RESUMO
Multiciliated ependymal cells line all brain cavities. The beating of their motile cilia contributes to the flow of cerebrospinal fluid, which is required for brain homoeostasis and functions. Motile cilia, nucleated from centrioles, persist once formed and withstand the forces produced by the external fluid flow and by their own cilia beating. Here, we show that a dense actin network around the centrioles is induced by cilia beating, as shown by the disorganisation of the actin network upon impairment of cilia motility. Moreover, disruption of the actin network, or specifically of the apical actin network, causes motile cilia and their centrioles to detach from the apical surface of ependymal cell. In conclusion, cilia beating controls the apical actin network around centrioles; the mechanical resistance of this actin network contributes, in turn, to centriole stability.
Assuntos
Actinas/fisiologia , Centríolos/fisiologia , Cílios/fisiologia , Epêndima/fisiologia , Actinas/química , Animais , Fenômenos Biomecânicos , Proteínas do Citoesqueleto , Epêndima/crescimento & desenvolvimento , Epêndima/ultraestrutura , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Proteínas dos Microfilamentos , Modelos Neurológicos , Mapas de Interação de Proteínas , Proteínas/genética , Proteínas/metabolismoRESUMO
A living cell's ability to assemble actin filaments in intracellular motile processes is directly dependent on the availability of polymerizable actin monomers, which feed polarized filament growth [1, 2]. Continued generation of the monomer pool by filament disassembly is therefore crucial. Disassemblers like actin depolymerizing factor (ADF)/cofilin and filament cappers like capping protein (CP) are essential agonists of motility [3-8], but the exact molecular mechanisms by which they accelerate actin polymerization at the leading edge and filament turnover has been debated for over two decades [9-12]. Whereas filament fragmentation by ADF/cofilin has long been demonstrated by total internal reflection fluorescence (TIRF) [13, 14], filament depolymerization was only inferred from bulk solution assays [15]. Using microfluidics-assisted TIRF microscopy, we provide the first direct visual evidence of ADF's simultaneous severing and rapid depolymerization of individual filaments. Using a conceptually novel assay to directly visualize ADF's effect on a population of pre-assembled filaments, we demonstrate how ADF's enhanced pointed-end depolymerization causes an increase in polymerizable actin monomers, thus promoting faster barbed-end growth. We further reveal that ADF-enhanced depolymerization synergizes with CP's long-predicted "monomer funneling" [16] and leads to skyrocketing of filament growth rates, close to estimated lamellipodial rates. The "funneling model" hypothesized, on thermodynamic grounds, that at high enough extent of capping, the few non-capped filaments transiently grow much faster [15], an effect proposed to be very important for motility. We provide the first direct microscopic evidence of monomer funneling at the scale of individual filaments. These results significantly enhance our understanding of the turnover of cellular actin networks.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Fatores de Despolimerização de Actina/metabolismo , Destrina/metabolismo , Animais , Movimento Celular , Humanos , Polimerização , CoelhosRESUMO
Various cellular processes (including cell motility) are driven by the regulated, polarized assembly of actin filaments into distinct force-producing arrays of defined size and architecture. Branched, linear, contractile and cytosolic arrays coexist in vivo, and cells intricately control the number, length and assembly rate of filaments in these arrays. Recent in vitro and in vivo studies have revealed novel molecular mechanisms that regulate the number of filament barbed and pointed ends and their respective assembly and disassembly rates, thus defining classes of dynamically different filaments, which coexist in the same cell. We propose that a global treadmilling process, in which a steady-state amount of polymerizable actin monomers is established by the dynamics of each network, is responsible for defining the size and turnover of coexisting actin networks. Furthermore, signal-induced changes in the partitioning of actin to distinct arrays (mediated by RHO GTPases) result in the establishment of various steady-state concentrations of polymerizable monomers, thereby globally influencing the growth rate of actin filaments.
