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1.
Nat Commun ; 12(1): 4502, 2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34301937

RESUMO

Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Fatores de Transcrição/metabolismo , Transcrição Genética/genética , Proteínas do Envelope Viral/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Transporte Biológico , Fusão Celular , Linhagem Celular , Linhagem Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Camundongos , RNA-Seq/métodos , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteínas do Envelope Viral/genética
2.
Science ; 363(6424)2019 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-30655415

RESUMO

Optical and electron microscopy have made tremendous inroads toward understanding the complexity of the brain. However, optical microscopy offers insufficient resolution to reveal subcellular details, and electron microscopy lacks the throughput and molecular contrast to visualize specific molecular constituents over millimeter-scale or larger dimensions. We combined expansion microscopy and lattice light-sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain. These included synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly brain region. The technology should enable statistically rich, large-scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.


Assuntos
Encéfalo/diagnóstico por imagem , Nanotecnologia , Neuroimagem/métodos , Imagem Óptica/métodos , Animais , Axônios , Espinhas Dendríticas , Drosophila , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Rim/diagnóstico por imagem , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microscopia de Fluorescência , Imagens de Fantasmas , Córtex Somatossensorial/diagnóstico por imagem , Sinapses
3.
J Cell Biol ; 214(3): 237-9, 2016 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-27482049

RESUMO

Tethered midbody remnants dancing across apical microvilli, encountering the centrosome, and beckoning forth a cilium-who would have guessed this is how polarized epithelial cells coordinate the end of mitosis and the beginning of ciliogenesis? New evidence from Bernabé-Rubio et al. (2016. J. Cell Biol http://dx.doi.org/10.1083/jcb.201601020) supports this emerging model.


Assuntos
Centrossomo/metabolismo , Cílios/metabolismo , Células Epiteliais/metabolismo , Animais , Citocinese , Cães , Células Madin Darby de Rim Canino , Modelos Biológicos
4.
Proc Natl Acad Sci U S A ; 112(5): 1410-5, 2015 Feb 03.
Artigo em Inglês | MEDLINE | ID: mdl-25605896

RESUMO

Primary cilia are ubiquitous, microtubule-based organelles that play diverse roles in sensory transduction in many eukaryotic cells. They interrogate the cellular environment through chemosensing, osmosensing, and mechanosensing using receptors and ion channels in the ciliary membrane. Little is known about the mechanical and structural properties of the cilium and how these properties contribute to ciliary perception. We probed the mechanical responses of primary cilia from kidney epithelial cells [Madin-Darby canine kidney-II (MDCK-II)], which sense fluid flow in renal ducts. We found that, on manipulation with an optical trap, cilia deflect by bending along their length and pivoting around an effective hinge located below the basal body. The calculated bending rigidity indicates weak microtubule doublet coupling. Primary cilia of MDCK cells lack interdoublet dynein motors. Nevertheless, we found that the organelles display active motility. 3D tracking showed correlated fluctuations of the cilium and basal body. These angular movements seemed random but were dependent on ATP and cytoplasmic myosin-II in the cell cortex. We conclude that force generation by the actin cytoskeleton surrounding the basal body results in active ciliary movement. We speculate that actin-driven ciliary movement might tune and calibrate ciliary sensory functions.


Assuntos
Cílios/fisiologia , Movimento , Animais , Centrossomo/fisiologia , Cães , Células Madin Darby de Rim Canino , Microscopia Eletrônica
5.
J Biol Chem ; 282(15): 11163-71, 2007 Apr 13.
Artigo em Inglês | MEDLINE | ID: mdl-17276986

RESUMO

The sequence of a transmembrane (TM) domain and the adjacent regions are important for recognition, orientation, and integration at the translocon during membrane protein biosynthesis. However, the sequences of individual TM domains vary considerably. Although some general effects of electrostatic and hydrophobic interactions have been observed, it is still not clear what features of diverse sequences influence TM domain orientation. Here we utilized the ability of the prion protein (PrP) to be synthesized in multiple topological forms to assay the effects of substitutions and mutations on TM domain orientation. Several of the TM domains we tested appear to contain no inherent information regulating orientation. In contrast, we found that the middle region of the PrP TM domain significantly reduces the ability of the chain to invert its orientation in the translocon. We also observed that the C-terminal region of the PrP TM domain influences orientation, and we characterized the orientation differences between two forms of a physiologically relevant polymorphism in this region. Specifically, we found that the identity of a single amino acid, that at position 129, can significantly alter PrP TM domain orientation. Because position 129 is the location of the disease-associated Met/Val polymorphism, we discuss both how this small change may affect TMD orientation and the larger biological implications of these results.


