Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 6 de 6
Filtrar
1.
Langmuir ; 27(1): 320-7, 2011 Jan 04.
Artigo em Inglês | MEDLINE | ID: mdl-21141848

RESUMO

A predominate question associated with supported bilayer assemblies containing proteins is whether or not the proteins remain active after incorporation. The major cause for concern is that strong interactions with solid supports can render the protein inactive. To address this question, a large transmembrane protein, the serotonin receptor, 5HT(3A), has been incorporated into several supported membrane bilayer assemblies of increasing complexity. The 5HT(3A) receptor has large extracellular domains on both sides of the membrane, which could cause strong interactions. The bilayer assemblies include a simple POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) supported planar bilayer, a "single-cushion" POPC bilayer with a PEG (poly(ethylene glycol)) layer between membrane and support, and a "double-cushion" POPC bilayer with both a PEG layer and a layer of BSA (bovine serum albumin). Single-cushion systems are designed to lift the bilayer from the surface, and double-cushion systems are designed to both lift the membrane and passivate the solid support. As in previously reported work, protein mobilities measured by ensemble fluorescence recovery after photobleaching (FRAP) are quite low, especially in the double-cushion system. But single-particle tracking of fluorescent 5HT(3A) molecules shows that individual proteins in the double-cushion system have quite high local mobilities but are spatially confined within small corralling domains ( 450 nm). Comparisons with the simple POPC membrane and the single-cushion POPC−PEG membrane reveal that BSA both serves to minimize interactions with the solid support and creates the corrals that reduce the long-range (ensemble averaged) mobility of large transmembrane proteins. These results suggest that in double-cushion assemblies proteins with large extra-membrane domains may remain active and unperturbed despite low bulk diffusion constants.


Assuntos
Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Bicamadas Lipídicas/química , Receptores 5-HT3 de Serotonina/química , Receptores 5-HT3 de Serotonina/metabolismo , Animais , Bovinos , Recuperação de Fluorescência Após Fotodegradação , Polietilenoglicóis/química , Soroalbumina Bovina/química
2.
Elife ; 102021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34236315

RESUMO

MGA, a transcription factor and member of the MYC network, is mutated or deleted in a broad spectrum of malignancies. As a critical test of a tumor suppressive role, we inactivated Mga in two mouse models of non-small cell lung cancer using a CRISPR-based approach. MGA loss significantly accelerated tumor growth in both models and led to de-repression of non-canonical Polycomb ncPRC1.6 targets, including genes involved in metastasis and meiosis. Moreover, MGA deletion in human lung adenocarcinoma lines augmented invasive capabilities. We further show that MGA-MAX, E2F6, and L3MBTL2 co-occupy thousands of promoters and that MGA stabilizes these ncPRC1.6 subunits. Lastly, we report that MGA loss also induces a pro-growth effect in human colon organoids. Our studies establish MGA as a bona fide tumor suppressor in vivo and suggest a tumor suppressive mechanism in adenocarcinomas resulting from widespread transcriptional attenuation of MYC and E2F target genes mediated by MGA-MAX associated with a non-canonical Polycomb complex.


Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Repressão Epigenética , Proteínas do Grupo Polycomb/genética , Adenocarcinoma de Pulmão/genética , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Humanos , Masculino , Camundongos , Invasividade Neoplásica/genética , Proteínas do Grupo Polycomb/metabolismo
3.
Dev Cell ; 45(6): 738-752.e6, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29920278

RESUMO

Normal cells acquire aggressive behavior by modifying signaling pathways. For instance, alteration of endocytosis profoundly impacts both proliferation and migration during tumorigenesis. Here we investigate the mechanisms that enable the endocytic machinery to coordinate these processes. We show that a membrane curvature-sensing protein, endophilin A3, promotes growth and migration of colon cancer cells through two competing mechanisms: an endocytosis pathway that is required for proliferation and a GTPase regulatory pathway that controls cell motility. EndoA3 stimulates cell migration by binding the Rac GEF TIAM1 leading to activation of small GTPases. Competing interactions of EndoA3 with membrane versus TIAM1 modulate hyperproliferative and metastatic phenotypes. Disruption of EndoA3-membrane interactions stimulates TIAM1 and small GTPases in vitro, and further promotes pro-metastatic phenotypes in vivo. Together, these results uncover a coupling mechanism, by which EndoA3 promotes growth and migration of colon cancers, by linking membrane dynamics to GTPase regulation.