Assuntos
Actinas/metabolismo , Actinas/genética , Animais , Humanos , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
Migration frequently involves Rac-mediated protrusion of lamellipodia, formed by Arp2/3 complex-dependent branching thought to be crucial for force generation and stability of these networks. The formins FMNL2 and FMNL3 are Cdc42 effectors targeting to the lamellipodium tip and shown here to nucleate and elongate actin filaments with complementary activities in vitro. In migrating B16-F1 melanoma cells, both formins contribute to the velocity of lamellipodium protrusion. Loss of FMNL2/3 function in melanoma cells and fibroblasts reduces lamellipodial width, actin filament density and -bundling, without changing patterns of Arp2/3 complex incorporation. Strikingly, in melanoma cells, FMNL2/3 gene inactivation almost completely abolishes protrusion forces exerted by lamellipodia and modifies their ultrastructural organization. Consistently, CRISPR/Cas-mediated depletion of FMNL2/3 in fibroblasts reduces both migration and capability of cells to move against viscous media. Together, we conclude that force generation in lamellipodia strongly depends on FMNL formin activity, operating in addition to Arp2/3 complex-dependent filament branching.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas/metabolismo , Pseudópodes/metabolismo , Animais , Fenômenos Biomecânicos , Sistemas CRISPR-Cas/genética , Movimento Celular , Fibroblastos/metabolismo , Forminas , Técnicas de Silenciamento de Genes , Melanoma Experimental/patologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Células NIH 3T3 , Fenótipo , Polimerização , Pseudópodes/ultraestrutura , Interferência de RNARESUMO
Cell migration and cell-cell communication involve the protrusion of actin-rich cell surface projections such as lamellipodia and filopodia. Lamellipodia are networks of actin filaments generated and turned over by filament branching through the Arp2/3 complex. Inhibition of branching is commonly agreed to eliminate formation and maintenance of lamellipodial actin networks, but the regulation of nucleation or elongation of Arp2/3-independent filament populations within the network by, for example, formins or Ena/VASP family members and its influence on the effectiveness of protrusion have been unclear. Here we analyzed the effects of a set of distinct formin fragments and VASP on site-specific, lamellipodial versus cytosolic actin assembly and resulting consequences on protrusion. Surprisingly, expression of formin variants but not VASP reduced lamellipodial protrusion in B16-F1 cells, albeit to variable extents. The rates of actin network polymerization followed a similar trend. Unexpectedly, the degree of inhibition of both parameters depended on the extent of cytosolic but not lamellipodial actin assembly. Indeed, excess cytosolic actin assembly prevented actin monomer from rapid translocation to and efficient incorporation into lamellipodia. Thus, as opposed to sole regulation by actin polymerases operating at their tips, the protrusion efficiency of lamellipodia is determined by a finely tuned balance between lamellipodial and cytosolic actin assembly.
Assuntos
Moléculas de Adesão Celular/metabolismo , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Pseudópodes/fisiologia , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Citosol/metabolismo , Humanos , Pseudópodes/metabolismoRESUMO
Cells respond to external stimuli by rapidly remodeling their actin cytoskeleton. At the heart of this function lies the intricately controlled regulation of individual filaments. The barbed end of an actin filament is the hotspot for the majority of the biochemical reactions that control filament assembly. Assays performed in bulk solution and with single filaments have enabled characterization of a plethora of barbed-end-regulating proteins. Interestingly, many of these regulators work in tandem with other proteins, which increase or decrease their affinity for the barbed end in a spatially and temporally controlled manner, often through simultaneous binding of two regulators at the barbed ends, in addition to standard mutually exclusive binding schemes. In this Cell Science at a Glance and the accompanying poster, we discuss key barbed-end-interacting proteins and the kinetic mechanisms by which they regulate actin filament assembly. We take F-actin capping protein, gelsolin, profilin and barbed-end-tracking polymerases, including formins and WH2-domain-containing proteins, as examples, and illustrate how their activity and competition for the barbed end regulate filament dynamics.
