scholarly journals Local Protein Dynamics during Microvesicle Exocytosis in Neuroendocrine Cells

2018 ◽  
Author(s):  
Agila Somasundaram ◽  
Justin Taraska
Keyword(s):  
Protein Dynamics ◽  
Spatial Dynamics ◽  
Neuroendocrine Cells ◽  
Live Cells ◽  
Rab Proteins ◽  
Vesicle Membrane ◽  
Bar Domain ◽  
Physiological Processes ◽  
Associated Proteins ◽  
Local Protein

ABSTRACTCalcium triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine transporter (VAChT) tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are pre-clustered at fusion sites and rapidly lost at fusion. The ATPase NSF is locally recruited at fusion. Interestingly, the endocytic BAR domain-containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites, and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the over-expression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

scholarly journals Local protein dynamics during microvesicle exocytosis in neuroendocrine cells

2018 ◽  
Vol 29 (15) ◽  
pp. 1891-1903 ◽  
Author(s):  
Agila Somasundaram ◽  
Justin W. Taraska
Keyword(s):  
Protein Dynamics ◽  
Spatial Dynamics ◽  
Neuroendocrine Cells ◽  
Live Cells ◽  
Rab Proteins ◽  
Vesicle Membrane ◽  
Physiological Processes ◽  
Associated Proteins ◽  
Membrane Bound ◽  
Local Protein

Calcium-triggered exocytosis is key to many physiological processes, including neurotransmitter and hormone release by neurons and endocrine cells. Dozens of proteins regulate exocytosis, yet the temporal and spatial dynamics of these factors during vesicle fusion remain unclear. Here we use total internal reflection fluorescence microscopy to visualize local protein dynamics at single sites of exocytosis of small synaptic-like microvesicles in live cultured neuroendocrine PC12 cells. We employ two-color imaging to simultaneously observe membrane fusion (using vesicular acetylcholine ACh transporter tagged to pHluorin) and the dynamics of associated proteins at the moments surrounding exocytosis. Our experiments show that many proteins, including the SNAREs syntaxin1 and VAMP2, the SNARE modulator tomosyn, and Rab proteins, are preclustered at fusion sites and rapidly lost at fusion. The ATPase N-ethylmaleimide–sensitive factor is locally recruited at fusion. Interestingly, the endocytic Bin-Amphiphysin-Rvs domain–containing proteins amphiphysin1, syndapin2, and endophilins are dynamically recruited to fusion sites and slow the loss of vesicle membrane-bound cargo from fusion sites. A similar effect on vesicle membrane protein dynamics was seen with the overexpression of the GTPases dynamin1 and dynamin2. These results suggest that proteins involved in classical clathrin-mediated endocytosis can regulate exocytosis of synaptic-like microvesicles. Our findings provide insights into the dynamics, assembly, and mechanistic roles of many key factors of exocytosis and endocytosis at single sites of microvesicle fusion in live cells.

scholarly journals Deimination, Intermediate Filaments and Associated Proteins

2020 ◽  
Vol 21 (22) ◽  
pp. 8746
Author(s):  
Julie Briot ◽  
Michel Simon ◽  
Marie-Claire Méchin
Keyword(s):  
Rheumatoid Arthritis ◽  
Multiple Sclerosis ◽  
Cell Differentiation ◽  
Intermediate Filaments ◽  
Cross Linking ◽  
Post Translational Modification ◽  
Innate And Adaptive Immunity ◽  
Calcium Dependent ◽  
Physiological Processes ◽  
Associated Proteins

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

scholarly journals The eisosome core is composed of BAR domain proteins

2011 ◽  
Vol 22 (13) ◽  
pp. 2360-2372 ◽  
Author(s):  
Agustina Olivera-Couto ◽  
Martin Graña ◽  
Laura Harispe ◽  
Pablo S. Aguilar
Keyword(s):  
Plasma Membrane ◽  
Mammalian Cells ◽  
Site Directed Mutagenesis ◽  
Bar Domain ◽  
Macromolecular Assemblies ◽  
Surface Patches ◽  
Cytoplasmic Proteins ◽  
Associated Proteins ◽  
Core Components ◽  
Membrane Bending

