Dynamic Regulation of Caveolin-1 Trafficking in the Germ Line and Embryo of Caenorhabditis elegans

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference–mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1–dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.

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Faculty Opinions recommendation of Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1092571.547000 ◽

2007 ◽

Author(s):

Michel Labouesse

Keyword(s):

Caenorhabditis Elegans ◽

Germ Line ◽

Dynamic Regulation ◽

Caveolin 1

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Src-mediated caveolin-1 phosphorylation affects the targeting of active Src to specific membrane sites

Molecular Biology of the Cell ◽

10.1091/mbc.e13-03-0163 ◽

2013 ◽

Vol 24 (24) ◽

pp. 3881-3895 ◽

Cited By ~ 35

Author(s):

Efrat Gottlieb-Abraham ◽

Dmitry E. Shvartsman ◽

John C. Donaldson ◽

Marcelo Ehrlich ◽

Orit Gutman ◽

...

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Cell Transformation ◽

Quantitative Imaging ◽

Focal Adhesions ◽

Sh2 Domain ◽

Size Analysis ◽

Caveolin 1 ◽

Green Fluorescent ◽

Specific Membrane

Src interactions with the plasma membrane are an important determinant of its activity. In turn, Src activity modulates its association with the membrane through binding of activated Src to phosphotyrosylated proteins. Caveolin-1 (Cav-1), a major component of caveolae, is a known Src phosphorylation target, and both were reported to regulate cell transformation. However, the nature of Src-Cav-1 interactions, a potential mechanism of their coregulation, remained unclear. Here we used fluorescence recovery after photobleaching beam-size analysis, coimmunoprecipitation, quantitative imaging, and far-Western studies with cells expressing wild type, as well as structural and activity mutants of Src–green fluorescent protein and Cav-1–monomeric red fluorescent protein, to measure their interactions with the membrane and with each other. We show dynamic Src–plasma membrane interactions, which are augmented and stabilized by Cav-1. The mechanism involves phosphorylation of Cav-1 at Tyr-14 by Src and subsequent binding of the Src SH2 domain to phospho–Cav-1, leading to accumulation of activated Src in focal adhesions. This novel Cav-1 function potentially modulates focal adhesion dynamics.

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Thioacylation is required for targeting G-protein subunit Go1α to detergent-insoluble caveolin-containing membrane domains

Biochemical Journal ◽

10.1042/bj3550323 ◽

2001 ◽

Vol 355 (2) ◽

pp. 323-331 ◽

Cited By ~ 2

Author(s):

Francesca GUZZI ◽

Deborah ZANCHETTA ◽

Bice CHINI ◽

Marco PARENTI

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Transmembrane Helix ◽

Membrane Domains ◽

Protein Subunit ◽

Caveolin 1 ◽

V2 Receptor ◽

Green Fluorescent ◽

Membrane Compartmentalization ◽

Α Subunits

α-Subunits of heterotrimeric Gi-like proteins (αi, αo and αz) associate with the cytoplasmic leaflet of the plasma membrane by means of N-terminally linked myristic acid and palmitic acid. An additional role for palmitate has been recently suggested by the observation that fusion with the palmitoylated N-terminus of αi1 relocalizes cytosolic green-fluorescent-protein reporter to low buoyancy, Triton-insoluble membrane domains (TIFF; Triton-insoluble floating fraction), enriched with caveolin-1 [Galbiati, Volonté, Meani, Milligan, Lublin, Lisanti and Parenti (1999) J. Biol. Chem 274, 5843-5850]. Here we show that, upon transient expression in transfected COS-7 cells, myristoylated and palmitoylated αo (αowt, where wt is wild-type) is exclusively found in TIFF, from where non-palmitoylated αowt and αoC3S (Cys3 → Ser) mutant are excluded. Moreover, αo fused to N-terminally truncated human vasopressin V2 receptor (V2TR-αo), lacking myristate and palmitate, still localizes at the plasma membrane by means of first transmembrane helix of V2R, but is excluded from TIFF. Likewise, αoC3S does not partition into TIFF, even when its membrane avidity is enhanced by co-expression of βγ-subunits. Thus membrane association, in the absence of added palmitate, is not sufficient to confer partitioning of αo within TIFF, suggesting that palmitoylation is a signal for membrane compartmentalization of dually acylated α-subunits

