RESUMEN
Many cytoskeletal networks consist of individual filaments that are organized into elaborate higher-order structures. While it is appreciated that the size and architecture of these networks are critical for their biological functions, much of the work investigating control over their assembly has focused on mechanisms that regulate the turnover of individual filaments through size-dependent feedback. Here, we propose a very different, feedback-independent mechanism to explain how yeast cells control the length of their actin cables. Our findings, supported by quantitative cell imaging and mathematical modeling, indicate that actin cable length control is an emergent property that arises from the cross-linked and bundled organization of the filaments within the cable. Using this model, we further dissect the mechanisms that allow cables to grow longer in larger cells and propose that cell length-dependent tuning of formin activity allows cells to scale cable length with cell length. This mechanism is a significant departure from prior models of cytoskeletal filament length control and presents a different paradigm to consider how cells control the size, shape, and dynamics of higher-order cytoskeletal structures.
Asunto(s)
Citoesqueleto , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Eukaryotic cells contain branched actin networks that are essential for endocytosis, motility, and other key cellular processes. These networks, which are formed by filamentous actin and the Arp2/3 complex, must subsequently be debranched to allow network remodeling and to recycle the Arp2/3 complex. Debranching appears to be catalyzed by two different members of the actin depolymerizing factor homology protein family: cofilin and glial maturation factor (GMF). However, their mechanisms of debranching are only partially understood. Here, we used single-molecule fluorescence imaging of Arp2/3 complex and actin filaments under physiological ionic conditions to observe debranching by GMF and cofilin. We demonstrate that cofilin, like GMF, is an authentic debrancher independent of its filament-severing activity and that the debranching activities of the two proteins are additive. While GMF binds directly to the Arp2/3 complex, cofilin selectively accumulates on branch-junction daughter filaments in tropomyosin-decorated networks just prior to debranching events. Quantitative comparison of debranching rates with the known kinetics of cofilin-actin binding suggests that cofilin occupancy of a particular single actin site at the branch junction is sufficient to trigger debranching. In rare cases in which the order of departure could be resolved during GMF- or cofilin-induced debranching, the Arp2/3 complex left the branch junction bound to the pointed end of the daughter filament, suggesting that both GMF and cofilin can work by destabilizing the mother filament-Arp2/3 complex interface. Taken together, these observations suggest that GMF and cofilin promote debranching by distinct yet complementary mechanisms.
Asunto(s)
Factores Despolimerizantes de la Actina , Factor de Maduración de la Glia , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Factor de Maduración de la Glia/metabolismo , Microscopía Fluorescente , Imagen Individual de MoléculaRESUMEN
We study a reconstituted composite system consisting of an active microtubule network interdigitated with a passive network of entangled F-actin filaments. Increasing the concentration of filamentous actin controls the emergent dynamics, inducing a transition from turbulent-like flows to bulk contractions. At intermediate concentrations, where the active stresses change their symmetry from anisotropic extensile to isotropic contracting, the composite separates into layered asters that coexist with the background turbulent fluid. Contracted onion-like asters have a radially extending microtubule-rich cortex that envelops alternating layers of microtubules and F-actin. These self-regulating structures undergo internal reorganization, which appears to minimize the surface area and maintain the ordered layering, even when undergoing aster merging events. Finally, the layered asters are metastable structures. Their lifetime, which ranges from minutes to hours, is encoded in the material properties of the composite. These results challenge the current models of active matter. They demonstrate self-organized dynamical states and patterns evocative of those observed in the cytoskeleton do not require precise biochemical regulation, but can arise from purely mechanical interactions of actively driven filamentous materials.