Assuntos
Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Actinas/genética , Actinas/metabolismo , Animais , Citoesqueleto/genética , Citoesqueleto/metabolismo , HumanosRESUMO
Cell motility and actin homeostasis depend on the control of polarized growth of actin filaments. Profilin, an abundant regulator of actin dynamics, supports filament assembly at barbed ends by binding G-actin. Here, we demonstrate how, by binding and destabilizing filament barbed ends at physiological concentrations, profilin also controls motility, cell migration, and actin homeostasis. Profilin enhances filament length fluctuations. Profilin competes with Capping Protein at barbed ends, which generates a lower amount of profilin-actin than expected if barbed ends were tightly capped. Profilin competes with barbed end polymerases, such as formins and VopF, and inhibits filament branching by WASP-Arp2/3 complex by competition for filament barbed ends, accounting for its as-yet-unknown effects on motility and metastatic cell migration observed in this concentration range. In conclusion, profilin is a major coordinator of polarized growth of actin filaments, controlled by competition between barbed end cappers, trackers, destabilizers, and filament branching machineries.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Citoesqueleto/metabolismo , Profilinas/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Movimento Celular/fisiologia , Ligação Proteica/fisiologia , CoelhosRESUMO
Polarized assembly of actin filaments forms the basis of actin-based motility and is regulated both spatially and temporally. Cells use a variety of mechanisms by which intrinsically slower processes are accelerated, and faster ones decelerated, to match rates observed in vivo. Here we discuss how kinetic studies of individual reactions and cycles that drive actin remodeling have provided a mechanistic and quantitative understanding of such processes. We specifically consider key barbed-end regulators such as capping protein and formins as illustrative examples. We compare and contrast different kinetic approaches, such as the traditional pyrene-polymerization bulk assays, as well as more recently developed single-filament and single-molecule imaging approaches. Recent development of novel biophysical methods for sensing and applying forces will in future allow us to address the very important relationship between mechanical stimulus and kinetics of actin-based motility.
Assuntos
Citoesqueleto de Actina/metabolismo , Movimento Celular/fisiologia , Citoesqueleto/metabolismo , Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto/química , Proteínas Fetais/química , Proteínas Fetais/metabolismo , Forminas , Cinética , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Profilinas/química , Profilinas/metabolismo , Ligação ProteicaRESUMO
Formin 2 (Fmn2), a member of the FMN family of formins, plays an important role in early development. This formin cooperates with profilin and Spire, a WASP homology domain 2 (WH2) repeat protein, to stimulate assembly of a dynamic cytoplasmic actin meshwork that facilitates translocation of the meiotic spindle in asymmetric division of mouse oocytes. The kinase-like non-catalytic domain (KIND) of Spire directly interacts with the C-terminal extension of the formin homology domain 2 (FH2) domain of Fmn2, called FSI. This direct interaction is required for the synergy between the two proteins in actin assembly. We have recently demonstrated how Spire, which caps barbed ends via its WH2 domains, activates Fmn2. Fmn2 by itself associates very poorly to filament barbed ends but is rapidly recruited to Spire-capped barbed ends via the KIND domain, and it subsequently displaces Spire from the barbed end to elicit rapid processive assembly from profilin·actin. Here, we address the mechanism by which Spire and Fmn2 compete at barbed ends and the role of FSI in orchestrating this competition as well as in the processivity of Fmn2. We have combined microcalorimetric, fluorescence, and hydrodynamic binding assays, as well as bulk solution and single filament measurements of actin assembly, to show that removal of FSI converts Fmn2 into a Capping Protein. This activity is mimicked by association of KIND to Fmn2. In addition, FSI binds actin at filament barbed ends as a weak capper and plays a role in displacing the WH2 domains of Spire from actin, thus allowing the association of actin-binding regions of FH2 to the barbed end.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Modelos Moleculares , Proteínas Nucleares/metabolismo , Citoesqueleto de Actina/química , Animais , Ligação Competitiva , Sequência Conservada , Forminas , Deleção de Genes , Humanos , Cinética , Camundongos , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Peso Molecular , Proteínas do Tecido Nervoso , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
The Arp2/3 complex consists of seven evolutionarily conserved subunits (Arp2, Arp3 and ARPC1-5) and plays an essential role in generating branched actin filament networks during many different cellular processes. In mammals, however, the ARPC1 and ARPC5 subunits are each encoded by two isoforms that are 67% identical. This raises the possibility that Arp2/3 complexes with different properties may exist. We found that Arp2/3 complexes containing ARPC1B and ARPC5L are significantly better at promoting actin assembly than those with ARPC1A and ARPC5, both in cells and in vitro. Branched actin networks induced by complexes containing ARPC1B or ARPC5L are also disassembled â¼2-fold slower than those formed by their counterparts. This difference reflects the ability of cortactin to stabilize ARPC1B- and ARPC5L- but not ARPC1A- and ARPC5-containing complexes against coronin-mediated disassembly. Our observations demonstrate that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteína 2 Relacionada a Actina/metabolismo , Proteína 3 Relacionada a Actina/metabolismo , Angiopoietinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteína 2 Semelhante a Angiopoietina , Proteínas Semelhantes a Angiopoietina , Animais , Linhagem Celular , Cortactina/metabolismo , Humanos , Camundongos , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
Proteins targeting actin filament barbed ends play a pivotal role in motile processes. While formins enhance filament assembly, capping protein (CP) blocks polymerization. On their own, they both bind barbed ends with high affinity and very slow dissociation. Their barbed-end binding is thought to be mutually exclusive. CP has recently been shown to be present in filopodia and controls their morphology and dynamics. Here we explore how CP and formins may functionally coregulate filament barbed-end assembly. We show, using kinetic analysis of individual filaments by microfluidics-assisted fluorescence microscopy, that CP and mDia1 formin are able to simultaneously bind barbed ends. This is further confirmed using single-molecule imaging. Their mutually weakened binding enables rapid displacement of one by the other. We show that formin FMNL2 behaves similarly, thus suggesting that this is a general property of formins. Implications in filopodia regulation and barbed-end structural regulation are discussed.
