scholarly journals Annexin A2 promotes phagophore assembly by enhancing Atg16L+ vesicle biogenesis and homotypic fusion

2015 ◽  
Vol 6 (1) ◽  
Author(s):  
Kateryna Morozova ◽  
Sunandini Sidhar ◽  
Valerio Zolla ◽  
Cristina C. Clement ◽  
Brian Scharf ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Annexin A2 ◽  
Membrane Curvature ◽  
Early Event ◽  
Vesicle Formation ◽  
Deficient Mouse ◽  
Lipidomic Analysis ◽  
Vesicle Biogenesis ◽  
Mouse Cells ◽  
Membrane Budding

Abstract Plasma membrane budding of Atg-16L-positive vesicles represents a very early event in the generation of the phagophore and in the process of macroautophagy. Here we show that the membrane curvature-inducing protein annexin A2 contributes to the formation of these vesicles and their fusion to form phagophores. Ultrastructural, proteomic and FACS analyses of Atg16L-positive vesicles reveal that 30% of Atg16L-positive vesicles are also annexin A2-positive. Lipidomic analysis of annexin A2-deficient mouse cells indicates that this protein plays a role in recruiting phosphatidylserine and phosphatidylinositides to Atg16L-positive vesicles. Absence of annexin A2 reduces both vesicle formation and homotypic Atg16L vesicle fusion. Ultimately, a reduction in LC3 flux and dampening of macroautophagy are observed in dendritic cells from Anxa2 −/− mice. Together, our analyses highlight the importance of annexin A2 in vesiculation of a population of Atg16L-positive structures from the plasma membrane, and in their homotypic fusion to form phagophore structures.

scholarly journals Membrane Remodeling by Arc/Arg3.1

2021 ◽  
Vol 8 ◽  
Author(s):  
Per Niklas Hedde ◽  
Leonel Malacrida ◽  
Barbara Barylko ◽  
Derk D. Binns ◽  
Joseph P. Albanesi ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Ampa Receptors ◽  
Long Term Potentiation ◽  
Terminal Segment ◽  
Membrane Curvature ◽  
Early Gene ◽  
Striking Similarity ◽  
Term Potentiation ◽  
Membrane Budding

The activity-regulated cytoskeletal-associated protein (Arc, also known as Arg3.1) is an immediate early gene product induced by activity/experience and required for multiple modes of synaptic plasticity. Both long-term potentiation (LTP) and long-term depression (LTD) are impaired upon Arc deletion, as well as the ability to form long-term spatial, taste and fear memories. The best-characterized cellular function of Arc is enhancement of the endocytic internalization of AMPA receptors (AMPARs) in dendritic spines. Solution of the crystal structure of a C-terminal segment of Arc revealed a striking similarity to the capsid domain of HIV Gag. It was subsequently shown that Arc assembles into viral capsid-like structures that enclose Arc mRNA, are released into the extracellular space, and are internalized by neighboring cells. Thus, Arc is unique in participating in plasma membrane budding both into and out of the cell. In this report we study the interaction of Arc with membranes using giant unilamellar vesicles (GUVs). Using the fluorescent lipid probe LAURDAN, we find that Arc promotes the formation of smaller vesicles that penetrate into the GUV interior. Our results suggest that Arc induces negative membrane curvature and may therefore facilitate the formation of mRNA-containing extracellular vesicles from the plasma membrane.

scholarly journals Annexin A4 trimers are recruited by high membrane curvatures in Giant Plasma Membrane Vesicles

2020 ◽  
Author(s):  
Christoffer Florentsen ◽  
Alexander Kamp-Sonne ◽  
Guillermo Moreno-Pescador ◽  
Weria Pezeshkian ◽  
Ali Asghar Hakami Zanjani ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Vesicles ◽  
Membrane Curvature ◽  
Biological Functions ◽  
Membrane Repair ◽  
Plasma Membrane Vesicles ◽  
Curvature Sensing ◽  
Curved Structures ◽  
Annexin A4 ◽  
Membrane Budding

AbstractThe plasma membrane of eukaryotic cells consists of a crowded environment comprised of a high diversity of proteins in a complex lipid matrix. The lateral organization of membrane proteins in the plasma membrane (PM) is closely correlated with biological functions such as endocytosis, membrane budding and other processes which involve protein mediated shaping of the membrane into highly curved structures. Annexin A4 (ANXA4) is a prominent player in a number of biological functions including plasma membrane repair. Its binding to membranes is activated by Ca2+ influx and it is therefore rapidly recruited to the cell surface near rupture sites where Ca2+ influx takes place. However, the free edges near rupture sites can easily bend into complex curvatures and hence may accelerate recruitment of curvature sensing proteins to facilitate rapid membrane repair. To analyze the curvature sensing behavior of curvature inducing proteins in crowded membranes, we quantifify the affinity of ANXA4 monomers and trimers for high membrane curvatures by extracting membrane nanotubes from giant plasma membrane vesicles (GPMVs). ANXA4 is found to be a sensor of negative membrane curvatures. Multiscale simulations furthermore predicted that ANXA4 trimers generate membrane curvature upon binding and have an affinity for highly curved membrane regions only within a well defined membrane curvature window. Our results indicate that curvature sensing and mobility of ANXA4 depend on the trimer structure of ANXA4 which could provide new biophysical insight into the role of ANXA4 in membrane repair and other biological processes.

