scholarly journals Clathrin-mediated endocytosis-independent function of VAN3 ARF GTPase Activating Protein at the plasma membrane

2021 ◽  
Author(s):  
Maciek Adamowski ◽  
Jiří Friml
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Small Gtpases ◽  
Independent Function ◽  
Cellular Functions ◽  
Knockout Mutant ◽  
Subcellular Trafficking ◽  
Coated Pits ◽  
Plant Body ◽  
Developmental Patterning

ARF small GTPases in plants serve important cellular functions in subcellular trafficking and developmental functions in auxin-mediated patterning of the plant body. The Arabidopsis thaliana ARF regulator ARF-GAP VAN3 has been implicated to act at the plasma membrane (PM) and linked functionally to the clathrin- and dynamin-mediated endocytosis. Here we re-evaluated the localization of VAN3 at the PM and its function in endocytosis. Using Total Internal Reflection Fluorescence microscopy we observed remarkably transient associations of VAN3 to the PM at discrete foci, however, devoid of clathrin, the dynamin isoform DRP1A, or the ARF regulator GNOM, which is also involved in a developmental patterning function mediated from the PM. Clathrin-coated pits are abundant and endocytosis appears to proceed normally in van3-1 knockout mutant. In turn, post-translational silencing of clathrin expression indicates that the localization of VAN3 at the PM depends on clathrin function, presumably on clathrin-mediated endocytosis.

scholarly journals Developmental patterning function of GNOM ARF-GEF mediated from the plasma membrane

2022 ◽  
Author(s):  
Maciek Adamowski ◽  
Ivana Matijević ◽  
Jiří Friml
Keyword(s):  
Plasma Membrane ◽  
Brefeldin A ◽  
Small Gtpases ◽  
Nucleotide Exchange ◽  
Coated Pits ◽  
Guanine Nucleotide Exchange ◽  
Nucleotide Exchange Factor ◽  
Mutant Variant ◽  
Developmental Patterning ◽  
Mechanistic Basis

The GNOM (GN) Guanine nucleotide Exchange Factor for ARF small GTPases (ARF-GEF) is among the best studied trafficking regulators in plants, playing crucial and unique developmental roles in patterning and polarity. The current models place GN at the Golgi apparatus (GA), where it mediates secretion/recycling, and at the plasma membrane (PM) presumably contributing to clathrin-mediated endocytosis (CME). The mechanistic basis of the developmental function of GN, distinct from the other ARF-GEFs including its homologue GNOM-LIKE1 (GNL1), remains elusive. Insights from this study redefine the current notions of GN function. We show that GN, but not GNL1, localizes to the PM at long-lived structures distinct from clathrin-coated pits, while CME and secretion proceed normally in gn knockouts. The functional GN mutant variant GNfewerroots, absent from the GA, suggests that PM is the major place of GN action responsible for its developmental function. Following inhibition by Brefeldin A, GN, but not GNL1, relocates to the PM likely on exocytic vesicles, suggesting selective molecular associations. A study of GN-GNL1 chimeric ARF-GEFs indicate that all GN domains contribute to the specific GN function in a partially redundant manner. Together, this study offers significant steps towards the elucidation of the mechanism underlying unique cellular and development functions of GN.

scholarly journals Optogenetic inhibition and activation of Rac and Rap1 using a modified iLID system

2020 ◽  
Author(s):  
Nick R. Elston ◽  
Michael Pablo ◽  
Fred Pimenta ◽  
Klaus M. Hahn ◽  
Takashi Watanabe
Keyword(s):  
Plasma Membrane ◽  
Living Cells ◽  
Small Gtpases ◽  
Spatiotemporal Dynamics ◽  
Membrane Localization ◽  
Cellular Functions ◽  
Activation Kinetics ◽  
System 1 ◽  
Control Plasma

The small GTPases Rac1 and Rap1 can fulfill multiple cellular functions because their activation kinetics and localization are precisely controlled. To probe the role of their spatiotemporal dynamics, we generated optogenetic tools that activate or inhibit endogenous Rac and Rap1 in living cells. An improved version of the light induced dimerization (iLID) system [1] was used to control plasma membrane localization of protein domains that specifically activate or inactivate Rap1 and Rac (Tiam1 and Chimerin for Rac, RasGRP2 and Rap1GAP for Rap1 [2, 3, 4, 5]). Irradiation yielded a 50% to 230% increase in the concentration of these domains at the membrane, leading to effects on cell morphodynamics consistent with the known roles of Rac1 and Rap1.

scholarly journals Role of plasma-membrane-bound sialidase NEU3 in clathrin-mediated endocytosis

2015 ◽  
Vol 470 (1) ◽  
pp. 131-144 ◽  
Author(s):  
Macarena Rodriguez-Walker ◽  
Aldo A. Vilcaes ◽  
Eduardo Garbarino-Pico ◽  
José L. Daniotti
Keyword(s):  
Plasma Membrane ◽  
Subcellular Distribution ◽  
Cellular Functions ◽  
Coated Pits ◽  
Key Enzyme ◽  
Membrane Bound

