RESUMO
Tuft cells are a rare epithelial cell type that play important roles in sensing and responding to luminal antigens. A defining morphological feature of this lineage is the actin-rich apical "tuft," which contains large fingerlike protrusions. However, details of the cytoskeletal ultrastructure underpinning the tuft, the molecules involved in building this structure, or how it supports tuft cell biology remain unclear. In the context of the small intestine, we found that tuft cell protrusions are supported by long-core bundles that consist of F-actin crosslinked in a parallel and polarized configuration; they also contain a tuft cell-specific complement of actin-binding proteins that exhibit regionalized localization along the bundle axis. Remarkably, in the sub-apical cytoplasm, the array of core actin bundles interdigitates and co-aligns with a highly ordered network of microtubules. The resulting cytoskeletal superstructure is well positioned to support subcellular transport and, in turn, the dynamic sensing functions of the tuft cell that are critical for intestinal homeostasis.
Assuntos
Actinas , Citoesqueleto , Células Epiteliais , Mucosa Intestinal , Microtúbulos , Animais , Mucosa Intestinal/ultraestrutura , Mucosa Intestinal/metabolismo , Mucosa Intestinal/citologia , Células Epiteliais/metabolismo , Células Epiteliais/ultraestrutura , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Actinas/metabolismo , Camundongos , Intestino Delgado/metabolismo , Intestino Delgado/ultraestrutura , Intestino Delgado/citologia , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Células em TufoRESUMO
In the in vitro motility assay (IVMA), actin filaments are observed while propelled by surface-adsorbed myosin motor fragments such as heavy meromyosin (HMM). In addition to fundamental studies, the IVMA is the basis for a range of lab-on-a-chip applications, e.g. transport of cargoes in nanofabricated channels in nanoseparation/biosensing or the solution of combinatorial mathematical problems in network-based biocomputation. In these applications, prolonged myosin function is critical as is the potential to repeatedly exchange experimental solutions without functional deterioration. We here elucidate key factors of importance in these regards. Our findings support a hypothesis that early deterioration in the IVMA is primarily due to oxygen entrance into in vitro motility assay flow cells. In the presence of a typically used oxygen scavenger mixture (glucose oxidase, glucose, and catalase), this leads to pH reduction by a glucose oxidase-catalyzed reaction between glucose and oxygen but also contributes to functional deterioration by other mechanisms. Our studies further demonstrate challenges associated with evaporation and loss of actin filaments with time. However, over 8 h at 21-26 °C, there is no significant surface desorption or denaturation of HMM if solutions are exchanged manually every 30 min. We arrive at an optimized protocol with repeated exchange of carefully degassed assay solution of 45 mM ionic strength, at 30 min intervals. This is sufficient to maintain the high-quality function in an IVMA over 8 h at 21-26 °C, provided that fresh actin filaments are re-supplied in connection with each assay solution exchange. Finally, we demonstrate adaptation to a microfluidic platform and identify challenges that remain to be solved for real lab-on-a-chip applications.
Assuntos
Actomiosina , Dispositivos Lab-On-A-Chip , Actomiosina/metabolismo , Actomiosina/química , Citoesqueleto de Actina/metabolismo , Oxigênio/metabolismo , Animais , Glucose Oxidase/metabolismo , Glucose Oxidase/química , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Catalase/metabolismoRESUMO
Actin filament turnover involves subunits binding to and dissociating from the filament ends, with the pointed end being the primary site of filament disassembly. Several molecules modulate filament turnover, but the underlying mechanisms remain incompletely understood. Here, we present three cryo-EM structures of the F-actin pointed end in the presence and absence of phalloidin or DNase I. The two terminal subunits at the undecorated pointed end adopt a twisted conformation. Phalloidin can still bind and bridge these subunits, inducing a conformational shift to a flattened, F-actin-like state. This explains how phalloidin prevents depolymerization at the pointed end. Interestingly, two DNase I molecules simultaneously bind to the phalloidin-stabilized pointed end. In the absence of phalloidin, DNase I binding would disrupt the terminal actin subunit packing, resulting in filament disassembly. Our findings uncover molecular principles of pointed end regulation and provide structural insights into the kinetic asymmetry between the actin filament ends.