Asunto(s)
Actinas/metabolismo , Microtúbulos/metabolismo , Movimiento/fisiología , Citoesqueleto de Actina/química , Citoesqueleto de Actina/fisiología , Actinas/química , Citoesqueleto/fisiología , Humanos , Microtúbulos/química , Microtúbulos/fisiología , Contracción Muscular/fisiologíaRESUMEN
Cyclase-associated protein (CAP) has emerged as a central player in cellular actin turnover, but its molecular mechanisms of action are not yet fully understood. Recent studies revealed that the N terminus of CAP interacts with the pointed ends of actin filaments to accelerate depolymerization in conjunction with cofilin. Here, we use in vitro microfluidics-assisted TIRF microscopy to show that the C terminus of CAP promotes depolymerization at the opposite (barbed) ends of actin filaments. In the absence of actin monomers, full-length mouse CAP1 and C-terminal halves of CAP1 (C-CAP1) and CAP2 (C-CAP2) accelerate barbed end depolymerization. Using mutagenesis and structural modeling, we show that these activities are mediated by the WH2 and CARP domains of CAP. In addition, we observe that CAP collaborates with profilin to accelerate barbed end depolymerization and that these effects depend on their direct interaction, providing the first known example of CAP-profilin collaborative effects in regulating actin. In the presence of actin monomers, CAP1 attenuates barbed end growth and promotes formin dissociation. Overall, these findings demonstrate that CAP uses distinct domains and mechanisms to interact with opposite ends of actin filaments and drive turnover. Further, they contribute to the emerging view of actin barbed ends as sites of dynamic molecular regulation, where numerous proteins compete and cooperate with each other to tune polymer dynamics, similar to the rich complexity seen at microtubule ends.
Asunto(s)
Citoesqueleto de Actina , Actinas , Proteínas del Citoesqueleto , Forminas , Proteínas de la Membrana , Animales , Ratones , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Factores Despolimerizantes de la Actina/genética , Factores Despolimerizantes de la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Forminas/metabolismo , Profilinas/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Polimerizacion , Dominios Proteicos/genética , Modelos Moleculares , Estructura Terciaria de ProteínaRESUMEN
The discovery of >60 monogenic causes of nephrotic syndrome (NS) has revealed a central role for the actin regulators RhoA/Rac1/Cdc42 and their effectors, including the formin INF2. By whole-exome sequencing (WES), we here discovered bi-allelic variants in the formin DAAM2 in four unrelated families with steroid-resistant NS. We show that DAAM2 localizes to the cytoplasm in podocytes and in kidney sections. Further, the variants impair DAAM2-dependent actin remodeling processes: wild-type DAAM2 cDNA, but not cDNA representing missense variants found in individuals with NS, rescued reduced podocyte migration rate (PMR) and restored reduced filopodia formation in shRNA-induced DAAM2-knockdown podocytes. Filopodia restoration was also induced by the formin-activating molecule IMM-01. DAAM2 also co-localizes and co-immunoprecipitates with INF2, which is intriguing since variants in both formins cause NS. Using in vitro bulk and TIRF microscopy assays, we find that DAAM2 variants alter actin assembly activities of the formin. In a Xenopus daam2-CRISPR knockout model, we demonstrate actin dysregulation in vivo and glomerular maldevelopment that is rescued by WT-DAAM2 mRNA. We conclude that DAAM2 variants are a likely cause of monogenic human SRNS due to actin dysregulation in podocytes. Further, we provide evidence that DAAM2-associated SRNS may be amenable to treatment using actin regulating compounds.
Asunto(s)
Actinas/metabolismo , Variación Genética , Proteínas de Microfilamentos/genética , Síndrome Nefrótico/genética , Proteínas de Unión al GTP rho/genética , Alelos , Animales , Animales Modificados Genéticamente , Movimiento Celular/genética , Citoplasma/metabolismo , Forminas/metabolismo , Humanos , Riñón/metabolismo , Glomérulos Renales/metabolismo , Mutación Missense , Podocitos/metabolismo , Seudópodos/metabolismo , ARN Interferente Pequeño/metabolismo , Secuenciación del Exoma , XenopusRESUMEN
The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.
