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1.
iScience ; 23(6): 101244, 2020 Jun 26.
Article in English | MEDLINE | ID: mdl-32629610

ABSTRACT

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.

2.
Sci Rep ; 9(1): 1116, 2019 02 04.
Article in English | MEDLINE | ID: mdl-30718762

ABSTRACT

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.


Subject(s)
Caveolin 1/genetics , Caveolin 1/metabolism , Cilia/metabolism , rhoA GTP-Binding Protein/metabolism , Actin Cytoskeleton/metabolism , Animals , Centrosome/metabolism , Dogs , Formins/metabolism , Gene Expression Regulation , Gene Knockout Techniques , Gene Silencing , Humans , Madin Darby Canine Kidney Cells , Mice , Protein Isoforms/genetics , Protein Isoforms/metabolism , rab GTP-Binding Proteins/metabolism , rho-Associated Kinases/metabolism
3.
Cell Mol Life Sci ; 75(17): 3181-3191, 2018 09.
Article in English | MEDLINE | ID: mdl-29947928

ABSTRACT

The acetylation of the lysine 40 residue of α-tubulin was described more than 30 years ago and has been the subject of intense research ever since. Although the exact function of this covalent modification of tubulin in the cell remains unknown, it has been established that tubulin acetylation confers resilience to mechanical stress on the microtubules. Formins have a dual role in the fate of the actin and tubulin cytoskeletons. On the one hand, they catalyze the formation of actin filaments, and on the other, they bind microtubules, act on their stability, and regulate their acetylation and alignment with actin fibers. Recent evidence indicates that formins coordinate the actin cytoskeleton and tubulin acetylation by modulating the levels of free globular actin (G-actin). G-actin, in turn, controls the activity of the myocardin-related transcription factor-serum response factor transcriptional complex that regulates the expression of the α-tubulin acetyltransferase 1 (α-TAT1) gene, which encodes the main enzyme responsible for tubulin acetylation. The effect of formins on tubulin acetylation is the combined result of their ability to activate α-TAT1 gene transcription and of their capacity to regulate microtubule stabilization. The contribution of these two mechanisms in different formins is discussed, particularly with respect to INF2, a formin that is mutated in hereditary human renal and neurodegenerative disorders.


Subject(s)
Actin Cytoskeleton/metabolism , Microfilament Proteins/metabolism , Microtubules/metabolism , Models, Biological , Acetylation , Formins , Humans , Kidney Diseases/genetics , Kidney Diseases/metabolism , Microfilament Proteins/genetics , Mutation , Neurodegenerative Diseases/genetics , Neurodegenerative Diseases/metabolism , Protein Binding
5.
J Cell Biol ; 217(3): 929-944, 2018 03 05.
Article in English | MEDLINE | ID: mdl-29321169

ABSTRACT

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.


Subject(s)
Acetyltransferases/biosynthesis , Actins/metabolism , Gene Expression Regulation, Enzymologic , RNA, Messenger/biosynthesis , Serum Response Factor/metabolism , Trans-Activators/metabolism , Tubulin/metabolism , Acetylation , Acetyltransferases/genetics , Actins/genetics , Formins , Humans , Jurkat Cells , Microfilament Proteins/genetics , Microfilament Proteins/metabolism , RNA, Messenger/genetics , Serum Response Factor/genetics , Trans-Activators/genetics , Tubulin/genetics
6.
J Cell Biol ; 214(3): 259-73, 2016 08 01.
Article in English | MEDLINE | ID: mdl-27458130

ABSTRACT

The primary cilium is a membrane protrusion that is crucial for vertebrate tissue homeostasis and development. Here, we investigated the uncharacterized process of primary ciliogenesis in polarized epithelial cells. We show that after cytokinesis, the midbody is inherited by one of the daughter cells as a remnant that initially locates peripherally at the apical surface of one of the daughter cells. The remnant then moves along the apical surface and, once proximal to the centrosome at the center of the apical surface, enables cilium formation. The physical removal of the remnant greatly impairs ciliogenesis. We developed a probabilistic cell population-based model that reproduces the experimental data. In addition, our model explains, solely in terms of cell area constraints, the various observed transitions of the midbody, the beginning of ciliogenesis, and the accumulation of ciliated cells. Our findings reveal a biological mechanism that links the three microtubule-based organelles-the midbody, the centrosome, and the cilium-in the same cellular process.