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Spatially Regulated Recruitment of Clathrin to the Plasma Membrane during Capping and Cell Translocation

Molecular Biology of the Cell ◽

10.1091/mbc.11.6.2151 ◽

2000 ◽

Vol 11 (6) ◽

pp. 2151-2159 ◽

Cited By ~ 27

Author(s):

Cynthia K. Damer ◽

Theresa J. O'Halloran

Keyword(s):

Plasma Membrane ◽

Fluorescent Protein ◽

Dynamic Properties ◽

Chimeric Protein ◽

Cellular Membranes ◽

Dynamic Regulation ◽

Surface Receptors ◽

Motile Cells ◽

Coated Membranes ◽

Green Fluorescent

Clathrin-coated vesicles bud from selected cellular membranes to traffic-specific intracellular proteins. To study the dynamic properties of clathrin-coated membranes, we expressed clathrin heavy chain tagged with green fluorescent protein (GFP) inDictyostelium cells. GFP-clathrin was functional and retained the native properties of clathrin: the chimeric protein formed classic clathrin lattices on cellular membranes and also rescued phenotypic defects of clathrin null cells. GFP-clathrin distributed into punctate loci found throughout the cytoplasm, on the plasma membrane, and concentrated to a perinuclear location. These clathrin-coated structures were remarkably motile and capable of rapid and bidirectional transport across the cell. We identified two local domains of the plasma membrane as sites for clathrin recruitment in motile cells. First, as cells translocated or changed shape and retracted their tails, clathrin was transiently concentrated on the membrane at the back of the cell tail. Second, as cells capped their cell surface receptors, clathrin was recruited locally to the membrane under the tight cap of cross-linked receptors. This suggests that local sites for clathrin polymerization on specific domains of the plasma membrane undergo rapid and dynamic regulation in motile cells.

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Functional expression, quantification and cellular localization of the Hxt2 hexose transporter of Saccharomyces cerevisiae tagged with the green fluorescent protein

Biochemical Journal ◽

10.1042/bj3390299 ◽

1999 ◽

Vol 339 (2) ◽

pp. 299-307 ◽

Cited By ~ 33

Author(s):

Arthur L. KRUCKEBERG ◽

Ling YE ◽

Jan A. BERDEN ◽

Karel van DAM

Keyword(s):

Saccharomyces Cerevisiae ◽

Plasma Membrane ◽

Green Fluorescent Protein ◽

Glucose Transport ◽

Cellular Localization ◽

Fluorescent Protein ◽

Hexose Transporter ◽

High Concentrations ◽

Green Fluorescent

The Hxt2 glucose transport protein of Saccharomyces cerevisiae was genetically fused at its C-terminus with the green fluorescent protein (GFP). The Hxt2-GFP fusion protein is a functional hexose transporter: it restored growth on glucose to a strain bearing null mutations in the hexose transporter genes GAL2 and HXT1 to HXT7. Furthermore, its glucose transport activity in this null strain was not markedly different from that of the wild-type Hxt2 protein. We calculated from the fluorescence level and transport kinetics that induced cells had 1.4×105 Hxt2-GFP molecules per cell, and that the catalytic-centre activity of the Hxt2-GFP molecule in vivo is 53 s-1 at 30 °C. Expression of Hxt2-GFP was induced by growth at low concentrations of glucose. Under inducing conditions the Hxt2-GFP fluorescence was localized to the plasma membrane. In a strain impaired in the fusion of secretory vesicles with the plasma membrane, the fluorescence accumulated in the cytoplasm. When induced cells were treated with high concentrations of glucose, the fluorescence was redistributed to the vacuole within 4 h. When endocytosis was genetically blocked, the fluorescence remained in the plasma membrane after treatment with high concentrations of glucose.