Asunto(s)
Dictyostelium/genética , MAP Quinasa Quinasa Quinasa 3/genética , Proteínas Protozoarias/genética , Seudópodos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Animales , Sistemas CRISPR-Cas , Adhesión Celular , Línea Celular Tumoral , Quimiotaxis/genética , Dictyostelium/metabolismo , Dictyostelium/ultraestructura , Edición Génica/métodos , Regulación de la Expresión Génica , MAP Quinasa Quinasa Quinasa 3/metabolismo , Melanocitos/metabolismo , Melanocitos/ultraestructura , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Mutación , Células 3T3 NIH , Fenotipo , Fosforilación , Ploidias , Proteínas Protozoarias/metabolismo , Seudópodos/genética , Seudópodos/ultraestructura , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismoRESUMEN
Formins are highly conserved proteins that have essential roles in remodelling the actin and microtubule cytoskeletons to influence eukaryotic cell shape and behaviour. Recent work has identified numerous cellular factors that locally recruit, activate or inactivate formins to bridle and unleash their potent effects on actin nucleation and elongation. The effects of formins on microtubules have also begun to be described, which places formins in a prime position to coordinate actin and microtubule dynamics. The emerging complexity in the mechanisms governing formins mirrors the wide range of essential functions that they perform in cell motility, cell division and cell and tissue morphogenesis.
Asunto(s)
Actinas/metabolismo , Citoesqueleto/metabolismo , Proteínas Fetales/metabolismo , Proteínas de Microfilamentos/metabolismo , Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Actinas/química , Animales , Citoesqueleto/química , Forminas , Humanos , Microtúbulos/químicaRESUMEN
Actins are major eukaryotic cytoskeletal proteins, and they are involved in many important cell functions, including cell division, cell polarity, wound healing and muscle contraction. Despite obvious drawbacks, muscle actin, which is easily purified, is used extensively for biochemical studies of the non-muscle actin cytoskeleton. Here, we report a rapid and cost-effective method to purify heterologous actins expressed in the yeast Pichia pastoris Actin is expressed as a fusion with the actin-binding protein thymosin ß4 and purified by means of an affinity tag introduced in the fusion. Following cleavage of thymosin ß4 and the affinity tag, highly purified functional full-length actin is liberated. We purify actins from Saccharomycescerevisiae and Schizosaccharomycespombe, and the ß- and γ-isoforms of human actin. We also report a modification of the method that facilitates expression and purification of arginylated actin, a form of actin thought to regulate dendritic actin networks in mammalian cells. The methods we describe can be performed in all laboratories equipped for molecular biology, and should greatly facilitate biochemical and cell biological studies of the actin cytoskeleton.
Asunto(s)
Actinas/metabolismo , Isoformas de Proteínas/metabolismo , Animales , Humanos , PichiaRESUMEN
Mutations in profilin 1 (PFN1) are associated with amyotrophic lateral sclerosis (ALS); however, the pathological mechanism of PFN1 in this fatal disease is unknown. We demonstrate that ALS-linked mutations severely destabilize the native conformation of PFN1 in vitro and cause accelerated turnover of the PFN1 protein in cells. This mutation-induced destabilization can account for the high propensity of ALS-linked variants to aggregate and also provides rationale for their reported loss-of-function phenotypes in cell-based assays. The source of this destabilization is illuminated by the X-ray crystal structures of several PFN1 proteins, revealing an expanded cavity near the protein core of the destabilized M114T variant. In contrast, the E117G mutation only modestly perturbs the structure and stability of PFN1, an observation that reconciles the occurrence of this mutation in the control population. These findings suggest that a destabilized form of PFN1 underlies PFN1-mediated ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Mutación , Profilinas/química , Línea Celular , Cristalografía por Rayos X , Humanos , Neuronas/metabolismo , Profilinas/genética , Profilinas/metabolismo , Conformación Proteica , Pliegue de ProteínaRESUMEN
Toll-like receptors (TLRs) function as initiators of inflammation through their ability to sense pathogen-associated molecular patterns and products of tissue damage. Transcriptional activation of many TLR-responsive genes requires an initial de-repression step in which nuclear receptor co-repressor (NCoR) complexes are actively removed from the promoters of target genes to relieve basal repression. Ligand-dependent SUMOylation of liver X receptors (LXRs) has been found to suppress TLR4-induced transcription potently by preventing the NCoR clearance step, but the underlying mechanisms remain enigmatic. Here we provide evidence that coronin 2A (CORO2A), a component of the NCoR complex of previously unknown function, mediates TLR-induced NCoR turnover by a mechanism involving interaction with oligomeric nuclear actin. SUMOylated LXRs block NCoR turnover by binding to a conserved SUMO2/SUMO3-interaction motif in CORO2A and preventing actin recruitment. Intriguingly, the LXR transrepression pathway can itself be inactivated by inflammatory signals that induce calcium/calmodulin-dependent protein kinase IIγ (CaMKIIγ)-dependent phosphorylation of LXRs, leading to their deSUMOylation by the SUMO protease SENP3 and release from CORO2A. These findings uncover a CORO2A-actin-dependent mechanism for the de-repression of inflammatory response genes that can be differentially regulated by phosphorylation and by nuclear receptor signalling pathways that control immunity and homeostasis.
