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1.
Proc Natl Acad Sci U S A ; 119(50): e2202803119, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36475946

RESUMEN

Cellular morphogenesis and processes such as cell division and migration require the coordination of the microtubule and actin cytoskeletons. Microtubule-actin crosstalk is poorly understood and largely regarded as the capture and regulation of microtubules by actin. Septins are filamentous guanosine-5'-triphosphate (GTP) binding proteins, which comprise the fourth component of the cytoskeleton along microtubules, actin, and intermediate filaments. Here, we report that septins mediate microtubule-actin crosstalk by coupling actin polymerization to microtubule lattices. Superresolution and platinum replica electron microscopy (PREM) show that septins localize to overlapping microtubules and actin filaments in the growth cones of neurons and non-neuronal cells. We demonstrate that recombinant septin complexes directly crosslink microtubules and actin filaments into hybrid bundles. In vitro reconstitution assays reveal that microtubule-bound septins capture and align stable actin filaments with microtubules. Strikingly, septins enable the capture and polymerization of growing actin filaments on microtubule lattices. In neuronal growth cones, septins are required for the maintenance of the peripheral actin network that fans out from microtubules. These findings show that septins directly mediate microtubule interactions with actin filaments, and reveal a mechanism of microtubule-templated actin growth with broader significance for the self-organization of the cytoskeleton and cellular morphogenesis.


Asunto(s)
Actinas , Septinas , Microtúbulos
2.
Entropy (Basel) ; 24(5)2022 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-35626481

RESUMEN

The Age of Information (AoI) measures the freshness of information and is a critic performance metric for time-sensitive applications. In this paper, we consider a radio frequency energy-harvesting cognitive radio network, where the secondary user harvests energy from the primary users' transmissions and opportunistically accesses the primary users' licensed spectrum to deliver the status-update data pack. We aim to minimize the AoI subject to the energy causality and spectrum constraints by optimizing the sensing and update decisions. We formulate the AoI minimization problem as a partially observable Markov decision process and solve it via dynamic programming. Simulation results verify that our proposed policy is significantly superior to the myopic policy under different parameter settings.

3.
Omega (Westport) ; 85(3): 554-573, 2022 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-32807007

RESUMEN

This study aims to identify the factors that influence Chinese professional caregivers' bereavement experiences after patient deaths. Through a content analysis, the study reanalyzed the qualitative data initially collected to understand the lived experiences of professional bereavement in Mainland China. Specifically, the study assessed semi-structured interview transcripts conducted with 24 Chinese physicians and nurses and generated 15 open codes, reflecting the influencing factors. These were further categorized into four themes: dying and death conditions, professional caregivers' characteristics, professional caregivers' involvement, and the bereaved family. The results revealed that professional bereavement experiences and the unveiled factors relate to both the personal and professional lives of the interviewees. Overall, the health care system and cultural backgrounds should be listed as influencing factors for professional bereavement experiences in addition to the aforementioned four.


Asunto(s)
Aflicción , Cuidadores , China , Pesar , Humanos , Investigación Cualitativa
4.
J Biol Chem ; 294(12): 4704-4722, 2019 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-30692198

RESUMEN

Spatial and temporal control of actin polymerization is fundamental for many cellular processes, including cell migration, division, vesicle trafficking, and response to agonists. Many actin-regulatory proteins interact with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and are either activated or inactivated by local PI(4,5)P2 concentrations that form transiently at the cytoplasmic face of cell membranes. The molecular mechanisms of these interactions and how the dozens of PI(4,5)P2-sensitive actin-binding proteins are selectively recruited to membrane PI(4,5)P2 pools remains undefined. Using a combination of biochemical, imaging, and cell biologic studies, combined with molecular dynamics and analytical theory, we test the hypothesis that the lateral distribution of PI(4,5)P2 within lipid membranes and native plasma membranes alters the capacity of PI(4,5)P2 to nucleate actin assembly in brain and neutrophil extracts and show that activities of formins and the Arp2/3 complex respond to PI(4,5)P2 lateral distribution. Simulations and analytical theory show that cholesterol promotes the cooperative interaction of formins with multiple PI(4,5)P2 headgroups in the membrane to initiate actin nucleation. Masking PI(4,5)P2 with neomycin or disrupting PI(4,5)P2 domains in the plasma membrane by removing cholesterol decreases the ability of these membranes to nucleate actin assembly in cytoplasmic extracts.


