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1.
Semin Cell Dev Biol ; 102: 90-104, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31862219

RESUMO

B cells must extract antigens attached to the surface of antigen presenting cells to generate high-affinity antibodies. Antigen extraction requires force, and recent studies have implicated actomyosin-dependent pulling forces generated within the B cell as the major driver of antigen extraction. These actomyosin-dependent pulling forces also serve to test the affinity of the B cell antigen receptor for antigen prior to antigen extraction. Such affinity discrimination is central to the process of antibody affinity maturation. Here we review the evidence that actomyosin-dependent pulling forces generated within the B cell promote affinity discrimination and power antigen extraction. Our take on these critical B cell functions is influenced significantly by the recent identification of formin-generated, myosin-rich, concentric actin arcs in the medial portion of the T cell immune synapse, as B cells appear to contain a similar contractile actomyosin structure.

2.
F1000Res ; 82019.
Artigo em Inglês | MEDLINE | ID: mdl-31497286

RESUMO

Myosin 2 plays a central role in numerous, fundamental, actin-based biological processes, including cell migration, cell division, and the adhesion of cells to substrates and other cells. Here, we highlight recent studies in which the forces created by actomyosin 2 have been shown to also impact tension-sensitive ion channels and cell metabolism.


Assuntos
Actomiosina/fisiologia , Canais Iônicos/fisiologia , Miosina Tipo II/fisiologia , Adesão Celular , Divisão Celular , Movimento Celular , Humanos
3.
J Cell Sci ; 132(16)2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31371487

RESUMO

The spine apparatus (SA) is an endoplasmic reticulum-related organelle that is present in a subset of dendritic spines in cortical and pyramidal neurons, and plays an important role in Ca2+ homeostasis and dendritic spine plasticity. The protein synaptopodin is essential for the formation of the SA and is widely used as a maker for this organelle. However, it is still unclear which factors contribute to its localization at selected synapses, and how it triggers local SA formation. In this study, we characterized development, localization and mobility of synaptopodin clusters in hippocampal primary neurons, as well as the molecular dynamics within these clusters. Interestingly, synaptopodin at the shaft-associated clusters is less dynamic than at spinous clusters. We identify the actin-based motor proteins myosin V (herein referring to both the myosin Va and Vb forms) and VI as novel interaction partners of synaptopodin, and demonstrate that myosin V is important for the formation and/or maintenance of the SA. We found no evidence of active microtubule-based transport of synaptopodin. Instead, new clusters emerge inside spines, which we interpret as the SA being assembled on-site.

4.
Elife ; 82019 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-31157616

RESUMO

When B cells encounter antigens on the surface of an antigen-presenting cell (APC), B cell receptors (BCRs) are gathered into microclusters that recruit signaling enzymes. These microclusters then move centripetally and coalesce into the central supramolecular activation cluster of an immune synapse. The mechanisms controlling BCR organization during immune synapse formation, and how this impacts BCR signaling, are not fully understood. We show that this coalescence of BCR microclusters depends on the actin-related protein 2/3 (Arp2/3) complex, which nucleates branched actin networks. Moreover, in murine B cells, this dynamic spatial reorganization of BCR microclusters amplifies proximal BCR signaling reactions and enhances the ability of membrane-associated antigens to induce transcriptional responses and proliferation. Our finding that Arp2/3 complex activity is important for B cell responses to spatially restricted membrane-bound antigens, but not for soluble antigens, highlights a critical role for Arp2/3 complex-dependent actin remodeling in B cell responses to APC-bound antigens.


Assuntos
Proteína 3 Relacionada a Actina/metabolismo , Proteínas Semelhantes a Angiopoietina/metabolismo , Linfócitos B/imunologia , Sinapses Imunológicas/metabolismo , Ativação Linfocitária , Receptores de Antígenos de Linfócitos B/metabolismo , Transdução de Sinais , Actinas/metabolismo , Animais , Camundongos Endogâmicos C57BL
5.
Cerebellum ; 18(3): 406-421, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30729383