Asunto(s)
Actinas/metabolismo , Regulación de la Expresión Génica , Inflamación/genética , Proteínas de Microfilamentos/metabolismo , Actinas/química , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Línea Celular , Cisteína Endopeptidasas , Regulación de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Células HeLa , Homeostasis/genética , Humanos , Lipopolisacáridos/farmacología , Receptores X del Hígado , Ratones , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/deficiencia , Proteínas de Microfilamentos/genética , Receptores Nucleares Huérfanos/metabolismo , Péptido Hidrolasas/metabolismo , Peritonitis/inducido químicamente , Peritonitis/metabolismo , Fosforilación , Regiones Promotoras Genéticas/genética , Estructura Terciaria de Proteína , Transducción de Señal , Sumoilación , Tioglicolatos/farmacología , Receptores Toll-Like/metabolismoRESUMEN
Rapid and polarized turnover of actin networks is essential for motility, endocytosis, cytokinesis, and other cellular processes. However, the mechanisms that provide tight spatiotemporal control of actin disassembly remain poorly understood. Here, we show that yeast coronin (Crn1) makes a unique contribution to this process by differentially interacting with and regulating the effects of cofilin on ATP/ADP+P(i) versus ADP actin filaments. Crn1 potently blocks cofilin severing of newly assembled (ATP/ADP+P(i)) filaments but synergizes with cofilin to sever older (ADP) filaments. Thus, Crn1 has qualitatively distinct/opposite effects on actin dynamics depending on the nucleotide state of actin. This bimodal mechanism requires two separate actin-binding domains in Crn1. Consistent with these activities, Crn1 excludes GFP-Cof1 from newly assembled regions of actin networks in vivo and accelerates cellular actin turnover by four fold. We conclude that coronin polarizes the spatial distribution and activity of cofilin to promote selective disassembly of older actin filaments.
Asunto(s)
Actinas/metabolismo , Proteínas de Microfilamentos/metabolismo , Nucleótidos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Actinas/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Sitios de Unión , Cofilina 1/genética , Cofilina 1/metabolismo , Proteínas de Microfilamentos/genética , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Actin cables are linear cytoskeletal structures that serve as tracks for myosin-based intracellular transport of vesicles and organelles in both yeast and mammalian cells. In a yeast cell undergoing budding, cables are in constant dynamic turnover yet some cables grow from the bud neck toward the back of the mother cell until their length roughly equals the diameter of the mother cell. This raises the question: how is the length of these cables controlled? Here we describe a novel molecular mechanism for cable length control inspired by recent experimental observations in cells. This "antenna mechanism" involves three key proteins: formins, which polymerize actin, Smy1 proteins, which bind formins and inhibit actin polymerization, and myosin motors, which deliver Smy1 to formins, leading to a length-dependent actin polymerization rate. We compute the probability distribution of cable lengths as a function of several experimentally tuneable parameters such as the formin-binding affinity of Smy1 and the concentration of myosin motors delivering Smy1. These results provide testable predictions of the antenna mechanism of actin-cable length control.
Asunto(s)
Actinas/química , Actinas/metabolismo , Modelos Moleculares , Biología Computacional , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Polimerizacion , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Actin filament nucleation by actin-related protein (Arp) 2/3 complex is a critical process in cell motility and endocytosis, yet key aspects of its mechanism are unknown due to a lack of real-time observations of Arp2/3 complex through the nucleation process. Triggered by the verprolin homology, central, and acidic (VCA) region of proteins in the Wiskott-Aldrich syndrome protein (WASp) family, Arp2/3 complex produces new (daughter) filaments as branches from the sides of preexisting (mother) filaments. We visualized individual fluorescently labeled Arp2/3 complexes dynamically interacting with and producing branches on growing actin filaments in vitro. Branch formation was strikingly inefficient, even in the presence of VCA: only ~1% of filament-bound Arp2/3 complexes yielded a daughter filament. VCA acted at multiple steps, increasing both the association rate of Arp2/3 complexes with mother filament and the fraction of filament-bound complexes that nucleated a daughter. The results lead to a quantitative kinetic mechanism for branched actin assembly, revealing the steps that can be stimulated by additional cellular factors.
Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/química , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/química , Actinas/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/ultraestructura , Actinas/ultraestructura , Animales , Cinética , Microscopía de Fluorescencia por Excitación Multifotónica , Modelos Moleculares , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestructura , Familia de Proteínas del Síndrome de Wiskott-Aldrich/química , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/ultraestructuraRESUMEN
Srv2/CAP is a conserved actin-binding protein with important roles in driving cellular actin dynamics in diverse animal, fungal, and plant species. However, there have been conflicting reports about whether the activities of Srv2/CAP are conserved, particularly between yeast and mammalian homologs. Yeast Srv2 has two distinct functions in actin turnover: its hexameric N-terminal-half enhances cofilin-mediated severing of filaments, while its C-terminal-half catalyzes dissociation of cofilin from ADP-actin monomers and stimulates nucleotide exchange. Here, we dissected the structure and function of mouse CAP1 to better understand its mechanistic relationship to yeast Srv2. Although CAP1 has a shorter N-terminal oligomerization sequence compared with Srv2, we find that the N-terminal-half of CAP1 (N-CAP1) forms hexameric structures with six protrusions, similar to N-Srv2. Further, N-CAP1 autonomously binds to F-actin and decorates the sides and ends of filaments, altering F-actin structure and enhancing cofilin-mediated severing. These activities depend on conserved surface residues on the helical-folded domain. Moreover, N-CAP1 enhances yeast cofilin-mediated severing, and conversely, yeast N-Srv2 enhances human cofilin-mediated severing, highlighting the mechanistic conservation between yeast and mammals. Further, we demonstrate that the C-terminal actin-binding ß-sheet domain of CAP1 is sufficient to catalyze nucleotide-exchange of ADP-actin monomers, while in the presence of cofilin this activity additionally requires the WH2 domain. Thus, the structures, activities, and mechanisms of mouse and yeast Srv2/CAP homologs are remarkably well conserved, suggesting that the same activities and mechanisms underlie many of the diverse actin-based functions ascribed to Srv2/CAP homologs in different organisms.
Asunto(s)
Actinas/química , Proteínas Portadoras/química , Factores Despolimerizantes de la Actina/química , Actinas/ultraestructura , Proteínas Adaptadoras Transductoras de Señales/química , Adenosina Difosfato/química , Animales , Proteínas Portadoras/fisiología , Proteínas del Citoesqueleto/química , Células HEK293 , Humanos , Ratones , Unión Proteica , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/química , Especificidad de la EspecieRESUMEN
Formins perform essential roles in actin assembly and organization in vivo, but they also require tight regulation of their activities to produce properly functioning actin structures. Saccharomyces cerevisiae Bud14 is one member of an emerging class of formin regulators that target the FH2 domain to inhibit actin polymerization, but little is known about how these regulators are themselves controlled in vivo. Kelch proteins are critical for cell polarity and morphogenesis in a wide range of organisms, but their mechanistic roles in these processes are still largely undefined. Here, we report that S. cerevisiae Kelch proteins, Kel1 and Kel2, associate with Bud14 in cell extracts to form a stable 520-kDa complex with an apparent stoichiometry of 2:2:1 Bud14/Kel1/Kel2. Using pairwise combinations of GFP- and red fluorescent protein-tagged proteins, we show that Kel1, Kel2, and Bud14 interdependently co-localize at polarity sites. By analyzing single, double, and triple mutants, we show that Kel1 and Kel2 function in the same pathway as Bud14 in regulating Bnr1-mediated actin cable formation. Loss of any component of the complex results in long, bent, and hyper-stable actin cables, accompanied by defects in secretory vesicle traffic during polarized growth and septum formation during cytokinesis. These observations directly link S. cerevisiae Kelch proteins to the control of formin activity, and together with previous observations made for S. pombe homologues tea1p and tea3p, they have broad implications for understanding Kelch function in other systems.