Asunto(s)
Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Animales , Sitios de Unión , Bovinos , Membrana Celular/metabolismo , Humanos , Membrana Dobles de Lípidos , Simulación de Dinámica Molecular
5.
Immunity ; 35(3): 388-99, 2011 Sep 23.
Artículo en Inglés | MEDLINE | ID: mdl-21835647

RESUMEN

Dendritic cells (DCs) flexibly adapt to different microenvironments by using diverse migration strategies that are ultimately dependent on the dynamics and structural organization of the actin cytoskeleton. Here, we have shown that DCs require the actin capping activity of the signaling adaptor Eps8 to polarize and to form elongated migratory protrusions. DCs from Eps8-deficient mice are impaired in directional and chemotactic migration in 3D in vitro and are delayed in reaching the draining lymph node (DLN) in vivo after inflammatory challenge. Hence, Eps8-deficient mice are unable to mount a contact hypersensitivity response. We have also shown that the DC migratory defect is cell autonomous and that Eps8 is required for the proper architectural organization of the actin meshwork and dynamics of cell protrusions. Yet, Eps8 is not necessary for antigen uptake, processing, and presentation. Thus, we have identified Eps8 as a unique actin capping protein specifically required for DC migration.


Asunto(s)
Proteínas de Capping de la Actina/inmunología , Proteínas Adaptadoras Transductoras de Señales/inmunología , Proteínas del Citoesqueleto/inmunología , Células Dendríticas/inmunología , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales/deficiencia , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Presentación de Antígeno , Movimiento Celular/inmunología , Proliferación Celular , Células Cultivadas , Proteínas del Citoesqueleto/deficiencia , Proteínas del Citoesqueleto/genética , Dermatitis por Contacto/inmunología , Citometría de Flujo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Linfocitos T/inmunología
6.
bioRxiv ; 2024 Jun 26.
Artículo en Inglés | MEDLINE | ID: mdl-38979322

RESUMEN

Clathrin-mediated endocytosis (CME) is essential for maintaining cellular homeostasis. Previous studies have reported more than 50 CME accessory proteins; however, the mechanism driving the invagination of clathrin-coated pits (CCPs) remains elusive. Quantitative live cell imaging reveals that CCDC32, a poorly characterized endocytic accessory protein, regulates CCP stabilization and is required for efficient CCP invagination. CCDC32 interacts with the α-appendage domain (AD) of AP2 via its coiled-coil domain to exert this function. Furthermore, we showed that the clinically observed nonsense mutations in CCDC32, which result in the development of cardio-facio-neuro-developmental syndrome (CFNDS), inhibit CME by abolishing CCDC32-AP2 interactions. Overall, our data demonstrates the function and molecular mechanism of a novel endocytic accessory protein, CCDC32, in CME regulation. Significance Statement: Clathrin-mediated endocytosis (CME) happens via the initiation, stabilization, and invagination of clathrin-coated pits (CCPs). In this study, we used a combination of quantitative live cell imaging, ultrastructure electron microscopy and biochemical experiments to show that CCDC32, a poorly studied and functional ambiguous protein, acts as an important endocytic accessory protein that regulates CCP stabilization and invagination. Specifically, CCDC32 exerts this function via its interactions with AP2, and the coiled-coil domain of CCDC32 and the α-appendage domain (AD) of AP2 are essential in mediating CCDC32-AP2 interactions. Importantly, we demonstrate that clinically observed loss-of-function mutations in CCDC32 lose AP2 interaction capacity and inhibit CME, resulting in the development of cardio-facio-neuro-developmental syndrome (CFNDS).