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Synaptotagmins at the endoplasmic reticulum–plasma membrane contact sites maintain diacylglycerol homeostasis during abiotic stress

The Plant Cell ◽

10.1093/plcell/koab122 ◽

2021 ◽

Author(s):

Noemi Ruiz-Lopez ◽

Jessica Pérez-Sancho ◽

Alicia Esteban del Valle ◽

Richard P Haslam ◽

Steffen Vanneste ◽

...

Keyword(s):

Endoplasmic Reticulum ◽

Plasma Membrane ◽

Abiotic Stress ◽

Fluorescent Protein ◽

Mechanical Damage ◽

Membrane Contact Sites ◽

Contact Sites ◽

Membrane Contact ◽

Green Fluorescent

Abstract Endoplasmic reticulum-plasma membrane contact sites (ER-PM CS) play fundamental roles in all eukaryotic cells. Arabidopsis thaliana mutants lacking the ER-PM protein tether synaptotagmin1 (SYT1) exhibit decreased plasma membrane (PM) integrity under multiple abiotic stresses such as freezing, high salt, osmotic stress and mechanical damage. Here, we show that, together with SYT1, the stress-induced SYT3 is an ER-PM tether that also functions in maintaining PM integrity. The ER-PM CS localization of SYT1 and SYT3 is dependent on PM phosphatidylinositol-4-phosphate and is regulated by abiotic stress. Lipidomic analysis revealed that cold stress increased the accumulation of diacylglycerol at the PM in a syt1/3 double mutant relative to wild type while the levels of most glycerolipid species remain unchanged. Additionally, the SYT1-green fluorescent protein (GFP) fusion preferentially binds diacylglycerol in vivo with little affinity for polar glycerolipids. Our work uncovers a SYT-dependent mechanism of stress adaptation counteracting the detrimental accumulation of diacylglycerol at the PM produced during episodes of abiotic stress.

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Activity of the GR in G2 and Mitosis

Molecular Endocrinology ◽

10.1210/mend.16.6.0842 ◽

2002 ◽

Vol 16 (6) ◽

pp. 1352-1366 ◽

Cited By ~ 17

Author(s):

G. Alexander Abel ◽

Gabriela M. Wochnik ◽

Joëlle Rüegg ◽

Audrey Rouyer ◽

Florian Holsboer ◽

...

Keyword(s):

Cell Cycle ◽

Fluorescent Protein ◽

Chromatin Condensation ◽

Tyrosine Aminotransferase ◽

Cycle Phase ◽

M Cell ◽

Tumor Virus ◽

Mmtv Promoter ◽

Cycle Dependent ◽

Green Fluorescent

Abstract To elucidate the mechanisms mediating the reported transient physiological glucocorticoid resistance in G2/M cell cycle phase, we sought to establish a model system of glucocorticoid-resistant cells in G2. We synchronized various cell lines in G2 to measure dexamethasone (DEX)-induced transactivation of either two endogenous promoters (rat tyrosine aminotransferase and mouse metallothionein I) or the mouse mammary tumor virus (MMTV) promoter stably or transiently transfected. To circumvent the need for synchronization drugs, we stably transfected an MMTV-driven green fluorescent protein to directly correlate DEX-induced transactivation with the cell cycle position for each cell of an asynchronous population using flow cytometry. Surprisingly, all promoters tested were DEX-inducible in G2. Even in mitotic cells, only the stably transfected MMTV promoter was repressed, whereas the same promoter transiently transfected was inducible. The use of Hoechst 33342 for synchronization in previous studies probably caused a misinterpretation, because we detected interference of this drug with GR-dependent transcription independent of the cell cycle. Finally, GR activated a simple promoter in G2, excluding a functional effect of cell cycle-dependent phosphorylation of GR, as implied previously. We conclude that GR itself is fully functional throughout the entire cell cycle, but GR responsiveness is repressed in mitosis due to chromatin condensation rather than to specific modification of GR.

