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1.
Nat Cell Biol ; 25(10): 1426-1438, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37735598

RESUMO

Dedifferentiation is the process by which terminally differentiated cells acquire the properties of stem cells. During mouse skin wound healing, the differentiated Gata6-lineage positive cells of the sebaceous duct are able to dedifferentiate. Here we have integrated lineage tracing and single-cell mRNA sequencing to uncover the underlying mechanism. Gata6-lineage positive and negative epidermal stem cells in wounds are transcriptionally indistinguishable. Furthermore, in contrast to reprogramming of induced pluripotent stem cells, the same genes are expressed in the epidermal dedifferentiation and differentiation trajectories, indicating that dedifferentiation does not involve adoption of a new cell state. We demonstrate that dedifferentiation is not only induced by wounding, but also by retinoic acid treatment or mechanical expansion of the epidermis. In all three cases, dedifferentiation is dependent on the master transcription factor c-Myc. Mechanotransduction and actin-cytoskeleton remodelling are key features of dedifferentiation. Our study elucidates the molecular basis of epidermal dedifferentiation, which may be generally applicable to adult tissues.


Assuntos
Desdiferenciação Celular , Mecanotransdução Celular , Animais , Camundongos , Desdiferenciação Celular/genética , Diferenciação Celular , Células Epidérmicas , Epiderme
2.
Nat Commun ; 14(1): 919, 2023 02 17.
Artigo em Inglês | MEDLINE | ID: mdl-36808136

RESUMO

Cohort-wide sequencing studies have revealed that the largest category of variants is those deemed 'rare', even for the subset located in coding regions (99% of known coding variants are seen in less than 1% of the population. Associative methods give some understanding how rare genetic variants influence disease and organism-level phenotypes. But here we show that additional discoveries can be made through a knowledge-based approach using protein domains and ontologies (function and phenotype) that considers all coding variants regardless of allele frequency. We describe an ab initio, genetics-first method making molecular knowledge-based interpretations for exome-wide non-synonymous variants for phenotypes at the organism and cellular level. By using this reverse approach, we identify plausible genetic causes for developmental disorders that have eluded other established methods and present molecular hypotheses for the causal genetics of 40 phenotypes generated from a direct-to-consumer genotype cohort. This system offers a chance to extract further discovery from genetic data after standard tools have been applied.


Assuntos
Exoma , Predisposição Genética para Doença , Humanos , Fenótipo , Genótipo , Frequência do Gene
3.
Cell Mol Life Sci ; 79(5): 236, 2022 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-35399121

RESUMO

Proteolipids are proteins with unusual lipid-like properties. It has long been established that PLP and plasmolipin, which are two unrelated membrane-tetra-spanning myelin proteolipids, can be converted in vitro into a water-soluble form with a distinct conformation, raising the question of whether these, or other similar proteolipids, can adopt two different conformations in the cell to adapt their structure to distinct environments. Here, we show that MALL, another proteolipid with a membrane-tetra-spanning structure, distributes in membranes outside the nucleus and, within the nucleus, in membrane-less, liquid-like PML body biomolecular condensates. Detection of MALL in one or other environment was strictly dependent on the method of cell fixation used, suggesting that MALL adopts different conformations depending on its physical environment -lipidic or aqueous- in the cell. The acquisition of the condensate-compatible conformation requires PML expression. Excess MALL perturbed the distribution of the inner nuclear membrane proteins emerin and LAP2ß, and that of the DNA-binding protein BAF, leading to the formation of aberrant nuclei. This effect, which is consistent with studies identifying overexpressed MALL as an unfavorable prognostic factor in cancer, could contribute to cell malignancy. Our study establishes a link between proteolipids, membranes and biomolecular condensates, with potential biomedical implications.


Assuntos
Condensados Biomoleculares , Neoplasias , Núcleo Celular , Humanos , Conformação Molecular , Proteolipídeos/química
4.
Small Methods ; 5(2): e2000711, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-34927881