7.
Nat Commun ; 14(1): 6883, 2023 10 28.
Artículo en Inglés | MEDLINE | ID: mdl-37898620

RESUMEN

Exosomes are secreted to the extracellular milieu when multivesicular endosomes (MVEs) dock and fuse with the plasma membrane. However, MVEs are also known to fuse with lysosomes for degradation. How MVEs are directed to the plasma membrane for exosome secretion rather than to lysosomes is unclear. Here we report that a conversion of phosphatidylinositol-3-phosphate (PI(3)P) to phosphatidylinositol-4-phosphate (PI(4)P) catalyzed sequentially by Myotubularin 1 (MTM1) and phosphatidylinositol 4-kinase type IIα (PI4KIIα) on the surface of MVEs mediates the recruitment of the exocyst complex. The exocyst then targets the MVEs to the plasma membrane for exosome secretion. We further demonstrate that disrupting PI(4)P generation or exocyst function blocked exosomal secretion of Programmed death-ligand 1 (PD-L1), a key immune checkpoint protein in tumor cells, and led to its accumulation in lysosomes. Together, our study suggests that the PI(3)P to PI(4)P conversion on MVEs and the recruitment of the exocyst direct the exocytic trafficking of MVEs for exosome secretion.


Asunto(s)
Exosomas , Exosomas/metabolismo , Endosomas/metabolismo , Fosfatidilinositoles/metabolismo , Cuerpos Multivesiculares/metabolismo
8.
J Biol Chem ; 286(34): 30087-96, 2011 Aug 26.
Artículo en Inglés | MEDLINE | ID: mdl-21685497

RESUMEN

Fascin is the main actin filament bundling protein in filopodia. Because of the important role filopodia play in cell migration, fascin is emerging as a major target for cancer drug discovery. However, an understanding of the mechanism of bundle formation by fascin is critically lacking. Fascin consists of four ß-trefoil domains. Here, we show that fascin contains two major actin-binding sites, coinciding with regions of high sequence conservation in ß-trefoil domains 1 and 3. The site in ß-trefoil-1 is located near the binding site of the fascin inhibitor macroketone and comprises residue Ser-39, whose phosphorylation by protein kinase C down-regulates actin bundling and formation of filopodia. The site in ß-trefoil-3 is related by pseudo-2-fold symmetry to that in ß-trefoil-1. The two sites are ∼5 nm apart, resulting in a distance between actin filaments in the bundle of ∼8.1 nm. Residue mutations in both sites disrupt bundle formation in vitro as assessed by co-sedimentation with actin and electron microscopy and severely impair formation of filopodia in cells as determined by rescue experiments in fascin-depleted cells. Mutations of other areas of the fascin surface also affect actin bundling and formation of filopodia albeit to a lesser extent, suggesting that, in addition to the two major actin-binding sites, fascin makes secondary contacts with other filaments in the bundle. In a high resolution crystal structure of fascin, molecules of glycerol and polyethylene glycol are bound in pockets located within the two major actin-binding sites. These molecules could guide the rational design of new anticancer fascin inhibitors.


Asunto(s)
Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/genética , Animales , Sitios de Unión , Proteínas Portadoras/genética , Línea Celular Tumoral , Cristalografía por Rayos X , Humanos , Ratones , Proteínas de Microfilamentos/genética , Mutación , Fosforilación/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Estructura Terciaria de Proteína , Seudópodos/química , Seudópodos/genética , Seudópodos/metabolismo , Seudópodos/ultraestructura , Relación Estructura-Actividad
9.
Nat Commun ; 13(1): 6127, 2022 10 17.
Artículo en Inglés | MEDLINE | ID: mdl-36253374