Intracellular trafficking and cytoplasmic streaming under abiotic stress conditions

2019 ◽  
Vol 6 (04) ◽  
Author(s):  
JESHIMA KHAN YASIN ◽  
ANIL KUMAR SINGH
Keyword(s):  
Plasma Membrane ◽  
Abiotic Stress ◽  
Confocal Microscopy ◽  
Fusion Protein ◽  
Intracellular Trafficking ◽  
Cytoplasmic Streaming ◽  
Living Cells ◽  
Stress Conditions ◽  
Vesicle Formation ◽  
External Stimuli

Cytoplasmic streaming is one among the vital activities of the living cells. In plants cytolplasmic streaming could clearly be seen in hypocotyls of growing seedlings. To observe cytoplsmic streaming and its correlated intracellular trafficking an investigation was conducted in legumes in comparison with GFP-AtRab75 and 35S::GFP:δTIP tonoplast fusion protein expressing arabidopsis lines. These seedlings were observed under confocal microscopy with different buffer incubation treatments and under different stress conditions. GFP expressing 35S::GFP:δTIP tonoplast lines were looking similar to the control lines and differ under stress conditions. Movement of cytoplasmic invaginations within the tonoplast and cytoplasmic sub vesicle or bulb budding during cytoplasmic streaming was observed in hypocotyls of At-GFP tonoplast plants. We found the cytoplasmic bulbs/ vesicles or sub vesicle formation from the plasma membrane. The streaming speed also depends on the incubation medium in which the specimen was incubated, indicating that the external stimuli as well as internal stimuli can alter the speed of streaming

scholarly journals GTPase cross talk regulates TRAPPII activation of Rab11 homologues during vesicle biogenesis

2016 ◽  
Vol 215 (4) ◽  
pp. 499-513 ◽  
Author(s):  
Laura L. Thomas ◽  
J. Christopher Fromme
Keyword(s):  
Cross Talk ◽  
Vesicle Formation ◽  
Nucleotide Exchange ◽  
Endocytic Recycling ◽  
Direct Role ◽  
Definitive Evidence ◽  
Nucleotide Exchange Factor ◽  
Vesicle Biogenesis ◽  
Simultaneous Activation ◽  
Bona Fide

Rab guanosine triphosphatases (GTPases) control cellular trafficking pathways by regulating vesicle formation, transport, and tethering. Rab11 and its paralogs regulate multiple secretory and endocytic recycling pathways, yet the guanine nucleotide exchange factor (GEF) that activates Rab11 in most eukaryotic cells is unresolved. The large multisubunit transport protein particle (TRAPP) II complex has been proposed to act as a GEF for Rab11 based on genetic evidence, but conflicting biochemical experiments have created uncertainty regarding Rab11 activation. Using physiological Rab-GEF reconstitution reactions, we now provide definitive evidence that TRAPPII is a bona fide GEF for the yeast Rab11 homologues Ypt31/32. We also uncover a direct role for Arf1, a distinct GTPase, in recruiting TRAPPII to anionic membranes. Given the known role of Ypt31/32 in stimulating activation of Arf1, a bidirectional cross talk mechanism appears to drive biogenesis of secretory and endocytic recycling vesicles. By coordinating simultaneous activation of two essential GTPase pathways, this mechanism ensures recruitment of the complete set of effectors needed for vesicle formation, transport, and tethering.

Membrane curvature and the control of GTP hydrolysis in Arf1 during COPI vesicle formation

2005 ◽  
Vol 33 (4) ◽  
pp. 619-622 ◽  
Author(s):  
B. Antonny ◽  
J. Bigay ◽  
J.-F. Casella ◽  
G. Drin ◽  
B. Mesmin ◽  
...  
Keyword(s):  
Lipid Membranes ◽  
Membrane Curvature ◽  
Gtp Hydrolysis ◽  
Membrane Fission ◽  
Transport Vesicles ◽  
Highly Sensitive ◽  
Copi Vesicle ◽  
Membrane Budding ◽  
Gtpase Cycle ◽  
Adp Ribosylation Factor

The GTP switch of the small G-protein Arf1 (ADP-ribosylation factor 1) on lipid membranes promotes the polymerization of the COPI (coat protein complex I) coat, which acts as a membrane deforming shell to form transport vesicles. Real-time measurements for coat assembly on liposomes gives insights into how the GTPase cycle of Arf1 is coupled in time with the polymerization of the COPI coat and the resulting membrane deformation. One key parameter seems to be the membrane curvature. Arf-GAP1 (where GAP stands for GTPase-activating protein), which promotes GTP hydrolysis in the Arf1–COPI complex is highly sensitive to lipid packing. Its activity on Arf1-GTP increases by two orders of magnitude as the diameter of the liposomes approaches that of authentic transport vesicles (60 nm). This suggests that during membrane budding, Arf1-GTP molecules are progressively eliminated from the coated area where the membrane curvature is positive, but are protected from Arf-GAP1 at the bud neck due to the negative curvature of this region. As a result, the coat should be stable as long as the bud remains attached and should disassemble as soon as membrane fission occurs.