RESUMO

While mixed primary cerebellar cultures prepared from embryonic tissue have proven valuable for dissecting structure-function relationships in cerebellar Purkinje neurons (PNs), this technique is technically challenging and often yields few cells. Recently, mouse embryonic stem cells (mESCs) have been successfully differentiated into PNs, although the published methods are very challenging as well. The focus of this study was to simplify the differentiation of mESCs into PNs. Using a recently described neural differentiation media, we generate monolayers of neural progenitor cells from mESCs and differentiate them into PN precursors using specific extrinsic factors. These PN precursors are then differentiated into mature PNs by co-culturing them with granule neuron (GN) precursors also derived from neural progenitors using different extrinsic factors. The morphology of mESC-derived PNs is indistinguishable from PNs grown in primary culture in terms of gross morphology, spine length, and spine density. Furthermore, mESC-derived PNs express Calbindin D28K, IP3R1, IRBIT, PLCß4, PSD93, and myosin IIB-B2, all of which are either PN-specific or highly expressed in PNs. Moreover, we show that mESC-derived PNs form synapses with GN-like cells as in primary culture, express proteins driven by the PN-specific promoter Pcp2/L7, and exhibit the defect in spine ER inheritance seen in PNs isolated from dilute-lethal (myosin Va-null) mice when expressing a Pcp2/L7-driven miRNA directed against myosin Va. Finally, we define a novel extracellular matrix formulation that reproducibly yields monolayer cultures conducive for high-resolution imaging. Our improved method for differentiating mESCs into PNs should facilitate the dissection of molecular mechanisms and disease phenotypes in PNs.


Assuntos
Diferenciação Celular/fisiologia , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Neurais/citologia , Células de Purkinje/citologia , Animais , Técnicas de Cultura de Células , Camundongos , Camundongos Endogâmicos C57BL
6.
Annu Rev Immunol ; 37: 201-224, 2019 04 26.
Artigo em Inglês | MEDLINE | ID: mdl-30576253

RESUMO

The engagement of a T cell with an antigen-presenting cell (APC) or activating surface results in the formation within the T cell of several distinct actin and actomyosin networks. These networks reside largely within a narrow zone immediately under the T cell's plasma membrane at its site of contact with the APC or activating surface, i.e., at the immunological synapse. Here we review the origin, organization, dynamics, and function of these synapse-associated actin and actomyosin networks. Importantly, recent insights into the nature of these actin-based cytoskeletal structures were made possible in several cases by advances in light microscopy.


Assuntos
Actinas/metabolismo , Actomiosina/metabolismo , Células Apresentadoras de Antígenos/metabolismo , Citoesqueleto/metabolismo , Sinapses Imunológicas/metabolismo , Linfócitos T/metabolismo , Animais , Apresentação do Antígeno , Humanos , Ativação Linfocitária
7.
Structure ; 26(10): 1373-1383.e4, 2018 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-30174147

RESUMO

Melanoregulin (Mreg) is a small, highly charged, multiply palmitoylated protein present on the membrane of melanosomes. Mreg is implicated in the transfer of melanosomes from melanocytes to keratinocytes, and in promoting the microtubule minus end-directed transport of these organelles. The possible molecular function of Mreg was identified by solving its structure using nuclear magnetic resonance (NMR) spectroscopy. Mreg contains six α helices forming a fishhook-like fold in which positive and negative charges occupy opposite sides of the protein's surface and sandwich a putative, cholesterol recognition sequence (CRAC motif). Mreg containing a point mutation within its CRAC motif still targets to late endosomes/lysosomes, but no longer promotes their microtubule minus end-directed transport. Moreover, wild-type Mreg does not promote the microtubule minus end-directed transport of late endosomes/lysosomes in cells transiently depleted of cholesterol. Finally, reversing the charge of three clustered acidic residues partially inhibits Mreg's ability to drive these organelles to microtubule minus ends.


Assuntos
Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Colesterol/metabolismo , Dineínas/metabolismo , Mutação Puntual , Motivos de Aminoácidos , Animais , Proteínas de Transporte/genética , Linhagem Celular , Peptídeos e Proteínas de Sinalização Intracelular , Melanossomas/metabolismo , Camundongos , Microtúbulos/metabolismo , Modelos Moleculares , Estrutura Secundária de Proteína , Transporte Proteico
8.
Cytoskeleton (Hoboken) ; 75(9): 395-409, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29979496