RESUMEN

Clathrin-mediated endocytosis (CME) requires energy input from actin polymerization in mechanically challenging conditions. The roles of actin in CME are poorly understood due to inadequate knowledge of actin organization at clathrin-coated structures (CCSs). Using platinum replica electron microscopy of mammalian cells, we show that Arp2/3 complex-dependent branched actin networks, which often emerge from microtubule tips, assemble along the CCS perimeter, lack interaction with the apical clathrin lattice, and have barbed ends oriented toward the CCS. This structure is hardly compatible with the widely held "apical pulling" model describing actin functions in CME. Arp2/3 complex inhibition or epsin knockout produce large flat non-dynamic CCSs, which split into invaginating subdomains upon recovery from Arp2/3 inhibition. Moreover, epsin localization to CCSs depends on Arp2/3 activity. We propose an "edge pushing" model for CME, wherein branched actin polymerization promotes severing and invagination of flat CCSs in an epsin-dependent manner by pushing at the CCS boundary, thus releasing forces opposing the intrinsic curvature of clathrin lattices.


Asunto(s)
Actinas , Platino (Metal) , Complejo 2-3 Proteico Relacionado con la Actina , Animales , Clatrina , Vesículas Cubiertas por Clatrina , Endocitosis , Mamíferos , Polimerizacion
10.
Nat Commun ; 13(1): 7089, 2022 11 19.
Artículo en Inglés | MEDLINE | ID: mdl-36402771

RESUMEN

The formation and recovery of gaps in the vascular endothelium governs a wide range of physiological and pathological phenomena, from angiogenesis to tumor cell extravasation. However, the interplay between the mechanical and signaling processes that drive dynamic behavior in vascular endothelial cells is not well understood. In this study, we propose a chemo-mechanical model to investigate the regulation of endothelial junctions as dependent on the feedback between actomyosin contractility, VE-cadherin bond turnover, and actin polymerization, which mediate the forces exerted on the cell-cell interface. Simulations reveal that active cell tension can stabilize cadherin bonds, but excessive RhoA signaling can drive bond dissociation and junction failure. While actin polymerization aids gap closure, high levels of Rac1 can induce junction weakening. Combining the modeling framework with experiments, our model predicts the influence of pharmacological treatments on the junction state and identifies that a critical balance between RhoA and Rac1 expression is required to maintain junction stability. Our proposed framework can help guide the development of therapeutics that target the Rho family of GTPases and downstream active mechanical processes.


Asunto(s)
Actinas , Células Endoteliales , Células Endoteliales/metabolismo , Actinas/metabolismo , Retroalimentación , Transducción de Señal , Citoesqueleto de Actina/metabolismo
11.
Nat Commun ; 13(1): 4078, 2022 07 14.
Artículo en Inglés | MEDLINE | ID: mdl-35835783

RESUMEN

The lack of tumor infiltration by CD8+ T cells is associated with poor patient response to anti-PD-1 therapy. Understanding how tumor infiltration is regulated is key to improving treatment efficacy. Here, we report that phosphorylation of HRS, a pivotal component of the ESCRT complex involved in exosome biogenesis, restricts tumor infiltration of cytolytic CD8+ T cells. Following ERK-mediated phosphorylation, HRS interacts with and mediates the selective loading of PD-L1 to exosomes, which inhibits the migration of CD8+ T cells into tumors. In tissue samples from patients with melanoma, CD8+ T cells are excluded from the regions where tumor cells contain high levels of phosphorylated HRS. In murine tumor models, overexpression of phosphorylated HRS increases resistance to anti-PD-1 treatment, whereas inhibition of HRS phosphorylation enhances treatment efficacy. Our study reveals a mechanism by which phosphorylation of HRS in tumor cells regulates anti-tumor immunity by inducing PD-L1+ immunosuppressive exosomes, and suggests HRS phosphorylation blockade as a potential strategy to improve the efficacy of cancer immunotherapy.