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Epidermal Growth Factor Receptor Distribution during Chemotactic Responses

Molecular Biology of the Cell ◽

10.1091/mbc.11.11.3873 ◽

2000 ◽

Vol 11 (11) ◽

pp. 3873-3883 ◽

Cited By ~ 63

Author(s):

Maryse Bailly ◽

Jeffrey Wyckoff ◽

Boumediene Bouzahzah ◽

Ross Hammerman ◽

Vonetta Sylvestre ◽

...

Keyword(s):

Plasma Membrane ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Egf Receptor ◽

Fluorescent Protein ◽

Growth Factor Receptor ◽

Spatial Gradient ◽

Spatial Gradients ◽

Epidermal Growth ◽

Green Fluorescent

To determine the distribution of the epidermal growth factor (EGF) receptor (EGFR) on the surface of cells responding to EGF as a chemoattractant, an EGFR-green fluorescent protein chimera was expressed in the MTLn3 mammary carcinoma cell line. The chimera was functional and easily visualized on the cell surface. In contrast to other studies indicating that the EGFR might be localized to certain regions of the plasma membrane, we found that the chimera is homogeneously distributed on the plasma membrane and becomes most concentrated in vesicles after endocytosis. In spatial gradients of EGF, endocytosed receptor accumulates on the upgradient side of the cell. Visualization of the binding of fluorescent EGF to cells reveals that the affinity properties of the receptor, together with its expression level on cells, can provide an initial amplification step in spatial gradient sensing.

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Flagellar Morphogenesis: Protein Targeting and Assembly in the Paraflagellar Rod of Trypanosomes

Molecular and Cellular Biology ◽

10.1128/mcb.19.12.8191 ◽

1999 ◽

Vol 19 (12) ◽

pp. 8191-8200 ◽

Cited By ~ 63

Author(s):

Philippe Bastin ◽

Thomas H. MacRae ◽

Susan B. Francis ◽

Keith R. Matthews ◽

Keith Gull

Keyword(s):

Cell Cycle ◽

Trypanosoma Brucei ◽

Protein Targeting ◽

Fluorescent Protein ◽

Green Fluorescent Protein Reporter ◽

Mutant Proteins ◽

Green Fluorescent ◽

A Minor ◽

Fluorescent Protein Reporter

ABSTRACT The paraflagellar rod (PFR) of the African trypanosomeTrypanosoma brucei represents an excellent model to study flagellum assembly. The PFR is an intraflagellar structure present alongside the axoneme and is composed of two major proteins, PFRA and PFRC. By inducible expression of a functional epitope-tagged PFRA protein, we have been able to monitor PFR assembly in vivo. As T. brucei cells progress through their cell cycle, they possess both an old and a new flagellum. The induction of expression of tagged PFRA in trypanosomes growing a new flagellum provided an excellent marker of newly synthesized subunits. This procedure showed two different sites of addition: a major, polar site at the distal tip of the flagellum and a minor, nonpolar site along the length of the partially assembled PFR. Moreover, we have observed turnover of epitope-tagged PFRA in old flagella that takes place throughout the length of the PFR structure. Expression of truncated PFRA mutant proteins identified a sequence necessary for flagellum localization by import or binding. This sequence was not sufficient to confer full flagellum localization to a green fluorescent protein reporter. A second sequence, necessary for the addition of PFRA protein to the distal tip, was also identified. In the absence of this sequence, the mutant PFRA proteins were localized both in the cytosol and in the flagellum where they could still be added along the length of the PFR. This seven-amino-acid sequence is conserved in all PFRA and PFRC proteins and shows homology to a sequence in the flagellar dynein heavy chain of Chlamydomonas reinhardtii.

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