Sialidase NEU3 is a key enzyme in the catabolism of gangliosides. We demonstrated that NEU3 impairs cargo internalization via clathrin-coated pits, affecting AP-2 subcellular distribution. This study delineates previously unidentified cellular functions of NEU3.

scholarly journals The inositol 5-phosphatase SHIP2 regulates endocytic clathrin-coated pit dynamics

2010 ◽  
Vol 190 (3) ◽  
pp. 307-315 ◽  
Author(s):  
Fubito Nakatsu ◽  
Rushika M. Perera ◽  
Louise Lucast ◽  
Roberto Zoncu ◽  
Jan Domin ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Insulin Signaling ◽  
Negative Regulator ◽  
Phosphoinositide Metabolism ◽  
Cellular Functions ◽  
Coated Pits ◽  
Positive Role ◽  
Insulin Signal ◽  
Signal Output

Phosphatidylinositol (PI) 4,5-bisphosphate (PI(4,5)P2) and its phosphorylated product PI 3,4,5-triphosphate (PI(3,4,5)P3) are two major phosphoinositides concentrated at the plasma membrane. Their levels, which are tightly controlled by kinases, phospholipases, and phosphatases, regulate a variety of cellular functions, including clathrin-mediated endocytosis and receptor signaling. In this study, we show that the inositol 5-phosphatase SHIP2, a negative regulator of PI(3,4,5)P3-dependent signaling, also negatively regulates PI(4,5)P2 levels and is concentrated at endocytic clathrin-coated pits (CCPs) via interactions with the scaffold protein intersectin. SHIP2 is recruited early at the pits and dissociates before fission. Both knockdown of SHIP2 expression and acute production of PI(3,4,5)P3 shorten CCP lifetime by enhancing the rate of pit maturation, which is consistent with a positive role of both SHIP2 substrates, PI(4,5)P2 and PI(3,4,5)P3, on coat assembly. Because SHIP2 is a negative regulator of insulin signaling, our findings suggest the importance of the phosphoinositide metabolism at CCPs in the regulation of insulin signal output.

scholarly journals β-arrestin–dependent PI(4,5)P2 synthesis boosts GPCR endocytosis

2021 ◽  
Vol 118 (17) ◽  
pp. e2011023118
Author(s):  
Seung-Ryoung Jung ◽  
Yifei Jiang ◽  
Jong Bae Seo ◽  
Daniel T. Chiu ◽  
Bertil Hille ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Intracellular Signaling ◽  
Live Cells ◽  
Cellular Functions ◽  
Coated Pits ◽  
Endocytic Machinery ◽  
S 100 ◽  
Phosphatidylinositol 4 ◽  
Protease Activated Receptor 2 ◽  
G Protein Coupled

β-arrestins regulate many cellular functions including intracellular signaling and desensitization of G protein–coupled receptors (GPCRs). Previous studies show that β-arrestin signaling and receptor endocytosis are modulated by the plasma membrane phosphoinositide lipid phosphatidylinositol-(4, 5)-bisphosphate (PI(4,5)P2). We found that β-arrestin also helped promote synthesis of PI(4,5)P2 and up-regulated GPCR endocytosis. We studied these questions with the Gq-coupled protease-activated receptor 2 (PAR2), which activates phospholipase C, desensitizes quickly, and undergoes extensive endocytosis. Phosphoinositides were monitored and controlled in live cells using lipid-specific fluorescent probes and genetic tools. Applying PAR2 agonist initiated depletion of PI(4,5)P2, which then recovered during rapid receptor desensitization, giving way to endocytosis. This endocytosis could be reduced by various manipulations that depleted phosphoinositides again right after phosphoinositide recovery: PI(4)P, a precusor of PI(4,5)P2, could be depleted at either the Golgi or the plasma membrane (PM) using a recruitable lipid 4-phosphatase enzyme and PI(4,5)P2 could be depleted at the PM using a recruitable 5-phosphatase. Endocytosis required the phosphoinositides. Knock-down of β-arrestin revealed that endogenous β-arrestin normally doubles the rate of PIP5-kinase (PIP5K) after PAR2 desensitization, boosting PI(4,5)P2-dependent formation of clathrin-coated pits (CCPs) at the PM. Desensitized PAR2 receptors were swiftly immobilized when they encountered CCPs, showing a dwell time of ∼90 s, 100 times longer than for unactivated receptors. PAR2/β-arrestin complexes eventually accumulated around the edges or across the surface of CCPs promoting transient binding of PIP5K-Iγ. Taken together, β-arrestins can coordinate potentiation of PIP5K activity at CCPs to induce local PI(4,5)P2 generation that promotes recruitment of PI(4,5)P2-dependent endocytic machinery.

