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1.
Invest Ophthalmol Vis Sci ; 60(14): 4583-4595, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31675075

RESUMO

Purpose: Trabecular meshwork (TM) cells detect and coordinate responses to intraocular pressure (IOP) in the eye. TM cells become dysfunctional in glaucoma where IOP is often elevated. Recently, we showed that normal TM (NTM) cells communicate by forming tubular connections called tunneling nanotubes (TNTs). Here, we investigated TNTs in glaucomatous TM (GTM) cells. Methods: Primary GTM and NTM cells were established from cadaver eyes. Transfer of Vybrant DiO and DiD-labeled vesicles via TNT connections was measured. Imaris software measured the number and length of cell protrusions from immunofluorescent confocal images. Live-cell imaging of the actin cytoskeleton was performed. The distribution of myosin-X, a regulator of TNTs/filopodia, was investigated in TM cells and tissue. Results: GTM cells contained significantly more transferred fluorescent vesicles than NTM cells (49.6% vs. 35%). Although NTM cells had more protrusions at the cell surface than GTM cells (7.61 vs. 4.65 protrusions/cell), GTM protrusions were significantly longer (12.1 µm vs. 9.76 µm). Live-cell imaging demonstrated that the GTM actin cytoskeleton was less dynamic, and vesicle transfer between cells was significantly slower than NTM cells. Furthermore, rearrangement of the actin cortex adjacent to the TNT may influence TNT formation. Myosin-X immunostaining was punctate and disorganized in GTM cells and tissue compared to age-matched NTM controls. Conclusions: Together, our data demonstrate that GTM cells have phenotypic and functional differences in their TNTs. Significantly slower vesicle transfer via TNTs in GTM cells may delay the timely propagation of cellular signals when pressures become elevated in glaucoma.


Assuntos
Citoesqueleto de Actina/metabolismo , Glaucoma de Ângulo Aberto/patologia , Miosinas/metabolismo , Nanotubos , Pseudópodes/metabolismo , Malha Trabecular/patologia , Western Blotting , Tamanho Celular , Células Cultivadas , Senescência Celular/fisiologia , Densitometria , Glaucoma de Ângulo Aberto/metabolismo , Humanos , Microscopia Confocal , Fagocitose/fisiologia , Fenótipo , Transdução de Sinais/fisiologia , Malha Trabecular/metabolismo
2.
PLoS Biol ; 17(10): e3000457, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31600188

RESUMO

Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments.


Assuntos
Complexo 2-3 de Proteínas Relacionadas à Actina/genética , Actinas/genética , Polaridade Celular/genética , Quimiotaxia/genética , Família de Proteínas da Síndrome de Wiskott-Aldrich/genética , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Fenômenos Biomecânicos , Sistemas CRISPR-Cas , Adesão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Polaridade Celular/efeitos dos fármacos , Fatores Quimiotáticos/farmacologia , Quimiotaxia/efeitos dos fármacos , Edição de Genes , Regulação da Expressão Gênica , Células HEK293 , Células HL-60 , Humanos , Microscopia de Força Atômica , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Pseudópodes/ultraestrutura , Transdução de Sinais , Propriedades de Superfície , Família de Proteínas da Síndrome de Wiskott-Aldrich/deficiência
3.
Gastroenterology ; 157(6): 1544-1555.e3, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31473225