Asunto(s)
Exosomas , Melanoma , Animales , Antígeno B7-H1 , Linfocitos T CD8-positivos , Línea Celular Tumoral , Exosomas/metabolismo , Humanos , Inmunoterapia , Ratones , Fosforilación , Receptor de Muerte Celular Programada 1 , Microambiente Tumoral
12.
Mol Biol Cell ; 32(7): 579-589, 2021 04 01.
Artículo en Inglés | MEDLINE | ID: mdl-33502904

RESUMEN

Human fibroblasts can switch between lamellipodia-dependent and -independent migration mechanisms on two-dimensional surfaces and in three-dimensional (3D) matrices. RhoA GTPase activity governs the switch from low-pressure lamellipodia to high-pressure lobopodia in response to the physical structure of the 3D matrix. Inhibiting actomyosin contractility in these cells reduces intracellular pressure and reverts lobopodia to lamellipodial protrusions via an unknown mechanism. To test the hypothesis that high pressure physically prevents lamellipodia formation, we manipulated pressure by activating RhoA or changing the osmolarity of the extracellular environment and imaged cell protrusions. We find RhoA activity inhibits Rac1-mediated lamellipodia formation through two distinct pathways. First, RhoA boosts intracellular pressure by increasing actomyosin contractility and water influx but acts upstream of Rac1 to inhibit lamellipodia formation. Increasing osmotic pressure revealed a second RhoA pathway, which acts through nonmuscle myosin II (NMII) to disrupt lamellipodia downstream from Rac1 and elevate pressure. Interestingly, Arp2/3 inhibition triggered a NMII-dependent increase in intracellular pressure, along with lamellipodia disruption. Together, these results suggest that actomyosin contractility and water influx are coordinated to increase intracellular pressure, and RhoA signaling can inhibit lamellipodia formation via two distinct pathways in high-pressure cells.


Asunto(s)
Presión Osmótica/fisiología , Seudópodos/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/fisiología , Actomiosina/metabolismo , Técnicas de Cultivo de Célula , Movimiento Celular/fisiología , Proteínas del Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Fibroblastos/metabolismo , Humanos , Miosina Tipo II/metabolismo , Miosina Tipo II/fisiología , Transducción de Señal
13.
Dev Cell ; 9(2): 209-21, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16054028

RESUMEN

Actin polymerization in cells occurs via filament elongation at the barbed end. Proteins that cap the barbed end terminate this elongation. Heterodimeric capping protein (CP) is an abundant and ubiquitous protein that caps the barbed end. We find that the mouse homolog of the adaptor protein CARMIL (mCARMIL) binds CP with high affinity and decreases its affinity for the barbed end. Addition of mCARMIL to cell extracts increases the rate and extent of Arp2/3 or spectrin-actin seed-induced polymerization. In cells, GFP-mCARMIL concentrates in lamellipodia and increases the fraction of cells with large lamellipodia. Decreasing mCARMIL levels by siRNA transfection lowers the F-actin level and slows cell migration through a mechanism that includes decreased lamellipodia protrusion. This phenotype is reversed by full-length mCARMIL but not mCARMIL lacking the domain that binds CP. Thus, mCARMIL is a key regulator of CP and has profound effects on cell behavior.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Microfilamentos/metabolismo , Factores Despolimerizantes de la Actina , Actinas/metabolismo , Secuencia de Aminoácidos , Animales , Proteínas Portadoras/genética , Extractos Celulares , Línea Celular Tumoral , Movimiento Celular , Destrina , Glioblastoma , Humanos , Técnicas In Vitro , Ratones , Proteínas de Microfilamentos/genética , Datos de Secuencia Molecular , Mutación , Unión Proteica , Estructura Terciaria de Proteína , Seudópodos/fisiología , ARN Interferente Pequeño/genética , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido
14.
PLoS Biol ; 5(11): e317, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-18044991