Assuntos
Citoesqueleto de Actina , Actinas , Microscopia Crioeletrônica , Desoxirribonuclease I , Faloidina , Actinas/metabolismo , Desoxirribonuclease I/metabolismo , Desoxirribonuclease I/química , Citoesqueleto de Actina/metabolismo , Faloidina/metabolismo , Faloidina/química , Modelos Moleculares , Ligação Proteica , Animais , Coelhos , Conformação ProteicaRESUMO
Class I myosins (myosin-Is) colocalize with Arp2/3 complex-nucleated actin networks at sites of membrane protrusion and invagination, but the mechanisms by which myosin-I motor activity coordinates with branched actin assembly to generate force are unknown. We mimicked the interplay of these proteins using the "comet tail" bead motility assay, where branched actin networks are nucleated by the Arp2/3 complex on the surface of beads coated with myosin-I and nucleation-promoting factor. We observed that myosin-I increased bead movement efficiency by thinning actin networks without affecting growth rates. Myosin-I triggered symmetry breaking and comet tail formation in dense networks resistant to spontaneous fracturing. Even with arrested actin assembly, myosin-I alone could break the network. Computational modeling recapitulated these observations, suggesting myosin-I acts as a repulsive force shaping the network's architecture and boosting its force-generating capacity. We propose that myosin-I leverages its power stroke to amplify the forces generated by Arp2/3 complex-nucleated actin networks.
Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina , Actinas , Miosina Tipo I , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Miosina Tipo I/metabolismo , Animais , Citoesqueleto de Actina/metabolismo , Ligação ProteicaRESUMO
The excessive inflammation caused by the prolonged activation of Toll-like receptor 4 (TLR4) and its downstream signaling pathways leads to sepsis. CD14-mediated endocytosis of TLR4 is the key step to control the amount of TLR4 on cell membrane and the activity of downstream pathways. The actin cytoskeleton is necessary for receptor-mediated endocytosis, but its role in TLR4 endocytosis remains elusive. Here we show that Tropomodulin 1 (Tmod1), an actin capping protein, inhibited lipopolysaccharide (LPS)-induced TLR4 endocytosis and intracellular trafficking in macrophages. Thus it resulted in increased surface TLR4 and the upregulation of myeloid differentiation factor 88 (MyD88)-dependent pathway and the downregulation of TIR domain-containing adaptor-inducing interferon-ß (TRIF)-dependent pathway, leading to the enhanced secretion of inflammatory cytokines, such as TNF-α and IL-6, and the reduced secretion of cytokines, such as IFN-ß. Macrophages deficient with Tmod1 relieved the inflammatory response in LPS-induced acute lung injury mouse model. Mechanistically, Tmod1 negatively regulated LPS-induced TLR4 endocytosis and inflammatory response through modulating the activity of CD14/Syk/PLCγ2/IP3/Ca2+ signaling pathway, the reorganization of actin cytoskeleton, and the membrane tension. Therefore, Tmod1 is a key regulator of inflammatory response and immune functions in macrophages and may be a potential target for the treatment of excessive inflammation and sepsis.
Assuntos
Endocitose , Inflamação , Lipopolissacarídeos , Macrófagos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptor 4 Toll-Like , Tropomodulina , Animais , Humanos , Camundongos , Citoesqueleto de Actina/metabolismo , Lesão Pulmonar Aguda/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Citocinas/metabolismo , Inflamação/metabolismo , Inflamação/patologia , Receptores de Lipopolissacarídeos/metabolismo , Lipopolissacarídeos/farmacologia , Macrófagos/metabolismo , Macrófagos/imunologia , Camundongos Knockout , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/genética , Células RAW 264.7 , Receptor 4 Toll-Like/metabolismo , Tropomodulina/metabolismo , Tropomodulina/genéticaRESUMO
Germline variants in the NSD1 gene are responsible for Sotos syndrome, while somatic variants promote neoplastic cell transformation. Our previous studies revealed three alternative RNA isoforms of NSD1 present in fibroblast cell lines (FBs): the canonical full transcript and 2 alternative transcripts, termed AT2 (NSD1 Δ5Δ7) and AT3 (NSD1 Δ19-23 at the 5' end). The precise molecular pathways affected by each specific isoform of NSD1 are uncharacterized to date. To elucidate the role of these isoforms, their expression was suppressed by siRNA knockdown in FBs and protein expression and transcriptome data was explored. We demonstrate that one gene target of NSD1 isoform AT2 is ARP3 actin-related protein 3 homolog B (ACTR3B). We show that loss of both canonical NSD1 and AT2 isoforms impaired the ability of fibroblasts to regulate the actin cytoskeleton, and we observed that this caused selective loss of stress fibers. Our findings provide novel insights into NSD1 function by distinguishing isoform function and demonstrating an essential role of NSD1 in regulating the actin cytoskeleton and stress fiber formation in fibroblasts.