Assuntos
Membrana Celular/metabolismo , Príons/metabolismo , Sequência de Aminoácidos , Animais , Células CHO , Sequência Conservada , Cricetinae , Cricetulus , Humanos , Metionina/metabolismo , Camundongos , Dados de Sequência Molecular , Príons/química , Príons/genética
6.
Biochemistry ; 43(38): 11973-82, 2004 Sep 28.
Artigo em Inglês | MEDLINE | ID: mdl-15379537

RESUMO

Biosynthesis of the prion protein at the endoplasmic reticulum generates multiple topological forms. The topology of an individual chain is determined first by the localization of the N terminus and then by potential integration of the transmembrane domain into the lipid bilayer. Here, we provide the first evidence that signal sequences affect the latter of these events by demonstrating that some but not other signal sequences and signal sequence mutations result in significant increases in the fraction of prion protein nascent chains that integrate into the lipid bilayer. Through analysis of the prolactin signal sequence, an especially poor integration effector, we find that the N terminal and hydrophobic regions of the signal sequence affect integration most significantly. Mutations in either region result in a considerable increase in the number of chains that integrate. The effect of the signal sequence cannot be attributed to timing of signal cleavage or the state of the ribosome membrane junction, parameters previously found to affect protein biogenesis. We also present evidence that signal sequences that are poor integration effectors can promote integration under experimental conditions that allow the nascent chain more time to integrate. These findings reveal a previously unappreciated relationship between signal sequences and transmembrane integration.


Assuntos
Retículo Endoplasmático/metabolismo , Membranas Intracelulares/metabolismo , Príons/química , Príons/metabolismo , Sinais Direcionadores de Proteínas , Sequência de Aminoácidos , Retículo Endoplasmático/química , Interações Hidrofóbicas e Hidrofílicas , Membranas Intracelulares/química , Dados de Sequência Molecular , Mutação/genética , Príons/genética , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas/genética , Transporte Proteico , Ribossomos/química , Ribossomos/genética , Ribossomos/metabolismo
7.
J Biol Chem ; 278(32): 30365-72, 2003 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-12771148

RESUMO

An interaction between an N-terminal signal sequence and the translocon leads to the initiation of protein translocation into the endoplasmic reticulum lumen. Subsequently, folding and modification of the substrate rapidly ensue. The close temporal coordination of these processes suggests that they may be structurally and functionally coordinated as well. Here we show that information encoded in the hydrophobic domain of a signal sequence influences the timing and efficiency of at least two steps in maturation, namely N-linked glycosylation and signal sequence cleavage. We demonstrate that these consequences correlate with and likely stem from the nature of the initial association made between the signal sequence and the translocon during the initiation of translocation. We propose a model by which these maturational events are controlled by the signal sequence-translocon interaction. Our work demonstrates that the pathway taken by a nascent chain through post-translational maturation depends on information encoded in its signal sequence.


Assuntos
Retículo Endoplasmático/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Reagentes para Ligações Cruzadas/farmacologia , DNA Complementar/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicosilação , Dados de Sequência Molecular , Plasmídeos/metabolismo , Testes de Precipitina , Biossíntese de Proteínas , Conformação Proteica , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Sinais Direcionadores de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Homologia de Sequência de Aminoácidos , Transdução de Sinais , Tripsina/farmacologia
8.
J Cell Sci ; 115(Pt 10): 2003-9, 2002 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-11973342

RESUMO

Integral membrane protein biogenesis requires the coordination of several events: accurate targeting of the nascent chain to the membrane; recognition, orientation and integration of transmembrane (TM) domains; and proper formation of tertiary and quaternary structure. Initially unanticipated inter- and intra-protein interactions probably mediate each stage of biogenesis for single spanning, polytopic and C-terminally anchored membrane proteins. The importance of these regulated interactions is illustrated by analysis of topology prediction algorithm failures. Misassigned or misoriented TM domains occur because the primary sequence and overall hydrophobicity of a single TM domain are not the only determinants of membrane integration.


Assuntos
Membrana Celular/química , Membrana Celular/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Algoritmos , Animais , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Membrana/metabolismo , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Transporte Proteico
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