RESUMO

BACKGROUND & AIMS: Sperm flagellar 1 (also called CLAMP) is a microtubule-associated protein that regulates microtubule dynamics and planar cell polarity in multi-ciliated cells. We investigated the localization and function of sperm flagellar 1, or CLAMP, in human intestinal epithelia cells (IECs). METHODS: We performed studies with SKCO-15 and human intestinal enteroids established from biopsies from different intestinal segments (duodenal, jejunum, ileal, and colon) of a single donor. Enteroids were induced to differentiation after incubation with growth factors. The distribution of endogenous CLAMP in IECs was analyzed by immunofluorescence microscopy using total internal reflection fluorescence-ground state depletion and confocal microscopy. CLAMP localization was followed during the course of intestinal epithelial cell polarization as cells progressed from flat to compact, confluent monolayers. Protein interactions with endogenous CLAMP were determined in SKCO-15 cells using proximity ligation assays and co-immunoprecipitation. CLAMP was knocked down in SKCO-15 monolayers using small hairpin RNAs and cells were analyzed by immunoblot and immunofluorescence microscopy. The impact of CLAMP knock-down in migrating SKCO-15 cells was assessed using scratch-wound assays. RESULTS: CLAMP bound to actin and apical junctional complex proteins but not microtubules in IECs. In silico analysis predicted the calponin-homology domain of CLAMP to contain conserved amino acids required for actin binding. During IEC polarization, CLAMP distribution changed from primarily basal stress fibers and cytoplasm in undifferentiated cells to apical membranes and microvilli in differentiated monolayers. CLAMP accumulated in lamellipodia and filopodia at the leading edge of migrating cells in association with actin. CLAMP knock-down reduced the number of filopodia, perturbed filopodia polarity, and altered the organization of actin filaments within lamellipodia. CONCLUSIONS: CLAMP is an actin-binding protein, rather than a microtubule-binding protein, in IECs. CLAMP distribution changes during intestinal epithelial cell polarization, regulates the formation of filopodia, and appears to assist in the organization of actin bundles within lamellipodia of migrating IECs. Studies are needed to define the CLAMP domains that interact with actin and whether its loss from IECs affects intestinal function.


Assuntos
Actinas/metabolismo , Movimento Celular , Mucosa Intestinal/citologia , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Colo/citologia , Colo/metabolismo , Células Epiteliais , Humanos , Mucosa Intestinal/metabolismo , Microtúbulos/metabolismo
4.
Elife ; 82019 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486771

RESUMO

Cultured mouse peritoneal macrophages release large numbers of ~30-nm cholesterol-rich particles. Here, we show that those particles represent fragments of the plasma membrane that are pulled away and left behind during the projection and retraction of filopodia and lamellipodia. Consistent with this finding, the particles are enriched in proteins found in focal adhesions, which attach macrophages to the substrate. The release of particles is abolished by blocking cell movement (either by depolymerizing actin with latrunculin A or by inhibiting myosin II with blebbistatin). Confocal microscopy and NanoSIMS imaging studies revealed that the plasma membrane-derived particles are enriched in 'accessible cholesterol' (a mobile pool of cholesterol detectable with the modified cytolysin ALO-D4) but not in sphingolipid-sequestered cholesterol [a pool detectable with ostreolysin A (OlyA)]. The discovery that macrophages release cholesterol-rich particles during cellular locomotion is likely relevant to cholesterol efflux and could contribute to extracellular cholesterol deposition in atherosclerotic plaques.


Assuntos
Membrana Celular/metabolismo , Movimento Celular , Micropartículas Derivadas de Células/química , Micropartículas Derivadas de Células/metabolismo , Colesterol/análise , Macrófagos Peritoneais/metabolismo , Pseudópodes/metabolismo , Animais , Células Cultivadas , Camundongos , Proteínas/análise
5.
Nat Commun ; 10(1): 3700, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31420552

RESUMO

Little is known about the role of islet delta cells in regulating blood glucose homeostasis in vivo. Delta cells are important paracrine regulators of beta cell and alpha cell secretory activity, however the structural basis underlying this regulation has yet to be determined. Most delta cells are elongated and have a well-defined cell soma and a filopodia-like structure. Using in vivo optogenetics and high-speed Ca2+ imaging, we show that these filopodia are dynamic structures that contain a secretory machinery, enabling the delta cell to reach a large number of beta cells within the islet. This provides for efficient regulation of beta cell activity and is modulated by endogenous IGF-1/VEGF-A signaling. In pre-diabetes, delta cells undergo morphological changes that may be a compensation to maintain paracrine regulation of the beta cell. Our data provides an integrated picture of how delta cells can modulate beta cell activity under physiological conditions.


Assuntos
Ilhotas Pancreáticas/ultraestrutura , Comunicação Parácrina , Estado Pré-Diabético/patologia , Pseudópodes/ultraestrutura , Células Secretoras de Somatostatina/ultraestrutura , Animais , Glicemia/metabolismo , Humanos , Fator de Crescimento Insulin-Like I/metabolismo , Células Secretoras de Insulina/citologia , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestrutura , Microscopia Intravital , Ilhotas Pancreáticas/citologia , Ilhotas Pancreáticas/metabolismo , Ilhotas Pancreáticas/patologia , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Imagem Óptica , Optogenética , Estado Pré-Diabético/metabolismo , Pseudópodes/metabolismo , Células Secretoras de Somatostatina/citologia , Células Secretoras de Somatostatina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo
6.
Cells ; 8(7)2019 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-31330900