Assuntos
Citoesqueleto de Actina , Fibroblastos , Histona-Lisina N-Metiltransferase , Isoformas de Proteínas , Fibroblastos/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Divisão Celular/genética , Linhagem Celular , Processamento Alternativo , Fibras de Estresse/metabolismoRESUMO
Mechanical forces are transmitted from the actin cytoskeleton to the membrane during clathrin-mediated endocytosis (CME) in the fission yeast Schizosaccharomyces pombe. End4p directly transmits force in CME by binding to both the membrane (through the AP180 N-terminal homology [ANTH] domain) and F-actin (through the talin-HIP1/R/Sla2p actin-tethering C-terminal homology [THATCH] domain). We show that 7 pN force is required for stable binding between THATCH and F-actin. We also characterized a domain in End4p, Rend (rod domain in End4p), that resembles R12 of talin. Membrane localization of Rend primes the binding of THATCH to F-actin, and force-induced unfolding of Rend at 15 pN terminates the transmission of force. We show that the mechanical properties (mechanical stability, unfolding extension, hysteresis) of Rend and THATCH are tuned to form a circuit for the initiation, transmission, and termination of force between the actin cytoskeleton and membrane. The mechanical circuit by Rend and THATCH may be conserved and coopted evolutionarily in cell adhesion complexes.
Assuntos
Actinas , Clatrina , Endocitose , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Endocitose/fisiologia , Schizosaccharomyces/metabolismo , Clatrina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Actinas/metabolismo , Domínios Proteicos , Citoesqueleto de Actina/metabolismo , Ligação Proteica , Membrana Celular/metabolismoRESUMO
Formin HOmology Domain 2-containing (FHOD) proteins are a subfamily of actin-organizing formins important for striated muscle development in many animals. We showed previously that absence of the sole FHOD protein, FHOD-1, from Caenorhabditis elegans results in thin body wall muscles with misshapen dense bodies that serve as sarcomere Z-lines. We demonstrate here that mutations predicted to specifically disrupt actin polymerization by FHOD-1 similarly disrupt muscle development, and that FHOD-1 cooperates with profilin PFN-3 for dense body morphogenesis, and with profilins PFN-2 and PFN-3 to promote body wall muscle growth. We further demonstrate that dense bodies in worms lacking FHOD-1 or PFN-2/PFN-3 are less stable than in wild-type animals, having a higher proportion of dynamic protein, and becoming distorted by prolonged muscle contraction. We also observe accumulation of actin and actin depolymerization factor/cofilin homologue UNC-60B in body wall muscle of these mutants. Such accumulations may indicate targeted disassembly of thin filaments dislodged from unstable dense bodies, possibly accounting for the abnormally slow growth and reduced body wall muscle strength in fhod-1 mutants. Overall, these results implicate FHOD protein-mediated actin assembly in forming stable sarcomere Z-lines, and identify profilin as a new contributor to FHOD activity in striated muscle development.