RESUMO

The actin-based motor myosin Va transports numerous cargos, including the smooth endoplasmic reticulum (SER) in cerebellar Purkinje neurons (PNs) and melanosomes in melanocytes. Identifying proteins that interact with this myosin is key to understanding its cellular functions. Toward that end, we used recombineering to insert via homologous recombination a tandem affinity purification (TAP) tag composed of the immunoglobulin G-binding domain of protein A, a tobacco etch virus cleavage site, and a FLAG tag into the mouse MYO5A locus immediately after the initiation codon. Importantly, we provide evidence that the TAP-tagged version of myosin Va (TAP-MyoVa) functions normally in terms of SER transport in PNs and melanosome positioning in melanocytes. Given this and other evidence that TAP-MyoVa is fully functional, we purified it together with associated proteins directly from juvenile mouse cerebella and subjected the samples to mass spectroscopic analyses. As expected, known myosin Va-binding partners like dynein light chain were identified. Importantly, numerous novel interacting proteins were also tentatively identified, including guanine nucleotide-binding protein G(o) subunit alpha (Gnao1), a biomarker for schizophrenia. Consistently, an antibody to Gnao1 immunoprecipitates myosin Va, and Gnao1's localization to PN dendritic spines depends on myosin Va. The mouse model created here should facilitate the identification of novel myosin Va-binding partners, which in turn should advance our understanding of the roles played by this important myosin in vivo.


Assuntos
Cerebelo/fisiologia , Camundongos Transgênicos/metabolismo , Miosina Tipo V/metabolismo , Animais , Camundongos
9.
Proc Natl Acad Sci U S A ; 115(19): 4813-4815, 2018 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-29691320

Assuntos
Plaquetas , Miosinas
10.
Curr Biol ; 28(4): R155-R157, 2018 02 19.
Artigo em Inglês | MEDLINE | ID: mdl-29462581

RESUMO

Cytotoxic T cells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.


Assuntos
Actinas , Amigos , Grânulos Citoplasmáticos , Sinapses Imunológicas , Células Matadoras Naturais , Sinapses
11.
Sci Rep ; 7(1): 17354, 2017 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-29229982

RESUMO

Myosin-X (Myo10) is an unconventional myosin best known for its striking localization to the tips of filopodia. Despite the broad expression of Myo10 in vertebrate tissues, its functions at the organismal level remain largely unknown. We report here the generation of KO-first (Myo10 tm1a/tm1a ), floxed (Myo10 tm1c/tm1c ), and KO mice (Myo10 tm1d/tm1d ). Complete knockout of Myo10 is semi-lethal, with over half of homozygous KO embryos exhibiting exencephaly, a severe defect in neural tube closure. All Myo10 KO mice that survive birth exhibit a white belly spot, all have persistent fetal vasculature in the eye, and ~50% have webbed digits. Myo10 KO mice that survive birth can breed and produce litters of KO embryos, demonstrating that Myo10 is not absolutely essential for mitosis, meiosis, adult survival, or fertility. KO-first mice and an independent spontaneous deletion (Myo10 m1J/m1J ) exhibit the same core phenotypes. During retinal angiogenesis, KO mice exhibit a ~50% decrease in endothelial filopodia, demonstrating that Myo10 is required to form normal numbers of filopodia in vivo. The Myo10 mice generated here demonstrate that Myo10 has important functions in mammalian development and provide key tools for defining the functions of Myo10 in vivo.


Assuntos
Miosinas/fisiologia , Neovascularização Patológica , Tubo Neural/fisiopatologia , Artéria Oftálmica/fisiopatologia , Pigmentação , Pseudópodes/patologia , Corpo Vítreo/patologia , Animais , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Artéria Oftálmica/metabolismo , Pseudópodes/metabolismo , Corpo Vítreo/irrigação sanguínea , Corpo Vítreo/metabolismo
12.
J Biol Chem ; 292(50): 20410-20411, 2017 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-29247130

RESUMO

The scaffolding protein AKAP350A is known to localize to the centrosome and the Golgi, but the molecular details of its function at the centrosome remain elusive. Using structure-function analyses, protein interaction assays, and super-resolution microscopy, Kolobova et al. now identify AKAP350A's specific location and protein partners at the centrosome. The authors further define an autoregulatory mechanism that likely controls AKAP350A's ability to nucleate microtubule growth.