scholarly journals Drosophila F-BAR protein Syndapin contributes to coupling the plasma membrane and contractile ring in cytokinesis

Open Biology ◽  
2013 ◽  
Vol 3 (8) ◽  
pp. 130081 ◽  
Author(s):  
Tetsuya Takeda ◽  
Iain M. Robinson ◽  
Matthew M. Savoian ◽  
John R. Griffiths ◽  
Anthony D. Whetton ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Membrane Curvature ◽  
Cellular Process ◽  
Cleavage Furrow ◽  
Contractile Ring ◽  
Functional Interaction ◽  
Cortical Dynamics ◽  
Central Spindle ◽  
Bar Domain ◽  
Spindle Microtubules

Cytokinesis is a highly ordered cellular process driven by interactions between central spindle microtubules and the actomyosin contractile ring linked to the dynamic remodelling of the plasma membrane. The mechanisms responsible for reorganizing the plasma membrane at the cell equator and its coupling to the contractile ring in cytokinesis are poorly understood. We report here that Syndapin, a protein containing an F-BAR domain required for membrane curvature, contributes to the remodelling of the plasma membrane around the contractile ring for cytokinesis. Syndapin colocalizes with phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ) at the cleavage furrow, where it directly interacts with a contractile ring component, Anillin. Accordingly, Anillin is mislocalized during cytokinesis in Syndapin mutants. Elevated or diminished expression of Syndapin leads to cytokinesis defects with abnormal cortical dynamics. The minimal segment of Syndapin, which is able to localize to the cleavage furrow and induce cytokinesis defects, is the F-BAR domain and its immediate C-terminal sequences. Phosphorylation of this region prevents this functional interaction, resulting in reduced ability of Syndapin to bind to and deform membranes. Thus, the dephosphorylated form of Syndapin mediates both remodelling of the plasma membrane and its proper coupling to the cytokinetic machinery.

scholarly journals Extracellular vesicle budding is inhibited by redundant regulators of TAT-5 flippase localization and phospholipid asymmetry

2018 ◽  
Vol 115 (6) ◽  
pp. E1127-E1136 ◽  
Author(s):  
Katharina B. Beer ◽  
Jennifer Rivas-Castillo ◽  
Kenneth Kuhn ◽  
Gholamreza Fazeli ◽  
Birgit Karmann ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Vesicle ◽  
Endosomal Trafficking ◽  
Vesicle Budding ◽  
Sorting Nexins ◽  
Dnaj Protein ◽  
Membrane Budding ◽  
Pleiotropic Functions

Cells release extracellular vesicles (EVs) that mediate intercellular communication and repair damaged membranes. Despite the pleiotropic functions of EVs in vitro, their in vivo function is debated, largely because it is unclear how to induce or inhibit their formation. In particular, the mechanisms of EV release by plasma membrane budding or ectocytosis are poorly understood. We previously showed that TAT-5 phospholipid flippase activity maintains the asymmetric localization of the lipid phosphatidylethanolamine (PE) in the plasma membrane and inhibits EV budding by ectocytosis in Caenorhabditis elegans. However, no proteins that inhibit ectocytosis upstream of TAT-5 were known. Here, we identify TAT-5 regulators associated with retrograde endosomal recycling: PI3Kinase VPS-34, Beclin1 homolog BEC-1, DnaJ protein RME-8, and the uncharacterized Dopey homolog PAD-1. PI3Kinase, RME-8, and semiredundant sorting nexins are required for the plasma membrane localization of TAT-5, which is important to maintain PE asymmetry and inhibit EV release. PAD-1 does not directly regulate TAT-5 localization, but is required for the lipid flipping activity of TAT-5. PAD-1 also has roles in endosomal trafficking with the GEF-like protein MON-2, which regulates PE asymmetry and EV release redundantly with sorting nexins independent of the core retromer. Thus, in addition to uncovering redundant intracellular trafficking pathways, our study identifies additional proteins that regulate EV release. This work pinpoints TAT-5 and PE as key regulators of plasma membrane budding, further supporting the model that PE externalization drives ectocytosis.

scholarly journals EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells

2016 ◽  
Vol 212 (3) ◽  
pp. 297-306 ◽  
Author(s):  
Atsuhiro Nakajo ◽  
Shin-ichiro Yoshimura ◽  
Hiroko Togawa ◽  
Masataka Kunii ◽  
Tomohiko Iwano ◽  
...  
Keyword(s):  
Small Intestine ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Basolateral Membrane ◽  
Membrane Curvature ◽  
Apical Plasma Membrane ◽  
Interacting Protein ◽  
Guanosine Triphosphatase ◽  
Biochemical Analyses ◽  
Interacting Protein Complex

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain–binding protein 1–like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1–dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

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