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1.
FASEB J ; 31(12): 5332-5341, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28778974

RESUMO

Cytoplasmic intermediate filaments (cIFs) are found in all eumetazoans, except arthropods. To investigate the compatibility of cIFs in arthropods, we expressed human vimentin (hVim), a cIF with filament-forming capacity in vertebrate cells and tissues, transgenically in Drosophila Transgenic hVim could be recovered from whole-fly lysates by using a standard procedure for intermediate filament (IF) extraction. When this procedure was used to test for the possible presence of IF-like proteins in flies, only lamins and tropomyosin were observed in IF-enriched extracts, thereby providing biochemical reinforcement to the paradigm that arthropods lack cIFs. In Drosophila, transgenic hVim was unable to form filament networks in S2 cells and mesenchymal tissues; however, cage-like vimentin structures could be observed around the nuclei in internal epithelia, which suggests that Drosophila retains selective competence for filament formation. Taken together, our results imply that although the filament network formation competence is partially lost in Drosophila, a rudimentary filament network formation ability remains in epithelial cells. As a result of the observed selective competence for cIF assembly in Drosophila, we hypothesize that internal epithelial cIFs were the last cIFs to disappear from arthropods.-Gullmets, J., Torvaldson, E., Lindqvist, J., Imanishi, S. Y., Taimen, P., Meinander, A., Eriksson, J. E. Internal epithelia in Drosophila display rudimentary competence to form cytoplasmic networks of transgenic human vimentin.


Assuntos
Citoplasma/metabolismo , Drosophila/metabolismo , Epitélio/metabolismo , Vimentina/metabolismo , Animais , Animais Geneticamente Modificados , Western Blotting , Linhagem Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Imunofluorescência , Humanos , Imuno-Histoquímica , Proteínas de Filamentos Intermediários/genética , Proteínas de Filamentos Intermediários/metabolismo , Filamentos Intermediários/enzimologia , Filamentos Intermediários/metabolismo , Laminas/genética , Laminas/metabolismo , Tropomiosina/genética , Tropomiosina/metabolismo , Vimentina/genética
2.
J Cell Sci ; 130(17): 2833-2842, 2017 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-28733456

RESUMO

Nestin, a member of the cytoskeletal family of intermediate filaments, regulates the onset of myogenic differentiation through bidirectional signaling with the kinase Cdk5. Here, we show that these effects are also reflected at the organism level, as there is a loss of skeletal muscle mass in nestin-/- (NesKO) mice, reflected as reduced lean (muscle) mass in the mice. Further examination of muscles in male mice revealed that these effects stemmed from nestin-deficient muscles being more prone to spontaneous regeneration. When the regeneration capacity of the compromised NesKO muscle was tested by muscle injury experiments, a significant healing delay was observed. NesKO satellite cells showed delayed proliferation kinetics in conjunction with an elevation in p35 (encoded by Cdk5r1) levels and Cdk5 activity. These results reveal that nestin deficiency generates a spontaneous regenerative phenotype in skeletal muscle that relates to a disturbed proliferation cycle that is associated with uncontrolled Cdk5 activity.


Assuntos
Homeostase , Músculo Esquelético/fisiologia , Nestina/metabolismo , Regeneração , Animais , Diferenciação Celular , Proliferação de Células , Células Cultivadas , Quinase 5 Dependente de Ciclina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fibras Musculares Esqueléticas/fisiologia , Mioblastos/metabolismo , Nestina/deficiência , Tamanho do Órgão , Fenótipo , Células Satélites de Músculo Esquelético/metabolismo , Cicatrização
3.
Methods Enzymol ; 568: 509-35, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26795482

RESUMO

Current research utilizes the specific expression pattern of intermediate filaments (IF) for identifying cellular state and origin, as well as for the purpose of disease diagnosis. Nestin is commonly utilized as a specific marker and driver for CNS progenitor cell types, but in addition, nestin can be found in several mesenchymal progenitor cells, and it is constitutively expressed in a few restricted locations, such as muscle neuromuscular junctions and kidney podocytes. Alike most other members of the IF protein family, nestin filaments are dynamic, constantly being remodeled through posttranslational modifications, which alter the solubility, protein levels, and signaling capacity of the nestin filaments. Through its interactions with kinases and other signaling executors, resulting in a complex and bidirectional regulation of cell signaling events, nestin has the potential to determine whether cells divide, differentiate, migrate, or stay in place. In this review, the broad and similar roles of IFs as dynamic signaling scaffolds, is exemplified by observations of nestin functions and its interaction with the cyclin- dependent kinase 5, the atypical kinase in the family of cyclin-dependent kinases.