RESUMO

The Rho GTPases comprise a subfamily of the Ras superfamily of small GTPases. Their importance in regulation of cell morphology and cell migration is well characterized. According to the prevailing paradigm, Cdc42 regulates the formation of filopodia, Rac1 regulates the formation of lamellipodia, and RhoA triggers the assembly of focal adhesions. However, this scheme is clearly an oversimplification, as the Rho subfamily encompasses 20 members with diverse effects on a number of vital cellular processes, including cytoskeletal dynamics and cell proliferation, migration, and invasion. This article highlights the importance of the catalytic activities of the classical Rho GTPases Cdc42 and Rac1, in terms of their specific effects on the dynamic reorganization of the actin filament system. GTPase-deficient mutants of Cdc42 and Rac1 trigger the formation of broad lamellipodia and stress fibers, and fast-cycling mutations trigger filopodia formation and stress fiber dissolution. The filopodia response requires the involvement of the formin family of actin nucleation promotors. In contrast, the formation of broad lamellipodia induced by GTPase-deficient Cdc42 and Rac1 is mediated through Arp2/3-dependent actin nucleation.


Assuntos
Fibras de Estresse/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Domínio Catalítico , Células Cultivadas , Humanos , Mutação , Pseudópodes/genética , Pseudópodes/metabolismo , Fibras de Estresse/genética , Proteína cdc42 de Ligação ao GTP/química , Proteína cdc42 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/química , Proteínas rac1 de Ligação ao GTP/genética
7.
Mol Biol Cell ; 30(16): 1938-1960, 2019 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-31188739

RESUMO

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell-cell junctions to allow dynamic remodeling while resisting forces generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional-cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction-cytoskeletal linkage during gastrulation. Canoe's mammalian homologue Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We take these insights back to the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found that Canoe helps cells maintain junctional-cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation, and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe, many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homologue Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest that Canoe and Polychaetoid stabilize Bazooka/Par3 at cell-cell junctions, helping maintain balanced apical contractility and tissue integrity.


Assuntos
Junções Aderentes/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Células Epiteliais/metabolismo , Proteínas de Junções Íntimas/metabolismo , Animais , Forma Celular , Citoesqueleto/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário , Epiderme/metabolismo , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Morfogênese , Mutação/genética , Fenótipo , Pseudópodes/metabolismo
8.
eNeuro ; 6(2)2019.
Artigo em Inglês | MEDLINE | ID: mdl-31068362

RESUMO

Mutations and copy number variants of the CUB and Sushi multiple domains 2 (CSMD2) gene are associated with neuropsychiatric disease. CSMD2 encodes a single-pass transmembrane protein with a large extracellular domain comprising repeats of CUB and Sushi domains. High expression of CSMD2 in the developing and mature brain suggests possible roles in neuron development or function, but the cellular functions of CSMD2 are not known. In this study, we show that mouse Csmd2 is expressed in excitatory and inhibitory neurons in the forebrain. Csmd2 protein exhibits a somatodendritic localization in the neocortex and hippocampus, with smaller puncta localizing to the neuropil. Using immunohistochemical and biochemical methods, we demonstrate that Csmd2 localizes to dendritic spines and is enriched in the postsynaptic density (PSD). Accordingly, we show that the cytoplasmic tail domain of Csmd2 interacts with synaptic scaffolding proteins of the membrane-associated guanylate kinase (MAGUK) family. The association between Csmd2 and MAGUK member PSD-95 is dependent on a PDZ-binding domain on the Csmd2 tail, which is also required for synaptic targeting of Csmd2. Finally, we show that knock-down of Csmd2 expression in hippocampal neuron cultures results in reduced complexity of dendritic arbors and deficits in dendritic spine density. Knock-down of Csmd2 in immature developing neurons results in reduced filopodia density, whereas Csmd2 knock-down in mature neurons causes significant reductions in dendritic spine density and dendrite complexity. Together, these results point toward a function for Csmd2 in development and maintenance of dendrites and synapses, which may account for its association with certain psychiatric disorders.


