Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 7 de 7
Filter
Add more filters










Database
Language
Publication year range
1.
Nat Commun ; 13(1): 3031, 2022 05 31.
Article in English | MEDLINE | ID: mdl-35641503

ABSTRACT

In response to vascular damage, P-selectin molecules are secreted onto the surface of cells that line our blood vessels. They then serve as mechanical anchors to capture leucocytes from the blood stream. Here, we track individual P-selectin molecules released at the surface of live endothelial cells following stimulated secretion. We find P-selectin initially shows fast, unrestricted diffusion but within a few minutes, movement becomes increasingly restricted and ~50% of the molecules become completely immobile; a process similar to a sol-gel transition. We find removal of the extracellular C-type lectin domain (ΔCTLD) and/or intracellular cytoplasmic tail domain (ΔCT) has additive effects on diffusive motion while disruption of the adapter complex, AP2, or removal of cell-surface heparan sulphate restores mobility of full-length P-selectin close to that of ΔCT and ΔCTLD respectively. We have found P-selectin spreads rapidly from sites of exocytosis and evenly decorates the cell surface, but then becomes less mobile and better-suited to its mechanical anchoring function.


Subject(s)
Endothelial Cells , P-Selectin , Cell Membrane/metabolism , Endothelial Cells/metabolism , Exocytosis , Leukocytes/metabolism , P-Selectin/metabolism
2.
Blood Adv ; 5(23): 5116-5127, 2021 12 14.
Article in English | MEDLINE | ID: mdl-34551092

ABSTRACT

von Willebrand factor (VWF) is an essential hemostatic protein that is synthesized and secreted by endothelial cells and stored in Weibel-Palade bodies (WPBs). The secretory Rab GTPases Rab27A, Rab3B, and Rab3D have been linked with WPB trafficking and secretion. How these Rabs are activated and recruited to WPBs remains elusive. In this study, we identified MAP kinase-activating death domain (MADD) as the guanine nucleotide exchange factor for Rab27A and both Rab3 isoforms in primary human endothelial cells. Rab activity assays revealed a reduction in Rab27A, Rab3B, and Rab3D activation upon MADD silencing. Rab activation, but not binding, was dependent on the differentially expressed in normal and neoplastic cells (DENN) domain of MADD, indicating the potential existence of 2 Rab interaction modules. Furthermore, immunofluorescent analysis showed that Rab27A, Rab3B, and Rab3D recruitment to WPBs was dramatically decreased upon MADD knockdown, revealing that MADD drives Rab membrane targeting. Artificial mistargeting of MADD using a TOMM70 tag abolished Rab27A localization to WPB membranes in a DENN domain-dependent manner, indicating that normal MADD localization in the cytosol is crucial. Activation of Rab3B and Rab3D was reduced upon Rab27A silencing, suggesting that activation of these Rabs is enhanced through previous activation of Rab27A by MADD. MADD silencing did not affect WPB morphology, but it did reduce VWF intracellular content. Furthermore, MADD-depleted cells exhibited decreased histamine-evoked VWF release, similar to Rab27A-depleted cells. In conclusion, MADD acts as a master regulator of VWF secretion by coordinating the activation and membrane targeting of secretory Rabs to WPBs.


Subject(s)
Weibel-Palade Bodies , rab GTP-Binding Proteins , Death Domain Receptor Signaling Adaptor Proteins , Endothelial Cells/metabolism , Exocytosis , Guanine Nucleotide Exchange Factors/genetics , Guanine Nucleotide Exchange Factors/metabolism , Guanosine Triphosphate , Humans , Weibel-Palade Bodies/metabolism , rab GTP-Binding Proteins/genetics , rab GTP-Binding Proteins/metabolism
3.
J Cell Sci ; 129(3): 592-603, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26675235

ABSTRACT

Weibel-Palade body (WPB)-actin interactions are essential for the trafficking and secretion of von Willebrand factor; however, the molecular basis for this interaction remains poorly defined. Myosin Va (MyoVa or MYO5A) is recruited to WPBs by a Rab27A-MyRIP complex and is thought to be the prime mediator of actin binding, but direct MyRIP-actin interactions can also occur. To evaluate the specific contribution of MyRIP-actin and MyRIP-MyoVa binding in WPB trafficking and Ca(2+)-driven exocytosis, we used EGFP-MyRIP point mutants with disrupted MyoVa and/or actin binding and high-speed live-cell fluorescence microscopy. We now show that the ability of MyRIP to restrict WPB movement depends upon its actin-binding rather than its MyoVa-binding properties. We also show that, although the role of MyRIP in Ca(2+)-driven exocytosis requires both MyoVa- and actin-binding potential, it is the latter that plays a dominant role. In view of these results and together with the analysis of actin disruption or stabilisation experiments, we propose that the role of MyRIP in regulating WPB trafficking and exocytosis is mediated largely through its interaction with actin rather than with MyoVa.