Assuntos
Proteínas de Capeamento de Actina/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas/metabolismo , Proteínas de Capeamento de Actina/química , Citoesqueleto de Actina/química , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Forminas , Humanos , Cinética , Ligação Proteica , Proteínas/química , Proteínas/genética , CoelhosRESUMO
Actin cytoskeleton remodeling, which drives changes in cell shape and motility, is orchestrated by a coordinated control of polarized assembly of actin filaments. Signal responsive, membrane-bound protein machineries initiate and regulate polarized growth of actin filaments by mediating transient links with their barbed ends, which elongate from polymerizable actin monomers. The barbed end of an actin filament thus stands out as a hotspot of regulation of filament assembly. It is the target of both soluble and membrane-bound agonists as well as antagonists of filament assembly. Here, we review the molecular mechanisms by which various regulators of actin dynamics bind, synergize or compete at filament barbed ends. Two proteins can compete for the barbed end via a mutually exclusive binding scheme. Alternatively, two regulators acting individually at barbed ends may be bound together transiently to terminal actin subunits at barbed ends, leading to the displacement of one by the other. The kinetics of these reactions is a key in understanding how filament length and membrane-filament linkage are controlled. It is also essential for understanding how force is produced to shape membranes by mechano-sensitive, processive barbed end tracking machineries like formins and by WASP-Arp2/3 branched filament arrays. A combination of biochemical and biophysical approaches, including bulk solution assembly measurements using pyrenyl-actin fluorescence, single filament dynamics, single molecule fluorescence imaging and reconstituted self-organized filament assemblies, have provided mechanistic insight into the role of actin polymerization in motile processes.
Assuntos
Citoesqueleto de Actina/fisiologia , Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Modelos Biológicos , Modelos Moleculares , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteínas dos Microfilamentos/metabolismo , Imagem Óptica/métodos , Ligação Proteica , Conformação ProteicaRESUMO
VopF and VopL are highly similar virulence-factors of Vibrio cholerae and Vibrio parahaemolyticus respectively that disrupt the host's actin cytoskeleton, using a unique organization in dimerized WH2 repeats. Association of dimerized WH2 domains with the barbed face of actin confers multifunctional activities to VopF in vitro, including G-actin sequestration and filament nucleation, barbed end tracking and uncapping. Here, small angle X-ray scattering (SAXS) measurements of complexes of VopF with actin and structural modeling reveal that VopF stabilizes linear actin-strings that differ from canonical actin filament architectures but represent non-polymerizable sequestered forms of actin. The results exclude that VopL binds the pointed end of actin filaments in the template filament nucleation mechanism derived from crystallographic studies.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas da Membrana Bacteriana Externa/química , Modelos Moleculares , Vibrio cholerae/química , Fatores de Virulência/química , Proteínas da Membrana Bacteriana Externa/metabolismo , Dimerização , Conformação Proteica , Estrutura Terciária de Proteína , Espalhamento a Baixo Ângulo , Fatores de Virulência/metabolismoRESUMO
The actin cytoskeleton has the unique capability of producing pushing forces at the leading edge of motile cells without the implication of molecular motors. This phenomenon has been extensively studied theoretically, and molecular models, including the widely known Brownian ratchet, have been proposed. However, supporting experimental work is lacking, due in part to hardly accessible molecular length scales. We designed an experiment to directly probe the mechanism of force generation in a setup where a population of actin filaments grows against a load applied by magnetic microparticles. The filaments, arranged in stiff bundles by fascin, are constrained to point toward the applied load. In this protrusion-like geometry, we are able to directly measure the velocity of filament elongation and its dependence on force. Using numerical simulations, we provide evidence that our experimental data are consistent with a Brownian ratchet-based model. We further demonstrate the existence of a force regime far below stalling where the mechanical power transduced by the ratcheting filaments to the load is maximal. The actin machinery in migrating cells may tune the number of filaments at the leading edge to work in this force regime.