RESUMEN

Actin polymerization-driven protrusion of the leading edge is a key element of cell motility. The important actin nucleators formins and the Arp2/3 complex are believed to have nonoverlapping functions in inducing actin filament bundles in filopodia and dendritic networks in lamellipodia, respectively. We tested this idea by investigating the role of mDia2 formin in leading-edge protrusion by loss-of-function and gain-of-function approaches. Unexpectedly, mDia2 depletion by short interfering RNA (siRNA) severely inhibited lamellipodia. Structural analysis of the actin network in the few remaining lamellipodia suggested an mDia2 role in generation of long filaments. Consistently, constitutively active mDia2 (DeltaGBD-mDia2) induced accumulation of long actin filaments in lamellipodia and increased persistence of lamellipodial protrusion. Depletion of mDia2 also inhibited filopodia, whereas expression of DeltaGBD-mDia2 promoted their formation. Correlative light and electron microscopy showed that DeltaGBD-mDia2-induced filopodia were formed from lamellipodial network through gradual convergence of long lamellipodial filaments into bundles. Efficient filopodia induction required mDia2 targeting to the membrane, likely through a scaffolding protein Abi1. Furthermore, mDia2 and Abi1 interacted through the N-terminal regulatory sequences of mDia2 and the SH3-containing Abi1 sequences. We propose that mDia2 plays an important role in formation of lamellipodia by nucleating and/or protecting from capping lamellipodial actin filaments, which subsequently exhibit high tendency to converge into filopodia.


Asunto(s)
Proteínas Portadoras/genética , Movimiento Celular/fisiología , Seudópodos/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/genética , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Forminas , Expresión Génica , Silenciador del Gen , Células HeLa , Humanos , Ratones , Microscopía Electrónica de Rastreo , Seudópodos/ultraestructura , ARN Interferente Pequeño/genética
15.
JCI Insight ; 5(16)2020 08 20.
Artículo en Inglés | MEDLINE | ID: mdl-32814715

RESUMEN

Actin γ 2, smooth muscle (ACTG2) R257C mutation is the most common genetic cause of visceral myopathy. Individuals with ACTG2 mutations endure prolonged hospitalizations and surgical interventions, become dependent on intravenous nutrition and bladder catheterization, and often die in childhood. Currently, we understand little about how ACTG2 mutations cause disease, and there are no mechanism-based treatments. Our goal was to characterize the effects of ACTG2R257C on actin organization and function in visceral smooth muscle cells. We overexpressed ACTG2WT or ACTG2R257C in primary human intestinal smooth muscle cells (HISMCs) and performed detailed quantitative analyses to examine effects of ACTG2R257C on (a) actin filament formation and subcellular localization, (b) actin-dependent HISMC functions, and (c) smooth muscle contractile gene expression. ACTG2R257C resulted in 41% fewer, 13% thinner, 33% shorter, and 40% less branched ACTG2 filament bundles compared with ACTG2WT. Curiously, total F-actin probed by phalloidin and a pan-actin antibody was unchanged between ACTG2WT- and ACTG2R257C-expressing HISMCs, as was ultrastructural F-actin organization. ACTG2R257C-expressing HISMCs contracted collagen gels similar to ACTG2WT-expressing HISMCs but spread 21% more and were 11% more migratory. In conclusion, ACTG2R257C profoundly affects ACTG2 filament bundle structure, without altering global actin cytoskeleton in HISMCs.