Subject(s)
Actin Cytoskeleton/metabolism , Exocytosis/physiology , Vesicular Transport Proteins/metabolism , Weibel-Palade Bodies/metabolism , Weibel-Palade Bodies/physiology , Actins/metabolism , Calcium/metabolism , Cell Line , Cell Movement/physiology , Green Fluorescent Proteins/metabolism , Human Umbilical Vein Endothelial Cells , Humans , Myosin Heavy Chains/metabolism , Myosin Type V/metabolism , Protein Binding/physiology , Protein Transport/physiology
5.
J Cell Sci ; 126(Pt 23): 5490-9, 2013 Dec 01.
Article in English | MEDLINE | ID: mdl-24127569

ABSTRACT

Regulated secretion from endothelial cells is mediated by Weibel-Palade body (WPB) exocytosis. Plasma membrane cholesterol is implicated in regulating secretory granule exocytosis and fusion pore dynamics; however, its role in modulating WPB exocytosis is not clear. To address this we combined high-resolution electrochemical analysis of WPB fusion pore dynamics, by amperometry, with high-speed optical imaging of WPB exocytosis following cholesterol depletion or supplementation in human umbilical vein endothelial cells. We identified serotonin (5-HT) immunoreactivity in WPBs, and VMAT1 expression allowing detection of secreted 5-HT as discrete current spikes during exocytosis. A high proportion of spikes (∼75%) had pre-spike foot signals, indicating that WPB fusion proceeds via an initial narrow pore. Cholesterol depletion significantly reduced pre-spike foot signal duration and increased the rate of fusion pore expansion, whereas cholesterol supplementation had broadly the reverse effect. Cholesterol depletion slowed the onset of hormone-evoked WPB exocytosis, whereas its supplementation increased the rate of WPB exocytosis and hormone-evoked proregion secretion. Our results provide the first analysis of WPB fusion pore dynamics and highlight an important role for cholesterol in the regulation of WPB exocytosis.


Subject(s)
Cell Membrane/drug effects , Cholesterol/pharmacology , Exocytosis/drug effects , Weibel-Palade Bodies/drug effects , Biological Transport , Cell Membrane/metabolism , Cell Membrane/ultrastructure , Cell Membrane Permeability , Cells, Cultured , Cholesterol/metabolism , Electrochemical Techniques , Evoked Potentials/drug effects , Evoked Potentials/physiology , Gene Expression , Histamine/pharmacology , Human Umbilical Vein Endothelial Cells , Humans , Serotonin/metabolism , Serotonin/pharmacology , Vesicular Monoamine Transport Proteins/genetics , Vesicular Monoamine Transport Proteins/metabolism , Weibel-Palade Bodies/metabolism , Weibel-Palade Bodies/ultrastructure , beta-Cyclodextrins/pharmacology
6.
PLoS One ; 5(10)2010 Oct 05.
Article in English | MEDLINE | ID: mdl-20957192

ABSTRACT

For most secretory pathway proteins, crossing the endoplasmic reticulum (ER) membrane is an irreversible process. However, in some cases this flow can be reversed. For instance, misfolded proteins retained in the ER are retrotranslocated to the cytosol to be degraded by the proteasome. This mechanism, known as ER associated degradation (ERAD), is exploited by several bacterial toxins to gain access to the cytosol. Interestingly, some ER resident proteins can also be detected in the cytosol or nucleus, calreticulin (CRT) being the most studied. Here we show that in Trypanosoma cruzi a minor fraction of CRT localized to the cytosol. ER calcium depletion, but not increasing cytosolic calcium, triggered the retrotranslocation of CRT in a relatively short period of time. Cytosolic CRT was subsequently degraded by the proteasome. Interestingly, the single disulfide bridge of CRT is reduced when the protein is located in the cytosol. The effect exerted by ER calcium was strictly dependent on the C-terminal domain (CRT-C), since a CRT lacking it was totally retained in the ER, whereas the localization of an unrelated protein fused to CRT-C mirrored that of endogenous CRT. This finding expands the regulatory mechanisms of protein sorting and may represent a new crossroad between diverse physiological processes.


Subject(s)
Calcium/metabolism , Calreticulin/metabolism , Cytosol/metabolism , Endoplasmic Reticulum/metabolism , Trypanosoma cruzi/metabolism , Animals , Biological Transport
7.
Biochemistry ; 46(15): 4671-80, 2007 Apr 17.
Article in English | MEDLINE | ID: mdl-17385894

ABSTRACT

The ER resident protein calreticulin fulfills at least two important roles. It works as a chaperone preventing Golgi exit of non-native protein species and enhancing protein folding efficiency in either N-glycan-dependent, lectin chaperone, or classical chaperone, N-glycan-independent, modes and is one of the main calcium buffers in the cell. This last feature is independent from the lectin chaperone properties of the protein as this last activity is also observed in a CRT fragment lacking calcium buffer capacity. Here we study the interplay between calcium and the lectin and chaperone activities of CRT. The affinity of CRT for monoglucosylated glycans measured in solution by equilibrium dialysis and fluorescence anisotropy was not affected by the absence of calcium. Binding of CRT to monoglucosylated neoglycoproteins displaying either native or molten globule-like conformations was also independent of calcium concentration. Moreover, calcium and monoglucosylated glycans stabilized the CRT structure in an apparent additive, independent manner when the protein was subjected to thermal or urea-induced denaturation. In addition, the ability of CRT to decrease the level of aggregation of a chemically denatured monoglucosylated and nonglycosylated protein was also independent of calcium level.


Subject(s)
Calcium/chemistry , Calreticulin/chemistry , Lectins/chemistry , Animals , Binding Sites , Calcium/metabolism , Calreticulin/genetics , Calreticulin/metabolism , Chromatography, Gel , Circular Dichroism , Dimerization , Fluorescence Polarization , Glycoproteins/chemistry , Glycoproteins/metabolism , Lectins/metabolism , Molecular Chaperones/chemistry , Molecular Chaperones/metabolism , Oligosaccharides/chemistry , Oligosaccharides/metabolism , Peptides/chemistry , Peptides/metabolism , Plant Proteins/chemistry , Plant Proteins/metabolism , Polysaccharides/chemistry , Polysaccharides/metabolism , Protein Binding , Protein Denaturation/drug effects , Protein Structure, Tertiary , Rabbits , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Temperature , Time Factors , Urea/pharmacology
SELECTION OF CITATIONS
SEARCH DETAIL
...