Assuntos
Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large/metabolismo , Hipocampo/metabolismo , Proteínas de Membrana/metabolismo , Neocórtex/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/fisiologia , Densidade Pós-Sináptica/metabolismo , Animais , Células Cultivadas , Feminino , Hipocampo/citologia , Masculino , Proteínas de Membrana/deficiência , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/deficiência , Neurônios/metabolismo , Pseudópodes/metabolismo
9.
Dev Cell ; 49(3): 444-460.e9, 2019 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-31063759

RESUMO

Actin assembly supplies the structural framework for cell morphology and migration. Beyond structure, this actin framework can also be engaged to drive biochemical signaling programs. Here, we describe how the hyperactivation of Rac1 via the P29S mutation (Rac1P29S) in melanoma hijacks branched actin network assembly to coordinate proliferative cues that facilitate metastasis and drug resistance. Upon growth challenge, Rac1P29S-harboring melanoma cells massively upregulate lamellipodia formation by dendritic actin polymerization. These extended lamellipodia form a signaling microdomain that sequesters and phospho-inactivates the tumor suppressor NF2/Merlin, driving Rac1P29S cell proliferation in growth suppressive conditions. These biochemically active lamellipodia require cell-substrate attachment but not focal adhesion assembly and drive proliferation independently of the ERK/MAPK pathway. These data suggest a critical link between cell morphology and cell signaling and reconcile the dichotomy of Rac1's regulation of both proliferation and actin assembly by revealing a mutual signaling axis wherein actin assembly drives proliferation in melanoma.


Assuntos
Células Dendríticas/metabolismo , Melanoma/metabolismo , Pseudópodes/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Dendritos/metabolismo , Dendritos/patologia , Feminino , Xenoenxertos , Humanos , Sistema de Sinalização das MAP Quinases , Melanoma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Metástase Neoplásica , Pseudópodes/patologia , Proteínas rac1 de Ligação ao GTP/genética
10.
Mol Biol Cell ; 30(15): 1817-1833, 2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31116646

RESUMO

Cortactin is a Src tyrosine phosphorylation substrate that regulates multiple actin-related cellular processes. While frequently studied in nonneuronal cells, the functions of cortactin in neuronal growth cones are not well understood. We recently reported that cortactin mediates the effects of Src tyrosine kinase in regulating actin organization and dynamics in both lamellipodia and filopodia of Aplysia growth cones. Here, we identified a single cortactin tyrosine phosphorylation site (Y499) to be important for the formation of filopodia. Overexpression of a 499F phospho-deficient cortactin mutant decreased filopodia length and density, whereas overexpression of a 499E phospho-mimetic mutant increased filopodia length. Using an antibody against cortactin pY499, we showed that tyrosine-phosphorylated cortactin is enriched along the leading edge. The leading edge localization of phosphorylated cortactin is Src2-dependent, F-actin-independent, and important for filopodia formation. In vitro kinase assays revealed that Src2 phosphorylates cortactin at Y499, although Y505 is the preferred site in vitro. Finally, we provide evidence that Arp2/3 complex acts downstream of phosphorylated cortactin to regulate density but not length of filopodia. In conclusion, we have characterized a tyrosine phosphorylation site in Aplysia cortactin that plays a major role in the Src/cortactin/Arp2/3 signaling pathway controlling filopodia formation.


Assuntos
Cortactina/metabolismo , Cones de Crescimento/metabolismo , Neurônios/metabolismo , Fosfotirosina/metabolismo , Pseudópodes/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Animais , Aplysia/metabolismo , Membrana Celular/metabolismo , Fosforilação , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Quinases da Família src/metabolismo
11.
Mol Biol Cell ; 30(11): 1285-1297, 2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30893014

RESUMO

Filopodia are actin-filled membrane protrusions that play essential roles in cell motility and cell-cell communication and act as precursors of dendritic spines. IRSp53 is an essential regulator of filopodia formation, which couples Rho-GTPase signaling to actin cytoskeleton and membrane remodeling. IRSp53 has three major domains: an N-terminal inverse-BAR (I-BAR) domain, a Cdc42- and SH3-binding CRIB-PR domain, and an SH3 domain that binds downstream cytoskeletal effectors. Phosphorylation sites in the region between the CRIB-PR and SH3 domains mediate the binding of 14-3-3. Yet the mechanism by which 14--3-3 regulates filopodia formation and dynamics and its role in cell migration are poorly understood. Here, we show that phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters activation by Cdc42 and cytoskeletal effectors, resulting in down-regulation of filopodia dynamics and cancer cell migration. In serum-starved cells, increased IRSp53 phosphorylation triggers 14-3-3 binding, which inhibits filopodia formation and dynamics, irrespective of whether IRSp53 is activated by Cdc42 or downstream effectors (Eps8, Ena/VASP). Pharmacological activation or inhibition of AMPK, respectively, increases or decreases the phosphorylation of two of three sites in IRSp53 implicated in 14-3-3 binding. Mutating these phosphorylation sites reverses 14-3-3-dependent inhibition of filopodia dynamics and cancer cell chemotaxis.