Assuntos
Actinas , Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Forminas , Contração Muscular , Profilinas , Sarcômeros , Animais , Caenorhabditis elegans/metabolismo , Profilinas/metabolismo , Profilinas/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Sarcômeros/metabolismo , Contração Muscular/fisiologia , Forminas/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Proteínas dos Microfilamentos/genética , Mutação/genética , Desenvolvimento Muscular/fisiologia , Citoesqueleto de Actina/metabolismo , Músculo Estriado/metabolismo , Músculos/metabolismo , Fatores de Despolimerização de Actina/metabolismoRESUMO
Style penetration by pollen tubes is essential for reproductive success, a process requiring canonical Rab5s in Arabidopsis. However, functional loss of Arabidopsis Vps9a, the gene encoding for guanine nucleotide exchange factor (GEF) of Rab5s, did not affect male transmission, implying the presence of a compensation program or redundancy. By combining genetic, cytological, and molecular approaches, we report that Arabidopsis Vps9b is a pollen-preferential gene, redundantly mediating pollen tube penetration of style with Vps9a. Vps9b is functionally interchangeable with Vps9a, whose functional distinction results from distinct expression profiles. Functional loss of Vps9a and Vps9b results in the mis-targeting of Rab5-dependent tonoplast proteins, defective vacuolar biogenesis, disturbed distribution of post-Golgi vesicles, increased cellular turgor, cytosolic acidification, and disrupted organization of actin microfilaments (MF) in pollen tubes, which collectively lead to the failure of pollen tubes to grow through style.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Regulação da Expressão Gênica de Plantas , Tubo Polínico , Isoformas de Proteínas , Vacúolos , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/genética , Tubo Polínico/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Vacúolos/metabolismo , Citoesqueleto de Actina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , Complexo de Golgi/metabolismo , Mutação/genéticaRESUMO
Lead and cadmium are heavy metals widely distributed in the environment and contribute significantly to cardiovascular morbidity and mortality. Using Leadmium Green dye, we have shown that lead and cadmium enter cardiomyocytes, distributing throughout the cell. Using an in vitro motility assay, we have shown that sliding velocity of actin and native thin filaments over myosin decreases with increasing concentrations of Pb2+ and Cd2+. Significantly lower concentrations of Pb2+ and Cd2+ (0.6 mM) were required to stop sliding of thin filaments over myosin compared to the stopping actin sliding over the same myosin (1.1-1.6 mM). Lower concentration of Cd2+ (1.1 mM) needed to stop actin sliding over myosin compared to the Pb2++Cd2+ combination (1.3 mM) and lead alone (1.6 mM). There were no differences found in the effects of lead and cadmium cations on relative force developed by myosin heads or number of actin filaments bound to myosin. Sliding velocity of actin over myosin in the left atrium, right and left ventricles changed equally when exposed to the same dose of the same metal. Thus, we have demonstrated for the first time that Pb2+ and Cd2+ can directly affect myosin and thin filament function, with Cd2+ exerting a more toxic influence on myosin function compared to Pb2+.
Assuntos
Citoesqueleto de Actina , Cádmio , Cátions Bivalentes , Chumbo , Cádmio/farmacologia , Animais , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Miosinas Cardíacas/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Actinas/metabolismo , CoelhosRESUMO
The Caenorhabditis elegans HMP-2/HMP-1 complex, akin to the mammalian [Formula: see text]-catenin-[Formula: see text]-catenin complex, serves as a critical mechanosensor at cell-cell adherens junctions, transducing tension between HMR-1 (also known as cadherin in mammals) and the actin cytoskeleton. Essential for embryonic development and tissue integrity in C. elegans, this complex experiences tension from both internal actomyosin contractility and external mechanical microenvironmental perturbations. While offering a valuable evolutionary comparison to its mammalian counterpart, the impact of tension on the mechanical stability of HMP-1 and HMP-2/HMP-1 interactions remains unexplored. In this study, we directly quantified the mechanical stability of full-length HMP-1 and its force-bearing modulation domains (M1-M3), as well as the HMP-2/HMP-1 interface. Notably, the M1 domain in HMP-1 exhibits significantly higher mechanical stability than its mammalian analog, attributable to interdomain interactions with M2-M3. Introducing salt bridge mutations in the M3 domain weakens the mechanical stability of the M1 domain. Moreover, the intermolecular HMP-2/HMP-1 interface surpasses its mammalian counterpart in mechanical stability, enabling it to support the mechanical activation of the autoinhibited M1 domain for mechanotransduction. Additionally, the phosphomimetic mutation Y69E in HMP-2 weakens the mechanical stability of the HMP-2/HMP-1 interface, compromising the force-transmission molecular linkage and its associated mechanosensing functions. Collectively, these findings provide mechanobiological insights into the C. elegans HMP-2/HMP-1 complex, highlighting the impact of salt bridges on mechanical stability in [Formula: see text]-catenin and demonstrating the evolutionary conservation of the mechanical switch mechanism activating the HMP-1 modulation domain for protein binding at the single-molecule level.