RESUMO

Lipid liquid-liquid immiscibility and its consequent lateral heterogeneity have been observed under thermodynamic equilibrium in model and native membranes. However, cholesterol-rich membrane domains, sometimes referred to as lipid rafts, are difficult to observe spatiotemporally in live cells. Despite their importance in many biological processes, robust evidence for their existence remains elusive. This is mainly due to the difficulty in simultaneously determining their chemical composition and physicochemical nature, whilst spatiotemporally resolving their nanodomain lifetime and molecular dynamics. In this study, a bespoke method based on super-resolution stimulated emission depletion (STED) microscopy and raster imaging correlation spectroscopy (RICS) is used to overcome this issue. This methodology, laser interleaved confocal RICS and STED-RICS (LICSR), enables simultaneous tracking of lipid lateral packing and dynamics at the nanoscale. Previous work indicated that, in polarized epithelial cells, the midbody remnant licenses primary cilium formation through an unidentified mechanism. LICSR shows that lipid immiscibility and its adaptive collective nanoscale self-assembly are crucial for the midbody remnant to supply condensed membranes to the centrosome for the biogenesis of the ciliary membrane. Hence, this work poses a breakthrough in the field of lipid biology by providing compelling evidence of a functional role for liquid ordered-like membranes in primary ciliogenesis.


Assuntos
Membrana Celular/química , Cílios/fisiologia , Bicamadas Lipídicas/química , Animais , Linhagem Celular , Citocinese , Cães , Células Madin Darby de Rim Canino , Análise Espaço-Temporal
5.
Methods ; 193: 136-147, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34126167

RESUMO

The primary cilium is a specialized plasma membrane protrusion with important receptors for signalling pathways. In polarized epithelial cells, the primary cilium assembles after the midbody remnant (MBR) encounters the centrosome at the apical surface. The membrane surrounding the MBR, namely remnant-associated membrane patch (RAMP), once situated next to the centrosome, releases some of its lipid components to form a centrosome-associated membrane patch (CAMP) from which the ciliary membrane stems. The RAMP undergoes a spatiotemporal membrane refinement during the formation of the CAMP, which becomes highly enriched in condensed membranes with low lateral mobility. To better understand this process, we have developed a correlative imaging approach that yields quantitative information about the lipid lateral packing, its mobility and collective assembly at the plasma membrane at different spatial scales over time. Our work paves the way towards a quantitative understanding of the spatiotemporal lipid collective assembly at the plasma membrane as a functional determinant in cell biology and its direct correlation with the membrane physicochemical state. These findings allowed us to gain a deeper insight into the mechanisms behind the biogenesis of the ciliary membrane of polarized epithelial cells.


Assuntos
Membrana Celular , Células Epiteliais , Lipídeos
6.
iScience ; 23(6): 101244, 2020 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-32629610

RESUMO

The inheritance of the midbody remnant (MBR) breaks the symmetry of the two daughter cells, with functional consequences for lumen and primary cilium formation by polarized epithelial cells, and also for development and differentiation. However, despite its importance, neither the relationship between the plasma membrane and the inherited MBR nor the mechanism of MBR inheritance is well known. Here, the analysis by correlative light and ultra-high-resolution scanning electron microscopy reveals a membranous stalk that physically connects the MBR to the apical membrane of epithelial cells. The stalk, which derives from the uncleaved side of the midbody, concentrates the ESCRT machinery. The ESCRT CHMP4C subunit enables MBR inheritance, and its depletion dramatically reduces the percentage of ciliated cells. We demonstrate (1) that MBRs are physically connected to the plasma membrane, (2) how CHMP4C helps maintain the integrity of the connection, and (3) the functional importance of the connection.

7.
Front Cell Dev Biol ; 8: 622918, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33585461

RESUMO

Primary cilia are solitary, microtubule-based protrusions surrounded by a ciliary membrane equipped with selected receptors that orchestrate important signaling pathways that control cell growth, differentiation, development and homeostasis. Depending on the cell type, primary cilium assembly takes place intracellularly or at the cell surface. The intracellular route has been the focus of research on primary cilium biogenesis, whereas the route that occurs at the cell surface, which we call the "alternative" route, has been much less thoroughly characterized. In this review, based on recent experimental evidence, we present a model of primary ciliogenesis by the alternative route in which the remnant of the midbody generated upon cytokinesis acquires compact membranes, that are involved in compartmentalization of biological membranes. The midbody remnant delivers part of those membranes to the centrosome in order to assemble the ciliary membrane, thereby licensing primary cilium formation. The midbody remnant's involvement in primary cilium formation, the regulation of its inheritance by the ESCRT machinery, and the assembly of the ciliary membrane from the membranes originally associated with the remnant are discussed in the context of the literature concerning the ciliary membrane, the emerging roles of the midbody remnant, the regulation of cytokinesis, and the role of membrane compartmentalization. We also present a model of cilium emergence during evolution, and summarize the directions for future research.