Assuntos
Movimento Celular , Proteínas do Tecido Nervoso/metabolismo , Processamento de Proteína Pós-Traducional , Pseudópodes/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas 14-3-3/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Citoesqueleto de Actina , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células HEK293 , Humanos , Fosforilação , Pseudópodes/fisiologia
12.
Cells ; 8(3)2019 03 22.
Artigo em Inglês | MEDLINE | ID: mdl-30909497

RESUMO

CD44 is a multifunctional adhesion molecule typically upregulated in malignant, inflamed and injured tissues. Due to its ability to bind multiple ligands present in the tumor microenvironment, it promotes multiple cellular functions related to tumorigenesis. Recent data has shown that CD44 and its principal ligand hyaluronan (HA) are carried by extracellular vesicles (EV) derived from stem and tumor cells, but the role of CD44 in EV shedding has not been studied so far. To answer this question, we utilized CD44-negative human gastric carcinoma cell line MKN74 manipulated to stably express CD44 standard form (CD44s). The effect of CD44s expression on HA metabolism, EV secretion, morphology and growth of these cells was studied. Interestingly, HAS2 and HYAL2 expression levels were significantly upregulated in CD44s-expressing cells. Cell-associated HA levels were significantly increased, while HA levels in the culture medium of CD44s-positive cells was lower compared to CD44s-negative MOCK cells. CD44s expression had no significant effect on the proliferation capacity of cells, but cells showed diminished contact inhibition. Superresolution imaging revealed that CD44s and HA were accumulated on filopodia and EVs secreted from CD44s-positive cells, but no differences in total numbers of secreted EV between CD44s-negative and -positive cells was detected. In 3D cultures, CD44s-expressing cells had an enhanced invasion capacity in BME gel and increased spheroidal growth when cultured in collagen I gel. No significant differences in mitotic activity, tumor size or morphology were detected in CAM assays. However, a significant increase in HA staining coverage was detected in CD44s-positive tumors. Interestingly, CD44s-positive EVs embedded in HA-rich matrix were detected in the stromal areas of tumors. The results indicate that CD44s expression significantly increases the HA binding capacity of gastric cancer cells, while the secreted HA is downregulated. CD44s is also carried by EVs secreted by CD44s-expressing cells. These findings highlight the potential usefulness of CD44s and its ligands as multipurpose EV biomarkers, because they are upregulated in inflammatory, injured, and cancer cells and accumulate on the surface of EVs secreted in these situations.


Assuntos
Carcinogênese/metabolismo , Carcinogênese/patologia , Vesículas Extracelulares/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Pseudópodes/metabolismo , Neoplasias Gástricas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Galinhas , Membrana Corioalantoide/metabolismo , Colágeno/metabolismo , Vesículas Extracelulares/ultraestrutura , Humanos , Invasividade Neoplásica , Pseudópodes/ultraestrutura , Neoplasias Gástricas/ultraestrutura
13.
Breast Cancer ; 26(5): 581-593, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-30830684

RESUMO

PURPOSE: Pseudopodia are actin-rich ventral protrusions associated with cell motility and cancer cell invasion. We previously applied our established method of using excimer laser cell etching to isolate pseudopodial proteins from MDA-MB-231 breast cancer cells. We later identified 14-3-3γ as an oncogenic molecule among 46 candidate proteins that are specific to pseudopodia. The present study aimed to determine the function of 14-3-3γ in the motility of breast cancer cells. METHODS: MDA-MB-231 cells were cultured on 3-µm porous membranes and double stained to localize 14-3-3γ and phalloidin in pseudopodia using confocal imaging. We assessed pseudopodia numbers and length, as well as migration and wound healing in MDA-MB-231 cells with knockdown and forced expression of 14-3-3γ to determine 14-3-3γ involvement in cell motility. We also immunohistochemically analyzed 14-3-3γ in human breast cancer tissues with high-grade lymphatic invasion. RESULTS: We specifically located 14-3-3γ in pseudopodia of MDA-MB-231 cells. Knockdown and forced expression of 14-3-3γ, respectively, decreased and increased pseudopodial formation and elongation. Migration and wound healing assays also showed that 14-3-3γ knockdown and forced expression, respectively, decreased and increased the number of underside cells and acellular areas in MDA-MB-231 breast cancer cells. More 14-3-3γ was expressed in sites of lymphatic invasion, than in the center and periphery of human breast cancer tissues. CONCLUSION: The role of 14-3-3γ in breast cancer invasiveness might be to promote cell motility. Inhibition of 14-3-3γ could, therefore, become a novel target of therapy to prevent invasion and metastasis in patients with breast cancers expressing 14-3-3γ.