Assuntos
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Mecanotransdução Celular , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Animais , Caenorhabditis elegans/metabolismo , Mecanotransdução Celular/fisiologia , Imagem Individual de Molécula , Ligação Proteica , Caderinas/metabolismo , Caderinas/química , Caderinas/genética , Junções Aderentes/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/química , Proteínas do Citoesqueleto , alfa CateninaRESUMO
Confluent populations of the epithelial cell line, MDCK II, develop circumferential tight junctions joining adjacent cells to create a barrier to the paracellular movement of solutes and water. Treatment of MDCK II cell populations from the apical surface with 1 mM Na-caprate increased permeability to macromolecules (Leak Pathway) without increasing monolayer disruption or cell death. Graphical analysis of the apparent permeability versus solute Stokes radius for a size range of fluorescein-dextran species indicates apical 1 mM Na-caprate enhances Leak Pathway permeability by increasing the number of Leak Pathway openings without significantly affecting opening size. Na-caprate treatment did not alter the content of any tight junction protein examined. Treatment of MDCK II cell populations with apical 1 mM Na-caprate disrupted basal F-actin stress fibers and decreased the tortuosity of the tight junctions. Treatment of MDCK II cell populations with blebbistatin, a myosin ATPase inhibitor, alone had little effect on Leak Pathway permeability but synergistically increased Leak Pathway permeability when added with 1 mM Na-caprate. Na-caprate exhibited a similar ability to increase Leak Pathway permeability in wild-type MDCK II cell monolayers and ZO-1 knockdown MDCK II cell monolayers but an enhanced ability to increase Leak Pathway permeability in monolayers of TOCA-1 knockout MDCK II cells. These results demonstrate that Na-caprate increases MDCK II cell population Leak Pathway permeability by increasing the number of Leak Pathway openings. This action is likely mediated by alterations in F-actin organization, primarily involving disruption of basal F-actin stress fibers.NEW & NOTEWORTHY This study determines the underlying change in the openings in the epithelial tight junction permeability barrier structure that leads to a change in the paracellular permeability to macromolecules (the Leak Pathway) and connects this to disruption of specific F-actin structures within the cells. It provides important and novel insights into how tight junction permeability to macromolecules is modulated by specific changes to cellular and tight junction composition/organization.
Assuntos
Actinas , Células Epiteliais , Junções Íntimas , Cães , Animais , Actinas/metabolismo , Células Madin Darby de Rim Canino , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Junções Íntimas/metabolismo , Junções Íntimas/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Proteína da Zônula de Oclusão-1/metabolismo , Proteína da Zônula de Oclusão-1/genética , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Citoesqueleto de Actina/metabolismoRESUMO
The behavior and presence of actin-regulating proteins are characteristic of various clinical diseases. Changes in these proteins significantly impact the cytoskeletal and regenerative processes underlying pathological changes. Pituitary adenylate cyclase-activating polypeptide (PACAP), a cytoprotective neuropeptide abundant in the nervous system and endocrine organs, plays a key role in neuron differentiation and migration by influencing actin. This study aims to elucidate the role of PACAP as an actin-regulating polypeptide, its effect on actin filament formation, and the underlying regulatory mechanisms. We examined PACAP27, PACAP38, and PACAP6-38, measuring their binding to actin monomers via fluorescence spectroscopy and steady-state anisotropy. Functional polymerization tests were used to track changes in fluorescent intensity over time. Unlike PACAP27, PACAP38 and PACAP6-38 significantly reduced the fluorescence emission of Alexa488-labeled actin monomers and increased their anisotropy, showing nearly identical dissociation equilibrium constants. PACAP27 showed weak binding to globular actin (G-actin), while PACAP38 and PACAP6-38 exhibited robust interactions. PACAP27 did not affect actin polymerization, but PACAP38 and PACAP6-38 accelerated actin incorporation kinetics. Fluorescence quenching experiments confirmed structural changes upon PACAP binding; however, all studied PACAP fragments exhibited the same effect. Our findings indicate that PACAP38 and PACAP6-38 strongly bind to G-actin and significantly influence actin polymerization. Further studies are needed to fully understand the biological significance of these interactions.