Asunto(s)
Actinas/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/genética , Actinas/ultraestructura , Movimiento Celular/genética , Células Cultivadas , Colágeno/química , Regulación de la Expresión Génica , Humanos , Seudoobstrucción Intestinal/genética , Contracción Muscular/genética , Músculo Liso/citología , Mutación
16.
Nat Commun ; 11(1): 4818, 2020 09 23.
Artículo en Inglés | MEDLINE | ID: mdl-32968060

RESUMEN

Migrating cells move across diverse assemblies of extracellular matrix (ECM) that can be separated by micron-scale gaps. For membranes to protrude and reattach across a gap, actin filaments, which are relatively weak as single filaments, must polymerize outward from adhesion sites to push membranes towards distant sites of new adhesion. Here, using micropatterned ECMs, we identify T-Plastin, one of the most ancient actin bundling proteins, as an actin stabilizer that promotes membrane protrusions and enables bridging of ECM gaps. We show that T-Plastin widens and lengthens protrusions and is specifically enriched in active protrusions where F-actin is devoid of non-muscle myosin II activity. Together, our study uncovers critical roles of the actin bundler T-Plastin to promote protrusions and migration when adhesion is spatially-gapped.


Asunto(s)
Movimiento Celular/fisiología , Extensiones de la Superficie Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de Microfilamentos/metabolismo , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Sistemas CRISPR-Cas , Adhesión Celular , Línea Celular , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , Técnicas de Inactivación de Genes , Humanos , Cinética , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/ultraestructura , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/ultraestructura , Miosinas/metabolismo , Seudópodos/metabolismo , Receptor EphB2
17.
J Cell Biol ; 219(9)2020 09 07.
Artículo en Inglés | MEDLINE | ID: mdl-32597939

RESUMEN

Cell migration is driven by pushing and pulling activities of the actin cytoskeleton, but migration directionality is largely controlled by microtubules. This function of microtubules is especially critical for neuron navigation. However, the underlying mechanisms are poorly understood. Here we show that branched actin filament networks, the main pushing machinery in cells, grow directly from microtubule tips toward the leading edge in growth cones of hippocampal neurons. Adenomatous polyposis coli (APC), a protein with both tumor suppressor and cytoskeletal functions, concentrates at the microtubule-branched network interface, whereas APC knockdown nearly eliminates branched actin in growth cones and prevents growth cone recovery after repellent-induced collapse. Conversely, encounters of dynamic APC-positive microtubule tips with the cell edge induce local actin-rich protrusions. Together, we reveal a novel mechanism of cell navigation involving APC-dependent assembly of branched actin networks on microtubule tips.


Asunto(s)
Actinas/metabolismo , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Poliposis Adenomatosa del Colon/metabolismo , Microtúbulos/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Movimiento Celular/fisiología , Células Cultivadas , Conos de Crecimiento/metabolismo , Hipocampo/metabolismo , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley
18.
Mol Biol Cell ; 31(20): 2168-2178, 2020 09 15.
Artículo en Inglés | MEDLINE | ID: mdl-32697617

RESUMEN

SCAR/WAVE proteins and Arp2/3 complex assemble branched actin networks at the leading edge. Two isoforms of SCAR/WAVE, WAVE1 and WAVE2, reside at the leading edge, yet it has remained unclear whether they perform similar or distinct roles. Further, there have been conflicting reports about the Arp2/3-independent biochemical activities of WAVE1 on actin filament elongation. To investigate this in vivo, we knocked out WAVE1 and WAVE2 genes, individually and together, in B16-F1 melanoma cells. We demonstrate that WAVE1 and WAVE2 are redundant for lamellipodia formation and motility. However, there is a significant decrease in the rate of leading edge actin extension in WAVE2 KO cells, and an increase in WAVE1 KO cells. The faster rates of actin extension in WAVE1 KO cells are offset by faster retrograde flow, and therefore do not translate into faster lamellipodium protrusion. Thus, WAVE1 restricts the rate of actin extension at the leading edge, and appears to couple actin networks to the membrane to drive protrusion. Overall, these results suggest that WAVE1 and WAVE2 have redundant roles in promoting Arp2/3-dependent actin nucleation and lamellipodia formation, but distinct roles in controlling actin network extension and harnessing network growth to cell protrusion.