8.
Sci Rep ; 9(1): 1116, 2019 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718762

RESUMO

The primary cilium is a single non-motile protrusion of the plasma membrane of most types of mammalian cell. The structure, length and function of the primary cilium must be tightly controlled because their dysfunction is associated with disease. Caveolin 1 (Cav1), which is best known as a component of membrane invaginations called caveolae, is also present in non-caveolar membrane domains whose function is beginning to be understood. We show that silencing of α and ß Cav1 isoforms in different cell lines increases ciliary length regardless of the route of primary ciliogenesis. The sole expression of Cav1α, which is distributed at the apical membrane, restores normal cilium size in Cav1 KO MDCK cells. Cells KO for only Cav1α, which also show long cilia, have a disrupted actin cytoskeleton and reduced RhoA GTPase activity at the apical membrane, and a greater accumulation of Rab11 vesicles at the centrosome. Subsequent experiments showed that DIA1 and ROCK help regulate ciliary length. Since MDCK cells lack apical caveolae, our results imply that non-caveolar apical Cav1α is an important regulator of ciliary length, exerting its effect via RhoA and its effectors, ROCK and DIA1.


Assuntos
Caveolina 1/genética , Caveolina 1/metabolismo , Cílios/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Centrossomo/metabolismo , Cães , Forminas/metabolismo , Regulação da Expressão Gênica , Técnicas de Inativação de Genes , Inativação Gênica , Humanos , Células Madin Darby de Rim Canino , Camundongos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Quinases Associadas a rho/metabolismo
9.
J Cell Biol ; 217(3): 929-944, 2018 03 05.
Artigo em Inglês | MEDLINE | ID: mdl-29321169

RESUMO

The role of formins in microtubules is not well understood. In this study, we have investigated the mechanism by which INF2, a formin mutated in degenerative renal and neurological hereditary disorders, controls microtubule acetylation. We found that silencing of INF2 in epithelial RPE-1 cells produced a dramatic drop in tubulin acetylation, increased the G-actin/F-actin ratio, and impaired myocardin-related transcription factor (MRTF)/serum response factor (SRF)-dependent transcription, which is known to be repressed by increased levels of G-actin. The effect on tubulin acetylation was caused by the almost complete absence of α-tubulin acetyltransferase 1 (α-TAT1) messenger RNA (mRNA). Activation of the MRTF-SRF transcriptional complex restored α-TAT1 mRNA levels and tubulin acetylation. Several functional MRTF-SRF-responsive elements were consistently identified in the α-TAT1 gene. The effect of INF2 silencing on microtubule acetylation was also observed in epithelial ECV304 cells, but not in Jurkat T cells. Therefore, the actin-MRTF-SRF circuit controls α-TAT1 transcription. INF2 regulates the circuit, and hence microtubule acetylation, in cell types where it has a prominent role in actin polymerization.


Assuntos
Acetiltransferases/biossíntese , Actinas/metabolismo , Regulação Enzimológica da Expressão Gênica , RNA Mensageiro/biossíntese , Fator de Resposta Sérica/metabolismo , Transativadores/metabolismo , Tubulina (Proteína)/metabolismo , Acetilação , Acetiltransferases/genética , Actinas/genética , Forminas , Humanos , Células Jurkat , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , RNA Mensageiro/genética , Fator de Resposta Sérica/genética , Transativadores/genética , Tubulina (Proteína)/genética
10.
Cell Mol Life Sci ; 74(22): 4077-4095, 2017 11.
Artigo em Inglês | MEDLINE | ID: mdl-28624967

RESUMO

Primary cilia are solitary, microtubule-based protrusions of the cell surface that play fundamental roles as photosensors, mechanosensors and biochemical sensors. Primary cilia dysfunction results in a long list of developmental and degenerative disorders that combine to give rise to a large spectrum of human diseases affecting almost any major body organ. Depending on the cell type, primary ciliogenesis is initiated intracellularly, as in fibroblasts, or at the cell surface, as in renal polarized epithelial cells. In this review, we have focused on the routes of primary ciliogenesis placing particular emphasis on the recently described pathway in renal polarized epithelial cells by which the midbody remnant resulting from a previous cell division event enables the centrosome for initiation of primary cilium assembly. The protein machinery implicated in primary cilium formation in epithelial cells, including the machinery best known for its involvement in establishing cell polarity and polarized membrane trafficking, is also discussed.


Assuntos
Cílios/metabolismo , Animais , Síndrome de Bardet-Biedl/metabolismo , Síndrome de Bardet-Biedl/patologia , Membrana Celular/metabolismo , Polaridade Celular , Cílios/química , Humanos , Mecanotransdução Celular , Proteínas de Membrana/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Junções Íntimas/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
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