Assuntos
Actinas , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Espectrometria de Fluorescência , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/metabolismo , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/química , Actinas/metabolismo , Actinas/química , Animais , Espectrometria de Fluorescência/métodos , Citoesqueleto/metabolismo , Ligação Proteica , Citoesqueleto de Actina/metabolismo , Humanos , CinéticaRESUMO
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.
Assuntos
Citoesqueleto , Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Citoesqueleto/metabolismo , Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Modelos Biológicos , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Eukaryotic cells have been evolving for billions of years, giving rise to wildly diverse cell forms and functions. Despite their variability, all eukaryotic cells share key hallmarks, including membrane-bound organelles, heavily regulated cytoskeletal networks and complex signaling cascades. Because the actin cytoskeleton interfaces with each of these features, understanding how it evolved and diversified across eukaryotic phyla is essential to understanding the evolution and diversification of eukaryotic cells themselves. Here, we discuss what we know about the origin and diversity of actin networks in terms of their compositions, structures and regulation, and how actin evolution contributes to the diversity of eukaryotic form and function.
Assuntos
Citoesqueleto de Actina , Actinas , Células Eucarióticas , Actinas/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/citologia , Animais , Humanos , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/genética , Eucariotos/metabolismo , Eucariotos/genética , Evolução Molecular , Evolução Biológica , Transdução de SinaisRESUMO
In nephrotic syndrome, the podocyte filtration structures are damaged in a process called foot process effacement. This is mediated by the actin cytoskeleton; however, which actins are involved and how they interact with other filtration components, like the basement membrane, remains poorly understood. Here, we used the well-established Drosophila pericardial nephrocyte-the equivalent of podocytes in flies-knockdown models (RNAi) to study the interplay of the actin cytoskeleton (Act5C, Act57B, Act42A, and Act87E), alpha- and beta-integrin (basement membrane), and the slit diaphragm (Sns and Pyd). Knockdown of an actin gene led to variations of formation of actin stress fibers, the internalization of Sns, and a disrupted slit diaphragm cortical pattern. Notably, deficiency of Act5C, which resulted in complete absence of nephrocytes, could be partially mitigated by overexpressing Act42A or Act87E, suggesting at least partial functional redundancy. Integrin localized near the actin cytoskeleton as well as slit diaphragm components, but when the nephrocyte cytoskeleton or slit diaphragm was disrupted, this switched to colocalization, both at the surface and internalized in aggregates. Altogether, the data show that the interdependence of the slit diaphragm, actin cytoskeleton, and integrins is key to the structure and function of the Drosophila nephrocyte.
Assuntos
Citoesqueleto de Actina , Proteínas de Drosophila , Integrinas , Podócitos , Animais , Citoesqueleto de Actina/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Integrinas/metabolismo , Podócitos/metabolismo , Drosophila melanogaster/metabolismo , Drosophila/metabolismo , Actinas/metabolismo , ImunoglobulinasRESUMO
Synaptopodin 2-like protein (SYNPO2L) is localized in the sarcomere of cardiomyocytes and is involved in heart morphogenesis. However, the molecular function of SYNPO2L in the heart is not fully understood. We investigated the interaction of SYNPO2L with sarcomeric α-actinin and actin filaments in cultured mouse cardiomyocytes. Immunofluorescence studies showed that SYNPO2L colocalized with α-actinin and actin filaments at the Z-discs of the sarcomere. Recombinant SYNPO2La or SYNPO2Lb caused a bundling of the actin filaments in the absence of α-actinin and enhanced the α-actinin-dependent formation of actin bundles. In addition, high-speed atomic force microscopy revealed that SYNPO2La directly bound to α-actinin via its globular ends. The interaction between α-actinin and SYNPO2La fixed the movements of the two proteins on the actin filaments. These results strongly suggest that SYNPO2L cooperates with α-actinin during actin bundle formation to facilitate sarcomere formation and maintenance.
Assuntos
Actinina , Proteínas dos Microfilamentos , Proteínas Musculares , Miócitos Cardíacos , Ligação Proteica , Sarcômeros , Animais , Camundongos , Citoesqueleto de Actina/metabolismo , Actinina/metabolismo , Actinas/metabolismo , Proteínas dos Microfilamentos/metabolismo , Miócitos Cardíacos/metabolismo , Sarcômeros/metabolismo , Proteínas Musculares/metabolismoRESUMO
Human cancers express altered levels of actin-binding cytoskeletal filamin A (FLNA) protein. FLNA in mammals consists of an actin-binding domain at its N-terminus that is followed by 24 immunoglobulin-like repeat modules interrupted by two hinge regions between repeats 15-16 and 23-24. Cleavage of these hinge regions produces a naturally occurring C-terminal 90 kDa fragment of FLNA (FLNACT) that physically interacts with multiple proteins with diverse functions. This cleavage leads to actin cytoskeleton remodeling, which in turn contributes to cellular signaling, nucleocytoplasmic shuttling of transcriptional factors and nuclear receptors, and regulation of their transcriptional activities that are important for initiation and progression of cancers. Therefore, recent studies have proposed blocking FLNA cleavage as a means of cancer therapy. Here, we update how FLNA cleavage has been targeted by different approaches and their potential implications for future treatment of human cancers.
Assuntos
Filaminas , Neoplasias , Filaminas/metabolismo , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Animais , Citoesqueleto/metabolismo , Citoesqueleto de Actina/metabolismoRESUMO
Clathrin-mediated endocytosis has characteristic features in neuronal dendrites and presynapses, but how membrane proteins are internalized along the axon shaft remains unclear. We focused on clathrin-coated structures and endocytosis along the axon initial segment (AIS) and their relationship to the periodic actin-spectrin scaffold that lines the axonal plasma membrane. A combination of super-resolution microscopy and platinum-replica electron microscopy on cultured neurons revealed that AIS clathrin-coated pits form within "clearings", circular areas devoid of actin-spectrin mesh. Actin-spectrin scaffold disorganization increased clathrin-coated pit formation. Cargo uptake and live-cell imaging showed that AIS clathrin-coated pits are particularly stable. Neuronal plasticity-inducing stimulation triggered internalization of the clathrin-coated pits through polymerization of branched actin around them. Thus, spectrin and actin regulate clathrin-coated pit formation and scission to control endocytosis at the AIS.
Assuntos
Actinas , Axônios , Clatrina , Endocitose , Espectrina , Animais , Humanos , Ratos , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Axônios/metabolismo , Membrana Celular/metabolismo , Células Cultivadas , Clatrina/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Invaginações Revestidas da Membrana Celular/metabolismo , Células HEK293 , Plasticidade Neuronal , Neurônios/metabolismo , Espectrina/metabolismoRESUMO
The differentiation of bone marrow mesenchymal stem cells (BMSCs) toward osteogenesis can be induced by low-intensity pulsed ultrasound (LIPUS). However, the molecular mechanisms responsible for LIPUS stimulation are unclear. The possible molecular mechanisms by which LIPUS promotes osteogenic differentiation of BMSCs were investigated in this study. The quantification of alkaline phosphatase (ALP) activity, Alizarin Red S staining, ALP staining, and the establishment of a calvarial defect model were used to evaluate osteogenic effects. Immunofluorescence was performed to observe the expression of microfilaments and transient receptor potential melastatin 7 (TRPM7). The levels of F-actin/G-actin and osteogenesis-related proteins under LIPUS alone or LIPUS combined with cytoskeleton interfering drugs (Cytochalasin D [CytoD] or Jasplakinolide [JA]) were assayed by western blot. Quantitative real-time reverse transcription polymerase chain reaction was utilized to measure the expression of Trpm7 mRNA. Moreover, adenoviral Trpm7 knockdown was verified using western blot. The results demonstrated that LIPUS promoted bone formation in vivo. Under osteogenic induction in vitro, the osteogenesis of BMSCs induced by LIPUS was accompanied by the depolymerization and rearrangement of microfilaments and increased levels of TRPM7. By perturbing intracellular actin dynamics, CytoD enhanced the pro-osteogenicity of LIPUS and increased TRPM7 level, while JA inhibited the pro-osteogenicity of LIPUS and reduced TRPM7 level. Additionally, the knockdown of Trpm7 suppressed the osteogenic promotion of BMSCs induced by LIPUS. The transient depolymerization and rearrangement of the cytoskeleton microfilaments mediated by LIPUS can affect TRPM7 expression and subsequently promote the osteogenesis of BMSCs. This study provides further direction for exploring the molecular mechanism of LIPUS, as a mechanical stress, in facilitating the osteogenic differentiation of BMSCs.