Asunto(s)
Actinas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Actinas/química , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/genética , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Regulación Fúngica de la Expresión Génica , Proteínas de Microfilamentos/genética , Peso Molecular , Morfogénesis , Unión Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Formin proteins and their associated factors cooperate to assemble unbranched actin filaments in diverse cellular structures. The Saccharomyces cerevisiae formin Bni1 and its associated nucleation-promoting factor (NPF) Bud6 generate actin cables and mediate polarized cell growth. Bud6 binds to both the tail of the formin and G-actin, thereby recruiting monomeric actin to the formin to create a nucleation seed. Here, we structurally and functionally dissect the nucleation-promoting C-terminal region of Bud6 into a Bni1-binding "core" domain and a G-actin binding "flank" domain. The â¼2-Å resolution crystal structure of the Bud6 core domain reveals an elongated dimeric rod with a unique fold resembling a triple-helical coiled-coil. Binding and actin-assembly assays show that conserved residues on the surface of this domain mediate binding to Bni1 and are required for NPF activity. We find that the Bni1 dimer binds two Bud6 dimers and that the Bud6 flank binds a single G-actin molecule. These findings suggest a model in which a Bni1/Bud6 complex with a 2:4 subunit stoichiometry assembles a nucleation seed with Bud6 coordinating up to four actin subunits.
Asunto(s)
Proteínas de Microfilamentos/química , Proteínas de Saccharomyces cerevisiae/química , Actinas/química , Actinas/metabolismo , Secuencia de Aminoácidos , Unión Competitiva , Secuencia Conservada , Cristalografía por Rayos X , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Modelos Anatómicos , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Homología de Secuencia de Aminoácido , Electricidad EstáticaRESUMEN
BACKGROUND: Vertebrate APC collaborates with Dia through its Basic domain to assemble actin filaments. RESULTS: Despite limited sequence homology between the vertebrate and Drosophila APC Basic domains, Drosophila APC1 collaborates with Dia to stimulate actin assembly in vitro. CONCLUSION: The mechanism of actin assembly is highly conserved over evolution. SIGNIFICANCE: APC-Dia collaborations may be crucial in a wide range of animal cells. Adenomatous polyposis coli (APC) is a large multidomain protein that regulates the cytoskeleton. Recently, it was shown that vertebrate APC through its Basic domain directly collaborates with the formin mDia1 to stimulate actin filament assembly in the presence of nucleation barriers. However, it has been unclear whether these activities extend to homologues of APC and Dia in other organisms. Drosophila APC and Dia are each required to promote actin furrow formation in the syncytial embryo, suggesting a potential collaboration in actin assembly, but low sequence homology between the Basic domains of Drosophila and vertebrate APC has left their functional and mechanistic parallels uncertain. To address this question, we purified Drosophila APC1 and Dia and determined their individual and combined effects on actin assembly using both bulk fluorescence assays and total internal reflection fluorescence microscopy. Our data show that APC1, similar to its vertebrate homologue, bound to actin monomers and nucleated and bundled filaments. Further, Drosophila Dia nucleated actin assembly and protected growing filament barbed ends from capping protein. Drosophila APC1 and Dia directly interacted and collaborated to promote actin assembly in the combined presence of profilin and capping protein. Thus, despite limited sequence homology, Drosophila and vertebrate APCs exhibit highly related activities and mechanisms and directly collaborate with formins. These results suggest that APC-Dia interactions in actin assembly are conserved and may underlie important in vivo functions in a broad range of animal phyla.