Assuntos
Proteínas 14-3-3/metabolismo , Neoplasias da Mama/patologia , Movimento Celular , Pseudópodes/metabolismo , Proteínas 14-3-3/genética , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Técnicas de Silenciamento de Genes , Humanos , Metástase Linfática/fisiopatologia , Células MCF-7 , Camundongos , Microscopia Confocal , Pessoa de Meia-Idade , Células NIH 3T3 , Invasividade Neoplásica , Transfecção
14.
Cells ; 8(2)2019 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-30717410

RESUMO

Cell division cycle 42 (CDC42), a small Rho GTPase, plays a critical role in many cellular processes, including cell proliferation and survival. CDC42 interacts with the CRIB (Cdc42- and Rac-interactive binding) domain of CDC42SE1, a small effector protein of 9 kDa. We found that the expression of CDC42SE1 was reduced in human skin cancer samples relative to matched perilesional control. Exogenous expression of CDC42SE1 but not CDC42SE1H38A (mutation within CRIB domain) in A431 cells (A431SE1, A431SE1-H38A) reduced cell proliferation. Antibody microarray analysis of A431Ctrl and A431SE1 lysate suggested that reduced A431SE1 cells proliferation was due to inhibition of Akt pathway, which was confirmed by the reduced P-Akt and P-mTOR levels in A431SE1 cells compared to A431Ctrl cells. This suggests that CDC42SE1 modulates the CDC42-mediated Akt pathway by competing with other effector proteins to bind CDC42. A431SE1 cells formed smaller colonies in soft agar compared to A431Ctrl and A431SE1-H38A cells. These findings correlate with nude mice xenograft assays, where A431SE1 cells formed tumors with significantly-reduced volume compared to the tumors formed by A431Ctrl cells. Our results suggest that CDC42SE1 is downregulated in skin cancer to promote tumorigenesis, and thus CDC42SE1 might be an important marker of skin cancer progression.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas do Citoesqueleto/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Animais , Caderinas/metabolismo , Proteínas de Transporte/genética , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Citoplasma/metabolismo , Proteínas do Citoesqueleto/genética , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos Nus , Pseudópodes/metabolismo , Neoplasias Cutâneas/genética , Neoplasias Cutâneas/patologia
15.
Life Sci Alliance ; 2(1)2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30737247

RESUMO

Directed cell migration requires centrosome-mediated cell polarization and dynamical control of focal adhesions (FAs). To examine how FAs cooperate with centrosomes for directed cell migration, we used centrosome-deficient cells and found that loss of centrosomes enhanced the formation of acentrosomal microtubules, which failed to form polarized structures in wound-edge cells. In acentrosomal cells, we detected higher levels of Rac1-guanine nucleotide exchange factor TRIO (Triple Functional Domain Protein) on microtubules and FAs. Acentrosomal microtubules deliver TRIO to FAs for Rac1 regulation. Indeed, centrosome disruption induced excessive Rac1 activation around the cell periphery via TRIO, causing rapid FA turnover, a disorganized actin meshwork, randomly protruding lamellipodia, and loss of cell polarity. This study reveals the importance of centrosomes to balance the assembly of centrosomal and acentrosomal microtubules and to deliver microtubule-associated TRIO proteins to FAs at the cell front for proper spatial activation of Rac1, FA turnover, lamillipodial protrusion, and cell polarization, thereby allowing directed cell migration.


Assuntos
Movimento Celular/fisiologia , Polaridade Celular/fisiologia , Centrossomo/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Adesões Focais/metabolismo , Técnicas de Silenciamento de Genes , Fatores de Troca do Nucleotídeo Guanina/genética , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Pseudópodes/metabolismo , Epitélio Pigmentado da Retina/citologia , Transfecção
16.
Invest Ophthalmol Vis Sci ; 60(2): 843-851, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30807639

RESUMO

Purpose: The actin cytoskeleton plays a key role in outflow regulation through the trabecular meshwork (TM). Although actin stress fibers are a target of glaucoma therapies, the role of other actin cellular structures is unclear. Myosin-X (Myo10) is an actin-binding protein that is involved in tunneling nanotube (TNT) and filopodia formation. Here, we inhibited Myo10 pharmacologically or by gene silencing to investigate the role of filopodia/TNTs in the TM. Methods: Short hairpin RNA interference (RNAi) silencing lentivirus targeting myosin-X (shMyo10) was generated. Human anterior segments were perfused with shMyo10 or CK-666, an Arp2/3 inhibitor. Confocal microscopy investigated the colocalization of Myo10 with matrix metalloproteinase (MMPs). Western immunoblotting investigated the protein levels of MMPs and extracellular matrix (ECM) proteins. MMP activity and phagocytosis assays were performed. Results: CK-666 and shMyo10-silencing lentivirus caused a significant reduction in outflow rates in anterior segment perfusion culture, an ex vivo method to study intraocular pressure regulation. In human TM cells, Myo10 colocalized with MMP2, MMP14, and cortactin in podosome-like structures, which function as regions of focal ECM degradation. Furthermore, MMP activity, thrombospondin-1 and SPARC protein levels were significantly reduced in the media of CK-666-treated and shMyo10-silenced TM cells. However, neither Myo10 silencing or CK-666 treatment significantly affected phagocytic uptake. Conclusions: Inhibiting filopodia/TNTs caused opposite effects on outflow compared with inhibiting stress fibers. Moreover, Myo10 may also play a role in focal ECM degradation in TM cells. Our results provide additional insight into the function of actin supramolecular assemblies and actin-binding proteins in outflow regulation.


Assuntos
Humor Aquoso/fisiologia , Inativação Gênica , Microvasos/fisiologia , Miosinas/fisiologia , Malha Trabecular/metabolismo , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Proteínas da Matriz Extracelular/metabolismo , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Indóis/farmacologia , Lentivirus/genética , Masculino , Metaloproteinases da Matriz/metabolismo , Microscopia Confocal , Pessoa de Meia-Idade , Nanotubos , Fagocitose , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Interferência de RNA/fisiologia , Reação em Cadeia da Polimerase em Tempo Real
17.
PLoS One ; 14(2): e0212711, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30794657

RESUMO

Wnts are a family of secreted palmitoleated glycoproteins that play key roles in cell to cell communication during development and regulate stem cell compartments in adults. Wnt receptors, downstream signaling cascades and target pathways have been extensively studied while less is known about how Wnts are secreted and move from producing cells to receiving cells. We used the synchronization system called Retention Using Selective Hook (RUSH) to study Wnt trafficking from endoplasmic reticulum to Golgi and then to plasma membrane and filopodia in real time. Inhibition of porcupine (PORCN) or knockout of Wntless (WLS) blocked Wnt exit from the ER. Wnt-containing vesicles paused at sub-cortical regions of the plasma membrane before exiting the cell. Wnt-containing vesicles were associated with filopodia extending to adjacent cells. These data visualize and confirm the role of WLS and PORCN in ER exit of Wnts and support the role of filopodia in Wnt signaling.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Pseudópodes/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Wnt/metabolismo , Membrana Celular/genética , Retículo Endoplasmático/genética , Células HEK293 , Células HeLa , Humanos , Transporte Proteico/genética , Pseudópodes/genética , Vesículas Secretórias/genética , Proteínas Wnt/genética
18.
Nat Commun ; 10(1): 483, 2019 01 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696821

RESUMO

Filopodia are precursors of dendritic spines and polarized cell migration. The I-BAR-domain protein IRSp53 is a key regulator of filopodia dynamics that couples Rho-GTPase signaling to cytoskeleton and membrane remodeling, playing essential roles in neuronal development and cell motility. Here, we describe the structural-functional basis for 14-3-3-dependent inhibition of IRSp53. Phosphoproteomics, quantitative binding and crystallographic studies demonstrate that 14-3-3 binds to two pairs of phosphorylation sites in IRSp53. Using bicistronic expression, we obtain an IRSp53 heterodimer in which only one subunit is phosphorylated, and show that each subunit of IRSp53 independently binds one 14-3-3 dimer. A FRET-sensor assay using natively phosphorylated IRSp53 reveals opposite conformational changes upon binding of activatory (Cdc42, Eps8) or inhibitory (14-3-3) inputs. Finally, we show that 14-3-3 inhibits IRSp53 binding to membranes. Collectively, our findings support a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters membrane binding and interactions with Cdc42 and downstream cytoskeletal effectors.