Assuntos
Actinas/fisiologia , Movimento Celular/fisiologia , Modelos Biológicos , Animais , Fenômenos Biomecânicos/fisiologia , Simulação por Computador , Fluorescência , Cinética , Magnetismo , Polímeros , Coelhos , TermodinâmicaRESUMO
In mammalian oocytes, three actin binding proteins, Formin 2 (Fmn2), Spire, and profilin, synergistically organize a dynamic cytoplasmic actin meshwork that mediates translocation of the spindle toward the cortex and is required for successful fertilization. Here we characterize Fmn2 and elucidate the molecular mechanism for this synergy, using bulk solution and individual filament kinetic measurements of actin assembly dynamics. We show that by capping filament barbed ends, Spire recruits Fmn2 and facilitates its association with barbed ends, followed by rapid processive assembly and release of Spire. In the presence of actin, profilin, Spire, and Fmn2, filaments display alternating phases of rapid processive assembly and arrested growth, driven by a "ping-pong" mechanism, in which Spire and Fmn2 alternately kick off each other from the barbed ends. The results are validated by the effects of injection of Spire, Fmn2, and their interacting moieties in mouse oocytes. This original mechanism of regulation of a Rho-GTPase-independent formin, recruited by Spire at Rab11a-positive vesicles, supports a model for modulation of a dynamic actin-vesicle meshwork in the oocyte at the origin of asymmetric positioning of the meiotic spindle.
Assuntos
Actinas/química , Meiose , Proteínas dos Microfilamentos/fisiologia , Proteínas Nucleares/fisiologia , Actinas/metabolismo , Animais , Células Cultivadas , Retroalimentação Fisiológica , Forminas , Humanos , Cinética , Camundongos , Proteínas dos Microfilamentos/química , Proteínas do Tecido Nervoso , Proteínas Nucleares/química , Oócitos/metabolismo , Profilinas/química , Ligação Proteica , Multimerização ProteicaRESUMO
We describe how combining microfluidics with TIRF and epifluorescence microscopy can greatly facilitate the quantitative analysis of actin assembly dynamics and its regulation, as well as the exploration of issues that were often out of reach with standard single-filament microscopy, such as the kinetics of processes linked to actin self-assembly or the kinetics of interaction with regulators. We also show how the viscous drag force exerted by fluid flowing on the filaments can be calibrated in order to assess the mechanosensitivity of end-binding protein machineries such as formins or adhesion proteins. We also discuss how microfluidics, in conjunction with other techniques, could be used to address the mechanism of coordination between heterogeneous populations of filaments, or the behavior of individual filaments during regulated treadmilling. These techniques also can be applied to study the assembly and regulation of other cytoskeletal polymers such as microtubules, septins, intermediate filaments, as well as the transport of cargoes by molecular motors under a flow-produced load.