Eisosomes define sites of plasma membrane organization. In Saccharomyces cerevisiae, eisosomes delimit furrow-like plasma membrane invaginations that concentrate sterols, transporters, and signaling molecules. Eisosomes are static macromolecular assemblies composed of cytoplasmic proteins, most of which have no known function. In this study, we used a bioinformatics approach to analyze a set of 20 eisosome proteins. We found that the core components of eisosomes, paralogue proteins Pil1 and Lsp1, are distant homologues of membrane-sculpting Bin/amphiphysin/Rvs (BAR) proteins. Consistent with this finding, purified recombinant Pil1 and Lsp1 tubulated liposomes and formed tubules when the proteins were overexpressed in mammalian cells. Structural homology modeling and site-directed mutagenesis indicate that Pil1 positively charged surface patches are needed for membrane binding and liposome tubulation. Pil1 BAR domain mutants were defective in both eisosome assembly and plasma membrane domain organization. In addition, we found that eisosome-associated proteins Slm1 and Slm2 have F-BAR domains and that these domains are needed for targeting to furrow-like plasma membrane invaginations. Our results support a model in which BAR domain protein–mediated membrane bending leads to clustering of lipids and proteins within the plasma membrane.

scholarly journals Investigating Protein Dynamics at Sites of Exocytosis in Live Cells

2016 ◽  
Vol 110 (3) ◽  
pp. 429a
Author(s):  
Agila Somasundaram ◽  
Justin W. Taraska
Keyword(s):  
Protein Dynamics ◽  
Live Cells

scholarly journals Calpain-1 and Calpain-2 in the Brain: Dr. Jekill and Mr Hyde?

2019 ◽  
Vol 17 (9) ◽  
pp. 823-829 ◽  
Author(s):  
Michel Baudry
Keyword(s):  
Protein Synthesis ◽  
Signaling Pathways ◽  
Local Protein Synthesis ◽  
Cell Functions ◽  
Anchoring Proteins ◽  
Associated Proteins ◽  
Calpain 2 ◽  
The Brain ◽  
Calpain System ◽  
Local Protein

While the calpain system has now been discovered for over 50 years, there is still a paucity of information regarding the organization and functions of the signaling pathways regulated by these proteases, although calpains play critical roles in many cell functions. Moreover, calpain overactivation has been shown to be involved in numerous diseases. Among the 15 calpain isoforms identified, calpain-1 (aka µ-calpain) and calpain-2 (aka m-calpain) are ubiquitously distributed in most tissues and organs, including the brain. We have recently proposed that calpain-1 and calpain- 2 play opposite functions in the brain, with calpain-1 activation being required for triggering synaptic plasticity and neuroprotection (Dr. Jekill), and calpain-2 limiting the extent of plasticity and being neurodegenerative (Mr. Hyde). Calpain-mediated cleavage has been observed in cytoskeleton proteins, membrane-associated proteins, receptors/channels, scaffolding/anchoring proteins, and protein kinases and phosphatases. This review will focus on the signaling pathways related to local protein synthesis, cytoskeleton regulation and neuronal survival/death regulated by calpain-1 and calpain-2, in an attempt to explain the origin of the opposite functions of these 2 calpain isoforms. This will be followed by a discussion of the potential therapeutic applications of selective regulators of these 2 calpain isoforms.

scholarly journals Unbiased Thiol-Labeling and Top-Down Proteomic Analyses Implicate Multiple Proteins in the Late Steps of Regulated Secretion

Proteomes ◽  
2019 ◽  
Vol 7 (4) ◽  
pp. 34 ◽  
Author(s):  
Furber ◽  
Backlund ◽  
Yergey ◽  
Coorssen
Keyword(s):  
Lucifer Yellow ◽  
Biologically Active ◽  
Rab Proteins ◽  
Top Down ◽  
Vesicle Membrane ◽  
Two Dimensional Gel Electrophoresis ◽  
Regulated Exocytosis ◽  
Proteomic Approach ◽  
Proteomic Analyses ◽  
High Doses

Regulated exocytosis enables temporal and spatial control over the secretion of biologically active compounds; however, the mechanism by which Ca2+ modulates different stages of exocytosis is still poorly understood. For an unbiased, top-down proteomic approach, select thiol- reactive reagents were used to investigate this process in release-ready native secretory vesicles. We previously characterized a biphasic effect of these reagents on Ca2+-triggered exocytosis: low doses potentiated Ca2+ sensitivity, whereas high doses inhibited Ca2+ sensitivity and extent of vesicle fusion. Capitalizing on this novel potentiating effect, we have now identified fluorescent thiol- reactive reagents producing the same effects: Lucifer yellow iodoacetamide, monobromobimane, and dibromobimane. Top-down proteomic analyses of fluorescently labeled proteins from total and cholesterol-enriched vesicle membrane fractions using two-dimensional gel electrophoresis coupled with mass spectrometry identified several candidate targets, some of which have been previously linked to the late steps of regulated exocytosis and some of which are novel. Initial validation studies indicate that Rab proteins are involved in the modulation of Ca2+ sensitivity, and thus the efficiency of membrane fusion, which may, in part, be linked to their previously identified upstream roles in vesicle docking.

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