Asunto(s)
Actinas/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/metabolismo , Citoesqueleto de Actina/metabolismo , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Línea Celular Tumoral , Movimiento Celular/genética , Movimiento Celular/fisiología , Extensiones de la Superficie Celular/metabolismo , Humanos , Proteínas de Microfilamentos/metabolismo , Seudópodos/metabolismo , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética
19.
Nat Cell Biol ; 22(6): 674-688, 2020 06.
Artículo en Inglés | MEDLINE | ID: mdl-32451441

RESUMEN

The dynamin GTPase is known to bundle actin filaments, but the underlying molecular mechanism and physiological relevance remain unclear. Our genetic analyses revealed a function of dynamin in propelling invasive membrane protrusions during myoblast fusion in vivo. Using biochemistry, total internal reflection fluorescence microscopy, electron microscopy and cryo-electron tomography, we show that dynamin bundles actin while forming a helical structure. At its full capacity, each dynamin helix captures 12-16 actin filaments on the outer rim of the helix. GTP hydrolysis by dynamin triggers disassembly of fully assembled dynamin helices, releasing free dynamin dimers/tetramers and facilitating Arp2/3-mediated branched actin polymerization. The assembly/disassembly cycles of dynamin promote continuous actin bundling to generate mechanically stiff actin super-bundles. Super-resolution and immunogold platinum replica electron microscopy revealed dynamin along actin bundles at the fusogenic synapse. These findings implicate dynamin as a unique multifilament actin-bundling protein that regulates the dynamics and mechanical strength of the actin cytoskeletal network.


Asunto(s)
Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Comunicación Celular , Drosophila melanogaster/metabolismo , Dinaminas/metabolismo , Endocitosis , Complejo 2-3 Proteico Relacionado con la Actina/metabolismo , Actinas/genética , Secuencia de Aminoácidos , Animales , Drosophila melanogaster/genética , Dinaminas/genética , Femenino , Guanosina Trifosfato/metabolismo , Masculino , Mioblastos/citología , Mioblastos/metabolismo , Unión Proteica , Homología de Secuencia
20.
Nat Cell Biol ; 21(5): 603-613, 2019 05.
Artículo en Inglés | MEDLINE | ID: mdl-30988424

RESUMEN

Mitochondrial fission involves the preconstriction of an organelle followed by scission by dynamin-related protein Drp1. Preconstriction is facilitated by actin and non-muscle myosin II through a mechanism that remains unclear, largely due to the unknown cytoskeletal ultrastructure at mitochondrial constrictions. Here, using platinum replica electron microscopy, we show that mitochondria in cells are embedded in an interstitial cytoskeletal network that contains abundant unbranched actin filaments. Both spontaneous and induced mitochondrial constrictions typically associate with a criss-cross array of long actin filaments that comprise part of this interstitial network. Non-muscle myosin II is found adjacent to mitochondria but is not specifically enriched at the constriction sites. During ionomycin-induced mitochondrial fission, F-actin clouds colocalize with mitochondrial constriction sites, whereas dynamic myosin II clouds are present in the vicinity of constrictions. We propose that myosin II promotes mitochondrial constriction by inducing stochastic deformations of the interstitial actin network, which applies pressure on the mitochondrial surface and thus initiates curvature-sensing mechanisms that complete mitochondrial constriction.


Asunto(s)
Actinas/genética , Citoesqueleto/ultraestructura , Mitocondrias/ultraestructura , Dinámicas Mitocondriales/genética , Miosina Tipo II/genética , Citoesqueleto de Actina/química , Citoesqueleto de Actina/ultraestructura , Actinas/metabolismo , Animales , Células COS , Chlorocebus aethiops , Constricción , Citoesqueleto/metabolismo , Ionomicina/farmacología , Mitocondrias/genética , Dinámicas Mitocondriales/efectos de los fármacos , Proteínas Mitocondriales/química , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Miosina Tipo II/química , Miosina Tipo II/metabolismo
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