Assuntos
Proteínas de Ancoragem à Quinase A/metabolismo , Centrossomo/metabolismo , Proteínas do Citoesqueleto/metabolismo , Centro Organizador dos Microtúbulos/metabolismo , Modelos Moleculares , Proteínas de Ancoragem à Quinase A/química , Animais , Proteínas de Ciclo Celular , Centrossomo/química , Proteínas do Citoesqueleto/química , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Proteínas Associadas aos Microtúbulos/química , Proteínas Associadas aos Microtúbulos/metabolismo , Centro Organizador dos Microtúbulos/química , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Transporte Proteico
13.
J Cell Biol ; 216(10): 3231-3247, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28893839

RESUMO

Within the mitochondrial matrix, protein aggregation activates the mitochondrial unfolded protein response and PINK1-Parkin-mediated mitophagy to mitigate proteotoxicity. We explore how autophagy eliminates protein aggregates from within mitochondria and the role of mitochondrial fission in mitophagy. We show that PINK1 recruits Parkin onto mitochondrial subdomains after actinonin-induced mitochondrial proteotoxicity and that PINK1 recruits Parkin proximal to focal misfolded aggregates of the mitochondrial-localized mutant ornithine transcarbamylase (ΔOTC). Parkin colocalizes on polarized mitochondria harboring misfolded proteins in foci with ubiquitin, optineurin, and LC3. Although inhibiting Drp1-mediated mitochondrial fission suppresses the segregation of mitochondrial subdomains containing ΔOTC, it does not decrease the rate of ΔOTC clearance. Instead, loss of Drp1 enhances the recruitment of Parkin to fused mitochondrial networks and the rate of mitophagy as well as decreases the selectivity for ΔOTC during mitophagy. These results are consistent with a new model that, instead of promoting mitophagy, fission protects healthy mitochondrial domains from elimination by unchecked PINK1-Parkin activity.


Assuntos
Dinâmica Mitocondrial/fisiologia , Mitofagia/fisiologia , Modelos Biológicos , Agregados Proteicos/fisiologia , GTP Fosfo-Hidrolases/genética , GTP Fosfo-Hidrolases/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Ornitina Carbamoiltransferase/genética , Ornitina Carbamoiltransferase/metabolismo , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Fator de Transcrição TFIIIA/genética , Fator de Transcrição TFIIIA/metabolismo , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
14.
PLoS One ; 12(8): e0183174, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28817635

RESUMO

Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS.


Assuntos
Actomiosina/metabolismo , Ativação Linfocitária , Linfócitos T/metabolismo , Animais , Humanos , Células Jurkat , Ligantes , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosforilação , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/imunologia
15.
Cytoskeleton (Hoboken) ; 74(5): 205-218, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-28329908

RESUMO

The peri-centrosomal localization and morphology of the Golgi apparatus depends largely on the microtubule cytoskeleton and the microtubule motor protein dynein. Recent studies proposed that myosin 18Aα (M18Aα) also contributes to Golgi morphology by binding the Golgi protein GOLPH3 and walking along adjacent actin filaments to stretch the Golgi into its classic ribbon structure. Biochemical analyses have shown, however, that M18A is not an actin-activated ATPase and lacks motor activity. Our goal, therefore, was to define the precise molecular mechanism by which M18Aα determines Golgi morphology. We show that purified M18Aα remains inactive in the presence of GOLPH3, arguing against the Golgi-specific activation of the myosin. Using M18A-specific antibodies and expression of GFP-tagged M18Aα, we find no evidence that it localizes to the Golgi. Moreover, several cell lines with reduced or eliminated M18Aα expression exhibited normal Golgi morphology. Interestingly, actin filament disassembly resulted in a marked reduction in lateral stretching of the Golgi in both control and M18Aα-deficient cells. Importantly, this reduction was accompanied by an expansion of the Golgi in the vertical direction, vertical movement of the centrosome, and increases in the height of both the nucleus and the cell. Collectively, our data indicate that M18Aα does not localize to the Golgi or play a significant role in determining its morphology, and suggest that global F-actin disassembly alters Golgi morphology indirectly by altering cell shape.


Assuntos
Actinas/metabolismo , Complexo de Golgi/metabolismo , Miosinas/metabolismo , Humanos
16.
Nat Cell Biol ; 19(2): 85-93, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28114272

RESUMO

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.