Assuntos
Quinase 5 Dependente de Ciclina/metabolismo , Nestina/metabolismo , Animais , Humanos , Filamentos Intermediários/metabolismo , Fosforilação
4.
Mol Biol Cell ; 26(11): 1971-84, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25851605

RESUMO

Contrary to cell cycle-associated cyclin-dependent kinases, CDK5 is best known for its regulation of signaling processes in differentiated cells and its destructive activation in Alzheimer's disease. Recently, CDK5 has been implicated in a number of different cancers, but how it is able to stimulate cancer-related signaling pathways remains enigmatic. Our goal was to study the cancer-promoting mechanisms of CDK5 in prostate cancer. We observed that CDK5 is necessary for proliferation of several prostate cancer cell lines. Correspondingly, there was considerable growth promotion when CDK5 was overexpressed. When examining the reasons for the altered proliferation effects, we observed that CDK5 phosphorylates S308 on the androgen receptor (AR), resulting in its stabilization and differential expression of AR target genes including several growth-priming transcription factors. However, the amplified cell growth was found to be separated from AR signaling, further corroborated by CDK5-dependent proliferation of AR null cells. Instead, we found that the key growth-promoting effect was due to specific CDK5-mediated AKT activation. Down-regulation of CDK5 repressed AKT phosphorylation by altering its intracellular localization, immediately followed by prominent cell cycle inhibition. Taken together, these results suggest that CDK5 acts as a crucial signaling hub in prostate cancer cells by controlling androgen responses through AR, maintaining and accelerating cell proliferation through AKT activation, and releasing cell cycle breaks.


Assuntos
Proliferação de Células , Quinase 5 Dependente de Ciclina/metabolismo , Regulação Neoplásica da Expressão Gênica , Neoplasias da Próstata/enzimologia , Neoplasias da Próstata/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Androgênicos/metabolismo , Quinase 5 Dependente de Ciclina/genética , Humanos , Masculino , Fosforilação , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/fisiopatologia , Receptores Androgênicos/genética , Transdução de Sinais
5.
Mol Biol Cell ; 22(9): 1539-49, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21346193

RESUMO

Many types of progenitor cells are distinguished by the expression of the intermediate filament protein nestin, a frequently used stem cell marker, the physiological roles of which are still unknown. Whereas myogenesis is characterized by dynamically regulated nestin levels, we studied how altering nestin levels affects myoblast differentiation. Nestin determined both the onset and pace of differentiation. Whereas depletion of nestin by RNAi strikingly accelerated the process, overexpression of nestin completely inhibited differentiation. Nestin down-regulation augmented the early stages of differentiation, at the level of cell-cycle withdrawal and expression of myogenic markers, but did not affect proliferation of undifferentiated dividing myoblasts. Nestin regulated the cleavage of the Cdk5 activator protein p35 to its degradation-resistant form, p25. In this way, nestin has the capacity to halt myoblast differentiation by inhibiting sustained activation of Cdk5 by p25, which is critical for the progress of differentiation. Our results imply that nestin regulates the early stages of myogenesis rather than maintains the undifferentiated state of progenitor cells. In the bidirectional interrelationship between nestin and Cdk5, Cdk5 regulates the organization and stability of its own nestin scaffold, which in turn controls the effects of Cdk5. This nestin-Cdk5 cross-talk sets the pace of muscle differentiation.


Assuntos
Diferenciação Celular , Quinase 5 Dependente de Ciclina/metabolismo , Proteínas de Filamentos Intermediários/metabolismo , Mioblastos/citologia , Mioblastos/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Células-Tronco/metabolismo , Animais , Ciclo Celular , Proliferação de Células , Células Cultivadas , Quinase 5 Dependente de Ciclina/genética , Humanos , Proteínas de Filamentos Intermediários/genética , Camundongos , Desenvolvimento Muscular/genética , Proteínas do Tecido Nervoso/genética , Nestina , Fosfotransferases/metabolismo , Reação em Cadeia da Polimerase , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/genética , Células-Tronco/citologia
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