Assuntos
Citoesqueleto de Actina/metabolismo , Técnicas Analíticas Microfluídicas/instrumentação , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestrutura , Animais , Desenho de Equipamento , Cinética , Técnicas Analíticas Microfluídicas/métodos , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Propriedades de SuperfícieRESUMO
Several pathogens induce propulsive actin comet tails in cells they invade to disseminate their infection. They achieve this by recruiting factors for actin nucleation, the Arp2/3 complex, and polymerization regulators from the host cytoplasm. Owing to limited information on the structural organization of actin comets and in particular the spatial arrangement of filaments engaged in propulsion, the underlying mechanism of pathogen movement is currently speculative and controversial. Using electron tomography we have resolved the three-dimensional architecture of actin comet tails propelling baculovirus, the smallest pathogen yet known to hijack the actin motile machinery. Comet tail geometry was also mimicked in mixtures of virus capsids with purified actin and a minimal inventory of actin regulators. We demonstrate that propulsion is based on the assembly of a fishbone-like array of actin filaments organized in subsets linked by branch junctions, with an average of four filaments pushing the virus at any one time. Using an energy-minimizing function we have simulated the structure of actin comet tails as well as the tracks adopted by baculovirus in infected cells in vivo. The results from the simulations rule out gel squeezing models of propulsion and support those in which actin filaments are continuously tethered during branch nucleation and polymerization. Since Listeria monocytogenes, Shigella flexneri, and Vaccinia virus among other pathogens use the same common toolbox of components as baculovirus to move, we suggest they share the same principles of actin organization and mode of propulsion.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Complexo 2-3 de Proteínas Relacionadas à Actina/ultraestrutura , Baculoviridae/ultraestrutura , Modelos Estatísticos , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Baculoviridae/química , Baculoviridae/fisiologia , Ensaio Cometa , Tomografia com Microscopia Eletrônica , Expressão Gênica , Genes Reporter , Carpa Dourada , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Melanoma Experimental , Células Sf9 , Spodoptera , Proteína Vermelha FluorescenteRESUMO
Cordon-Bleu (Cobl) is a regulator of actin dynamics in neural development and ciliogenesis. Its function is associated with three adjacent actin binding WASP Homology 2 (WH2) domains. We showed that these WH2 repeats confer multifunctional regulation of actin dynamics, which makes Cobl a « dynamizer ¼ of actin assembly, inducing fast turnover of actin filaments and oscillatory polymerization regime via nucleation, severing, and rapid depolymerization activities. Cobl is the most efficient severer of actin filaments characterized so far. To understand which primary sequence elements determine the filament severing activity of the WH2 repeats, here we combine a mutagenetic/domain swapping approach of the minimal fully active Cobl-KAB construct, which comprises the lysine rich region K preceding the two first WH2 domains A and B. The mutated Cobl constructs display variable loss of the original filament nucleating activities of native Cobl-KAB, without any strict correlation with a loss in actin binding, which emphasizes the functional importance of the electrostatic environment of WH2 domains. Filament severing displayed the greatest stringency and was abolished in all mutated forms of Cobl-KAB. Filament severing and re-annealing by Cobl-KAB, which is key in its rapid remodeling of a population of actin filaments, and most likely responsible for its function in ciliogenesis, was analyzed by electron microscopy in comparison with Spire and ADF.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/metabolismo , Microscopia Eletrônica , Mutagênese/genética , Citoesqueleto de Actina/metabolismo , Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia em Gel , Humanos , Hidrodinâmica , Dados de Sequência Molecular , Proteínas Mutantes/metabolismo , Estrutura Terciária de Proteína , Coelhos , Alinhamento de Sequência , Proteína da Síndrome de Wiskott-Aldrich/químicaRESUMO
Actin is a major actor in the determination of cell shape. On the one hand, site-directed assembly/disassembly cycles of actin filaments drive protrusive force leading to lamellipodia and filopodia dynamics. Force produced by actin similarly contributes in membrane scission in endocytosis or Golgi remodeling. On the other hand, cellular processes like adhesion, immune synapse, cortex dynamics or cytokinesis are achieved by combining acto-myosin contractility and actin assembly in a complex and not fully understood manner. New chemical compounds are therefore needed to disentangle acto-myosin and actin dynamics. We have found that synthetic, cell permeant, short polyamines are promising new actin regulators in this context. They generate growth and stabilization of lamellipodia within minutes by slowing down the actin assembly/disassembly cycle and facilitating nucleation. We now report that these polyamines also slow down cytokinetic ring closure in fission yeast. This shows that these synthetic compounds are active also in yeasts, and these experiments specifically highlight that actin depolymerization is involved in the ring closure. Thus, synthetic polyamines appear to be potentially powerful agents in a quantitative approach to the role of actin in complex processes in cell biology, developmental biology and potentially cancer research.