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Sci Rep ; 6: 38063, 2016 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-27905489


Mechanical integration of the nucleus with the extracellular matrix (ECM) is established by linkage between the cytoskeleton and the nucleus. This integration is hypothesized to mediate sensing of ECM rigidity, but parsing the function of nucleus-cytoskeleton linkage from other mechanisms has remained a central challenge. Here we took advantage of the fact that the LINC (linker of nucleoskeleton and cytoskeleton) complex is a known molecular linker of the nucleus to the cytoskeleton, and asked how it regulates the sensitivity of genome-wide transcription to substratum rigidity. We show that gene mechanosensitivity is preserved after LINC disruption, but reversed in direction. Combined with myosin inhibition studies, we identify genes that depend on nuclear tension for their regulation. We also show that LINC disruption does not attenuate nuclear shape sensitivity to substrate rigidity. Our results show for the first time that the LINC complex facilitates mechano-regulation of expression across the genome.

Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Matriz Extracelular/metabolismo , RNA Mensageiro/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Humanos , Camundongos , Células NIH 3T3 , Análise de Sequência de RNA , Transcrição Genética
J Cell Physiol ; 231(6): 1269-75, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26496460


Despite being densely packed with chromatin, nuclear bodies and a nucleoskeletal network, the nucleus is a remarkably dynamic organelle. Chromatin loops form and relax, RNA transcripts and transcription factors move diffusively, and nuclear bodies move. We show here that RNA splicing speckled domains (splicing speckles) fluctuate in constrained nuclear volumes and remodel their shapes. Small speckles move in a directed way toward larger speckles with which they fuse. This directed movement is reduced upon decreasing cellular ATP levels or inhibiting RNA polymerase II activity. The random movement of speckles is reduced upon decreasing cellular ATP levels, moderately reduced after inhibition of SWI/SNF chromatin remodeling and modestly increased upon inhibiting RNA polymerase II activity. To define the paths through which speckles can translocate in the nucleus, we generated a pressure gradient to create flows in the nucleus. In response to the pressure gradient, speckles moved along curvilinear paths in the nucleus. Collectively, our results demonstrate a new type of ATP-dependent motion in the nucleus. We present a model where recycling splicing factors return as part of small sub-speckles from distal sites of RNA processing to larger splicing speckles by a directed ATP-driven mechanism through interchromatin spaces.

Núcleo Celular/metabolismo , Células Epiteliais/metabolismo , Glândulas Mamárias Humanas/metabolismo , Processamento de RNA , Transporte de RNA , RNA/metabolismo , Trifosfato de Adenosina/metabolismo , Antígenos Nucleares/genética , Antígenos Nucleares/metabolismo , Linhagem Celular , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Glândulas Mamárias Humanas/citologia , Modelos Biológicos , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Pressão , RNA/genética , Interferência de RNA , RNA Polimerase II/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Fatores de Tempo , Transfecção
J Cell Sci ; 128(10): 1901-11, 2015 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-25908852


Nuclear positioning is a crucial cell function, but how a migrating cell positions its nucleus is not understood. Using traction-force microscopy, we found that the position of the nucleus in migrating fibroblasts closely coincided with the center point of the traction-force balance, called the point of maximum tension (PMT). Positioning of the nucleus close to the PMT required nucleus-cytoskeleton connections through linker of nucleoskeleton-to-cytoskeleton (LINC) complexes. Although the nucleus briefly lagged behind the PMT following spontaneous detachment of the uropod during migration, the nucleus quickly repositioned to the PMT within a few minutes. Moreover, traction-generating spontaneous protrusions deformed the nearby nucleus surface to pull the nuclear centroid toward the new PMT, and subsequent retraction of these protrusions relaxed the nuclear deformation and restored the nucleus to its original position. We propose that the protruding or retracting cell boundary transmits a force to the surface of the nucleus through the intervening cytoskeletal network connected by the LINC complexes, and that these forces help to position the nucleus centrally and allow the nucleus to efficiently propagate traction forces across the length of the cell during migration.

Núcleo Celular/fisiologia , Citoesqueleto/fisiologia , Fibroblastos/citologia , Animais , Movimento Celular , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Células NIH 3T3