Assuntos
Aciltransferases/metabolismo , Neoplasias do Colo/metabolismo , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T/metabolismo , Animais , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Proliferação de Células/fisiologia , Transformação Celular Neoplásica , Neoplasias do Colo/patologia , Endocitose/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Camundongos , Metástase Neoplásica , Transdução de Sinais , Peixe-Zebra , Proteínas rac1 de Ligação ao GTP/metabolismo
4.
Dev Cell ; 43(5): 549-562.e6, 2017 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-29207258

RESUMO

Interactions between tumor cells and tumor-associated macrophages play critical roles in the initiation of tumor cell motility. To capture the cellular interactions of the tumor microenvironment with high-resolution imaging, we directly visualized tumor cells and their interactions with macrophages in zebrafish. Live imaging in zebrafish revealed that macrophages are dynamic, yet maintain sustained contact with tumor cells. In addition, the recruitment of macrophages to tumor cells promotes tumor cell dissemination. Using a Cre/LoxP strategy, we found that macrophages transfer cytoplasm to tumor cells in zebrafish and mouse models. Remarkably, macrophage cytoplasmic transfer correlated with melanoma cell dissemination. We further found that macrophages transfer cytoplasm to tumor cells upon cell contact in vitro. Thus, we present a model in which macrophage/tumor cell contact allows for the transfer of cytoplasmic molecules from macrophages to tumor cells corresponding to increased tumor cell motility and dissemination.


Assuntos
Comunicação Celular/fisiologia , Macrófagos/patologia , Melanoma/patologia , Microambiente Tumoral/fisiologia , Animais , Linhagem Celular Tumoral , Movimento Celular/fisiologia , Citoplasma/metabolismo , Camundongos , Invasividade Neoplásica , Peixe-Zebra
5.
Mol Biol Cell ; 27(13): 2119-32, 2016 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-27170174

RESUMO

Numerous proteins act in concert to sculpt membrane compartments for cell signaling and metabolism. These proteins may act as curvature sensors, membrane benders, and scaffolding molecules. Here we show that endophilin, a critical protein for rapid endocytosis, quickly transforms from a curvature sensor into an active bender upon membrane association. We find that local membrane deformation does not occur until endophilin inserts its amphipathic helices into lipid bilayers, supporting an active bending mechanism through wedging. Our time-course studies show that endophilin continues to drive membrane changes on a seconds-to-minutes time scale, indicating that the duration of endocytosis events constrains the mode of endophilin action. Finally, we find a requirement of coordinated activities between wedging and scaffolding for endophilin to produce stable membrane tubules in vitro and to promote synaptic activity in vivo. Together these data demonstrate that endophilin is a multifaceted molecule that precisely integrates activities of sensing, bending, and stabilizing curvature to sculpt membranes with speed.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Animais , Membrana Celular/metabolismo , Endocitose/genética , Endocitose/fisiologia , Bicamadas Lipídicas/metabolismo , Membranas/metabolismo , Camundongos , Modelos Moleculares , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Fatores de Tempo
6.
Curr Opin Cell Biol ; 26: 28-33, 2014 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-24529243

RESUMO

Synaptic vesicles (SVs) are the repositories of neurotransmitters. They are locally recycled at nerve terminals following exocytosis. A unique feature of these vesicles is the uniformity of their morphology, which is well maintained even after rounds of exocytosis and endocytosis. Several studies suggest that disruption of clathrin adaptor proteins leads to defects in sorting cargoes and alterations in SV morphology. Here, we review the links between adaptor proteins and SV size, and highlight how protein sorting may impact SV architecture. Molecular players such as clathrin, adaptor proteins, accessory proteins, SV cargoes and lipid composition may act together to establish a stable regulatory network to maintain SV morphology.


Assuntos
Proteínas/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Endocitose/fisiologia , Ligação Proteica , Transporte Proteico , Vesículas Sinápticas/ultraestrutura
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA