a calcium ion with a pentagonal bipyramidal coordination as in the canonical EF-hand. The C-domain structure resembles a three-helical bundle and superposes well onto the rod-shaped helical structures of some cytoskeletal proteins. Residues 216-244 in the C-domain form part of the strongest actin-binding sites (193-254) and exhibit a conserved sequence with the actin-binding region of α-actinin and ABP120. Furthermore, the second helical region of the C-domain is kinked by a proline break, offering a convex surface towards the solvent area which is implicated in actin binding. The F-actin-binding model suggests that ABP34 binds to the side of the actin filament and residues 216-244 fit into a pocket between actin subdomains -1 and -2 through hydrophobic interactions. These studies provide insights into the calcium coordination in the EF-hand and F-actin-binding site in the C-domain of ABP34, which are associated through interdomain interactions.
Asunto(s)
Actinas/química , Dictyostelium/química , Proteínas Protozoarias/química , Secuencia de Aminoácidos , Secuencia de Bases , Cristalografía por Rayos X , ADN Complementario , Dictyostelium/genética , Dictyostelium/aislamiento & purificación , Modelos Moleculares , Datos de Secuencia Molecular , Peso Molecular , Conformación Proteica , Homología de Secuencia de AminoácidoRESUMEN
Formins are actin polymerization factors that are known to nucleate and elongate actin filaments at the barbed end. In the present study we show that human FHOD1 lacks actin nucleation and elongation capacity, but acts as an actin bundling factor with capping activity toward the filament barbed end. Constitutively active FHOD1 associates with actin filaments in filopodia and lamellipodia at the leading edge, where it moves with the actin retrograde flow. At the base of lamellipodia, FHOD1 is enriched in nascent, bundled actin arcs as well as in more mature stress fibers. This function requires actin-binding domains located N-terminally to the canonical FH1-FH2 element. The bundling phenotype is maintained in the presence of tropomyosin, confirmed by electron microscopy showing assembly of 5 to 10 actin filaments into parallel, closely spaced filament bundles. Taken together, our data suggest a model in which FHOD1 stabilizes actin filaments by protecting barbed ends from depolymerization with its dimeric FH2 domain, whereas the region N-terminal to the FH1 domain mediates F-actin bundling by simultaneously binding to the sides of adjacent F-actin filaments.