Subject(s)
Cell Polarity , Centrosome/metabolism , Cilia/metabolism , Epithelial Cells/cytology , Epithelial Cells/metabolism , Animals , Biomarkers/metabolism , Carrier Proteins/metabolism , Cell Survival , Cilia/ultrastructure , Dogs , Epithelial Cells/ultrastructure , Imaging, Three-Dimensional , Madin Darby Canine Kidney Cells , Microscopy, Video , Microvilli/metabolism , Mitosis , Models, Biological , Single-Cell Analysis , rab GTP-Binding Proteins/metabolism
7.
J Cell Sci ; 128(12): 2261-70, 2015 Jun 15.
Article in English | MEDLINE | ID: mdl-25967552

ABSTRACT

The base of the primary cilium contains a zone of condensed membranes whose importance is not known. Here, we have studied the involvement of MAL, a tetraspanning protein that exclusively partitions into condensed membrane fractions, in the condensation of membranes at the ciliary base and investigated the importance of these membranes in primary cilium formation. We show that MAL accumulates at the ciliary base of epithelial MDCK cells. Knockdown of MAL expression resulted in a drastic reduction in the condensation of membranes at the ciliary base, the percentage of ciliated cells and the length of the cilia, but did not affect the docking of the centrosome to the plasma membrane or produce missorting of proteins to the pericentriolar zone or to the membrane of the remaining cilia. Rab8 (for which there are two isoforms, Rab8A and Rab8b), IFT88 and IFT20, which are important components of the machinery of ciliary growth, were recruited normally to the ciliary base of MAL-knockdown cells but were unable to elongate the primary cilium correctly. MAL, therefore, is crucial for the proper condensation of membranes at the ciliary base, which is required for efficient primary cilium extension.


Subject(s)
Cell Membrane/physiology , Centrosome/physiology , Cilia/physiology , Cilia/ultrastructure , Myelin and Lymphocyte-Associated Proteolipid Proteins/metabolism , Animals , Blotting, Western , Cells, Cultured , Dogs , Fluorescent Antibody Technique , Humans , Madin Darby Canine Kidney Cells , Microscopy, Electron , Morphogenesis , Myelin and Lymphocyte-Associated Proteolipid Proteins/antagonists & inhibitors , Myelin and Lymphocyte-Associated Proteolipid Proteins/genetics , RNA, Messenger/genetics , RNA, Small Interfering/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
8.
J Biol Chem ; 290(2): 827-40, 2015 Jan 09.
Article in English | MEDLINE | ID: mdl-25425646

ABSTRACT

Chemokine receptor CCR7 directs mature dendritic cells (mDCs) to secondary lymph nodes where these cells regulate the activation of T cells. CCR7 also promotes survival in mDCs, which is believed to take place largely through Akt-dependent signaling mechanisms. We have analyzed the involvement of the AMP-dependent kinase (AMPK) in the control of CCR7-dependent survival. A pro-apoptotic role for AMPK is suggested by the finding that pharmacological activators induce apoptosis, whereas knocking down of AMPK with siRNA extends mDC survival. Pharmacological activation of AMPK also induces apoptosis of mDCs in the lymph nodes. Stimulation of CCR7 leads to inhibition of AMPK, through phosphorylation of Ser-485, which was mediated by G(i)/Gßγ, but not by Akt or S6K, two kinases that control the phosphorylation of AMPK on Ser-485 in other settings. Using selective pharmacological inhibitors, we show that CCR7-induced phosphorylation of AMPK on Ser-485 is mediated by MEK and ERK. Coimmunoprecipitation analysis and proximity ligation assays indicate that AMPK associates with ERK, but not with MEK. These results suggest that in addition to Akt-dependent signaling mechanisms, CCR7 can also promote survival of mDCs through a novel MEK1/2-ERK1/2-AMPK signaling axis. The data also suggest that AMPK may be a potential target to modulate mDC lifespan and the immune response.


Subject(s)
AMP-Activated Protein Kinases/genetics , Immunity, Innate/genetics , MAP Kinase Kinase 1/genetics , MAP Kinase Kinase 2/genetics , Receptors, CCR7/metabolism , AMP-Activated Protein Kinases/metabolism , Apoptosis/genetics , Cell Survival , Dendritic Cells/immunology , Dendritic Cells/metabolism , Humans , MAP Kinase Kinase 1/metabolism , MAP Kinase Kinase 2/metabolism , Mitogen-Activated Protein Kinase 3/genetics , Phosphorylation , Receptors, CCR7/genetics , Signal Transduction/genetics , T-Lymphocytes/immunology , T-Lymphocytes/metabolism
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