Assuntos
Proteínas 14-3-3/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Proteínas 14-3-3/química , Proteínas 14-3-3/genética , Sítios de Ligação , Espinhas Dendríticas/genética , Espinhas Dendríticas/metabolismo , Humanos , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/genética , Fosforilação , Ligação Proteica , Pseudópodes/genética , Pseudópodes/metabolismo
19.
Genes Cells ; 24(3): 202-213, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30664308

RESUMO

Fascin, an actin-bundling protein, is present in the filopodia and lamellipodia of growth cones. However, few studies have examined lamellipodial fascin because it is difficult to observe. In this study, we evaluated lamellipodial fascin. We visualized the actin meshwork of lamellipodia in live growth cones by super-resolution microscopy. Fascin was colocalized with the actin meshwork in lamellipodia. Ser39 of fascin is a well-known phosphorylation site that controls the binding of fascin to actin filaments. Fluorescence recovery after photobleaching experiments with confocal microscopy showed that binding of fascin was controlled by phosphorylation of Ser39 in lamellipodia. Moreover, TPA, an agonist of protein kinase C, induced phosphorylation of fascin and dissociation from actin filaments in lamellipodia. Time series images showed that dissociation of fascin from the actin meshwork was induced by TPA. As fascin dissociated from actin filaments, the orientation of the actin filaments became parallel to the leading edge. The angle of actin filaments against the leading edge was changed from 73° to 15°. This decreased the elasticity of the lamellipodia by 40%, as measured by atomic force microscopy. These data suggest that actin bundles made by fascin contribute to elasticity of the growth cone.


Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas de Transporte/metabolismo , Proteínas dos Microfilamentos/metabolismo , Pseudópodes/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Proteínas de Transporte/química , Linhagem Celular , Elasticidade , Recuperação de Fluorescência Após Fotodegradação , Camundongos , Proteínas dos Microfilamentos/química , Fosforilação , Pseudópodes/ultraestrutura
20.
Int J Mol Sci ; 20(2)2019 01 21.
Artigo em Inglês | MEDLINE | ID: mdl-30669557

RESUMO

Arf GTPase-activating proteins (Arf GAPs) control the activity of ADP-ribosylation factors (Arfs) by inducing GTP hydrolysis and participate in a diverse array of cellular functions both through mechanisms that are dependent on and independent of their Arf GAP activity. A number of these functions hinge on the remodeling of actin filaments. Accordingly, some of the effects exerted by Arf GAPs involve proteins known to engage in regulation of the actin dynamics and architecture, such as Rho family proteins and nonmuscle myosin 2. Circular dorsal ruffles (CDRs), podosomes, invadopodia, lamellipodia, stress fibers and focal adhesions are among the actin-based structures regulated by Arf GAPs. Arf GAPs are thus important actors in broad functions like adhesion and motility, as well as the specialized functions of bone resorption, neurite outgrowth, and pathogen internalization by immune cells. Arf GAPs, with their multiple protein-protein interactions, membrane-binding domains and sites for post-translational modification, are good candidates for linking the changes in actin to the membrane. The findings discussed depict a family of proteins with a critical role in regulating actin dynamics to enable proper cell function.


Assuntos
Fatores de Ribosilação do ADP/metabolismo , Citoesqueleto de Actina/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fatores de Ribosilação do ADP/química , Citoesqueleto de Actina/química , Actinas/química , Actinas/metabolismo , Animais , Apoptose , Movimento Celular , Adesões Focais , Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/genética , Interações Hospedeiro-Patógeno , Humanos , Família Multigênica , Crescimento Neuronal , Neurônios/metabolismo , Podossomos/metabolismo , Ligação Proteica , Pseudópodes/metabolismo , Relação Estrutura-Atividade , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismo
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