Assuntos
Actinas/metabolismo , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Cadeias Leves de Miosina/genética , Quinase de Cadeia Leve de Miosina/metabolismo , Miosinas/metabolismo , Fragmentos de Peptídeos/metabolismo , Actomiosina/metabolismo , Animais , Técnicas de Introdução de Genes , Camundongos
17.
J Cell Biol ; 215(3): 383-399, 2016 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-27799367

RESUMO

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.


Assuntos
Actomiosina/metabolismo , Movimento Celular , Sinapses Imunológicas/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Animais , Células Apresentadoras de Antígenos/metabolismo , Linfócitos T CD8-Positivos/metabolismo , Agregação Celular , Proteínas Fetais , Fluorescência , Forminas , Humanos , Células Jurkat , Antígeno-1 Associado à Função Linfocitária/metabolismo , Camundongos , Proteínas dos Microfilamentos , Microscopia , Miosina Tipo II/metabolismo , Proteínas Nucleares , Linfócitos T/metabolismo
18.
Proc Natl Acad Sci U S A ; 113(43): E6610-E6619, 2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27791032

RESUMO

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1's ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."


Assuntos
Proteínas de Capeamento de Actina/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Dictyostelium/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Proteínas de Protozoários/genética , Pseudópodes/metabolismo , Proteínas de Capeamento de Actina/metabolismo , Proteína 2 Relacionada a Actina/genética , Proteína 2 Relacionada a Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Proteína 3 Relacionada a Actina/genética , Proteína 3 Relacionada a Actina/metabolismo , Actinas/genética , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Quimiotaxia/genética , Sequência Conservada , Dictyostelium/genética , Dictyostelium/ultraestrutura , Regulação da Expressão Gênica , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Cinética , Camundongos , Mutação , Fosforilação , Pinocitose/genética , Ligação Proteica , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/metabolismo , Pseudópodes/genética , Pseudópodes/ultraestrutura , Alinhamento de Sequência , Transdução de Sinais
19.
Science ; 349(6251): aab3500, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26315442

RESUMO

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.


Assuntos
Citoesqueleto/ultraestrutura , Endocitose , Imageamento Tridimensional/métodos , Microscopia de Fluorescência/métodos , Organelas/ultraestrutura , Actinina/análise , Actinas/análise , Animais , Linhagem Celular , Clatrina/análise , Vesículas Revestidas por Clatrina/química , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/química , Invaginações Revestidas da Membrana Celular/ultraestrutura , Citoesqueleto/química , Citoesqueleto/metabolismo , Endossomos/química , Endossomos/ultraestrutura , Complexo de Golgi/ultraestrutura , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional/instrumentação , Microscopia de Fluorescência/instrumentação , Mitocôndrias/química , Mitocôndrias/ultraestrutura , Organelas/química , Organelas/metabolismo , Proteínas rab5 de Ligação ao GTP/análise
20.
J Cell Sci ; 128(17): 3210-22, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26183180

RESUMO

The lipid phosphate phosphatase-related proteins (LPPRs), also known as plasticity-related genes (PRGs), are classified as a new brain-enriched subclass of the lipid phosphate phosphatase (LPP) superfamily. They induce membrane protrusions, neurite outgrowth or dendritic spine formation in cell lines and primary neurons. However, the exact roles of LPPRs and the mechanisms underlying their effects are not certain. Here, we present the results of a large-scale proteome analysis to determine LPPR1-interacting proteins using co-immunoprecipitation coupled to mass spectrometry. We identified putative LPPR1-binding proteins involved in various biological processes. Most interestingly, we identified the interaction of LPPR1 with its family member LPPR3, LPPR4 and LPPR5. Their interactions were characterized by co-immunoprecipitation and colocalization analysis using confocal and super-resolution microscopy. Moreover, co-expressing two LPPR members mutually elevated their protein levels, facilitated their plasma membrane localization and resulted in an increased induction of membrane protrusions as well as the phosphorylation of S6 ribosomal protein. Taken together, we revealed a new functional cooperation between LPPR family members and discovered for the first time that LPPRs likely exert their function through forming complex with its family members.


Assuntos
Membrana Celular/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Animais , Células COS , Membrana Celular/genética , Chlorocebus aethiops , Células HEK293 , Humanos , Camundongos , Proteínas do Tecido Nervoso/genética , Monoéster Fosfórico Hidrolases/genética , Fosforilação/fisiologia
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