Asunto(s)
Actinas/química , Proteínas Portadoras/química , Proteínas de Drosophila/química , Drosophila melanogaster , Multimerización de Proteína , Proteínas Supresoras de Tumor/química , Animales , Proteína CapZ/química , Proteínas del Citoesqueleto , Forminas , Cinética , Fragmentos de Péptidos/química , Profilinas/química , Estructura Terciaria de Proteína , Homología de Secuencia de AminoácidoRESUMEN
For over 20 years, the most effective probe for live imaging of yeast actin cables has been Abp140-GFP. Here, we report that endogenously-tagged Abp140-GFP poorly decorates actin patches and cables in the bud compartment of yeast cells, while robustly decorating these structures in the mother cell. Using mutagenesis, we found that asymmetric decoration by Abp140 requires F-actin binding. By expressing integrated Bni1-Bnr1 and Bnr1-Bni1 chimeras, we demonstrate that asymmetric cable decoration by Abp140 also does not depend on which formin assembles the cables in each compartment. In contrast, the short actin-binding fragment of Abp140 (known as "Lifeact"), fused to 1x or 3xmNeonGreen and expressed from the endogenous ABP140 promoter, uniformly decorates patches and cables in both compartments. Further, this probe dramatically improves live imaging detection of cables (and patches) without altering their in vivo dynamics or cell growth. Improved detection allows us to visualize cables growing inward from the cell cortex and dynamically interacting with the vacuole. This probe also robustly decorates the cytokinetic actomyosin ring. Because Lifeact-3xmNeon expressed at relatively low levels provides intense labeling of cellular F-actin structures, this tool may improve live imaging in other organisms where higher levels of Lifeact expression are detrimental.
Asunto(s)
Actinas , Proteínas de Microfilamentos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Actinas/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Microfilamentos/metabolismo , Proteínas de Microfilamentos/genética , Proteínas Fluorescentes Verdes/metabolismo , Citoesqueleto de Actina/metabolismo , Unión Proteica , Proteínas del CitoesqueletoRESUMEN
How cells tightly control the formation and turnover of branched actin filament arrays to drive cell motility, endocytosis, and other cellular processes is still not well understood. Here, we investigated the mechanistic relationship between two binding partners of the Arp2/3 complex, glia maturation factor (GMF) and cortactin. Individually, GMF and cortactin have opposite effects on the stability of actin filament branches, but it is unknown how they work in concert with each other to govern branch turnover. Using TIRF microscopy, we observe that GMF's branch destabilizing activities are potently blocked by cortactin (IC50 = 1.3 nM) and that this inhibition requires direct interactions of cortactin with Arp2/3 complex. The simplest model that would explain these results is competition for binding Arp2/3 complex. However, we find that cortactin and GMF do not compete for free Arp2/3 complex in solution. Further, we use single molecule analysis to show that cortactin's on-rate (3 ×107 s-1 M-1) and off-rate (0.03 s-1) at branch junctions are minimally affected by excess GMF. Together, these results show that cortactin binds with high affinity to branch junctions, where it blocks the destabilizing effects of GMF, possibly by a mechanism that is allosteric in nature. In addition, the affinities we measure for cortactin at actin filament branch junctions (Kd = 0.9 nM) and filament sides (Kd = 206 nM) are approximately 20-fold stronger than previously reported. These observations contribute to an emerging view of molecular complexity in how Arp2/3 complex is regulated through the integration of multiple inputs.
Asunto(s)
Cortactina , Factor de Maduración de la Glia , Factor de Maduración de la Glia/genética , Factor de Maduración de la Glia/química , Factor de Maduración de la Glia/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismoRESUMEN
In response to pheromone Saccharomyces cerevisiae extend a mating projection. This process depends on the formation of polarized actin cables which direct secretion to the mating tip and translocate the nucleus for karyogamy. Here, we demonstrate that proper mating projection formation requires the formin Bni1, as well as the actin nucleation promoting activities of Bud6, but not the formin Bnr1. Further, Bni1 is required for pheromone gradient tracking. Our work also reveals unexpected new functions for Bil2 in the pheromone response. Previously we identified Bil2 as a direct inhibitor of Bnr1 during vegetative cell growth. Here, we show that Bil2 has Bnr1-independent functions in spatially focusing Bni1-GFP at mating projection tips, and in vitro Bil2 and its binding partner Bud6 organize Bni1 into clusters that nucleate actin assembly. bil2∆ cells also display entangled Bni1-generated actin cable arrays and defects in secretory vesicle transport and nuclear positioning. At low pheromone concentrations, bil2∆ cells are delayed in establishing a polarity axis, and at high concentrations they prematurely form a second and a third mating projection. Together, these results suggest that Bil2 promotes the proper formation and timing of mating projections by organizing Bni1 and maintaining a persistent axis of polarized growth.