Changes Occur in Plasma Membrane Turnover when K562 Leukemia Cells are Induced to Differentiate

1982 ◽  
Vol 40 ◽  
pp. 286-287
Author(s):  
L. M. Marshall
Keyword(s):  
Plasma Membrane ◽  
Membrane Proteins ◽  
Intracellular Transport ◽  
Parent Cell ◽  
Membrane Turnover ◽  
Coated Pits ◽  
Surface Distribution ◽  
Specific Proteins ◽  
Erythroleukemic Cell ◽  
Specific Membrane

A human erythroleukemic cell line, metabolically blocked in a late stage of erythropoiesis, becomes capable of differentiation along the normal pathway when grown in the presence of hemin. This process is characterized by hemoglobin synthesis followed by rearrangement of the plasma membrane proteins and culminates in asymmetrical cytokinesis in the absence of nuclear division. A reticulocyte-like cell buds from the nucleus-containing parent cell after erythrocyte specific membrane proteins have been sequestered into its membrane. In this process the parent cell faces two obstacles. First, to organize its erythrocyte specific proteins at one pole of the cell for inclusion in the reticulocyte; second, to reduce or abolish membrane protein turnover since hemoglobin is virtually the only protein being synthesized at this stage. A means of achieving redistribution and cessation of turnover could involve movement of membrane proteins by a directional lipid flow. Generation of a lipid flow towards one pole and accumulation of erythrocyte-specific membrane proteins could be achieved by clathrin coated pits which are implicated in membrane endocytosis, intracellular transport and turnover. In non-differentiating cells, membrane proteins are turned over and are random in surface distribution. If, however, the erythrocyte specific proteins in differentiating cells were excluded from endocytosing coated pits, not only would their turnover cease, but they would also tend to drift towards and collect at the site of endocytosis. This hypothesis requires that different protein species are endocytosed by the coated vesicles in non-differentiating than by differentiating cells.

Caveolin-1, galectin-3 and lipid raft domains in cancer cell signalling

2015 ◽  
Vol 57 ◽  
pp. 189-201 ◽  
Author(s):  
Jay Shankar ◽  
Cecile Boscher ◽  
Ivan R. Nabi
Keyword(s):  
Plasma Membrane ◽  
Lipid Rafts ◽  
Cancer Cell ◽  
Cellular Response ◽  
Spatial Organization ◽  
Essential Feature ◽  
Cell Signalling ◽  
Coated Pits ◽  
Signalling Molecules ◽  
Raft Domains

Spatial organization of the plasma membrane is an essential feature of the cellular response to external stimuli. Receptor organization at the cell surface mediates transmission of extracellular stimuli to intracellular signalling molecules and effectors that impact various cellular processes including cell differentiation, metabolism, growth, migration and apoptosis. Membrane domains include morphologically distinct plasma membrane invaginations such as clathrin-coated pits and caveolae, but also less well-defined domains such as lipid rafts and the galectin lattice. In the present chapter, we will discuss interaction between caveolae, lipid rafts and the galectin lattice in the control of cancer cell signalling.

scholarly journals Revising Endosomal Trafficking under Insulin Receptor Activation

2021 ◽  
Vol 22 (13) ◽  
pp. 6978
Author(s):  
Maria J. Iraburu ◽  
Tommy Garner ◽  
Cristina Montiel-Duarte
Keyword(s):  
Plasma Membrane ◽  
Tyrosine Kinase ◽  
Insulin Receptor ◽  
Molecular Mechanisms ◽  
Receptor Activation ◽  
Small Gtpases ◽  
Early Endosome ◽  
Tyrosine Kinase Receptors ◽  
Endosomal Trafficking ◽  
Cell Functions

The endocytosis of ligand-bound receptors and their eventual recycling to the plasma membrane (PM) are processes that have an influence on signalling activity and therefore on many cell functions, including migration and proliferation. Like other tyrosine kinase receptors (TKR), the insulin receptor (INSR) has been shown to be endocytosed by clathrin-dependent and -independent mechanisms. Once at the early endosome (EE), the sorting of the receptor, either to the late endosome (LE) for degradation or back to the PM through slow or fast recycling pathways, will determine the intensity and duration of insulin effects. Both the endocytic and the endosomic pathways are regulated by many proteins, the Arf and Rab families of small GTPases being some of the most relevant. Here, we argue for a specific role for the slow recycling route, whilst we review the main molecular mechanisms involved in INSR endocytosis, sorting and recycling, as well as their possible role in cell functions.

Visualization of Membrane Sorting and Fusion in Living Cells using Total Internal Reflection (TIR) and Multicolor Video Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 34-35
Author(s):  
Derek Toomre ◽  
Patrick Keller ◽  
Elena Diaz ◽  
Jamie White ◽  
Kai Simons
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Fluorescent Protein ◽  
Time Lapse ◽  
Living Cells ◽  
Video Microscopy ◽  
Internal Reflection ◽  
Fast Time ◽  
Cellular Polarity ◽  
Microscopy Technique

Post-Golgi sorting of different classes of newly synthesized proteins and lipids is central to the generation and maintenance of cellular polarity. to directly visualize the dynamics and location of apical/basolateral sorting and trafficking we used fast time-lapse multicolor video microscopy in living cells. Specifically, green fluorescent protein color variants (cyan, CFP; yellow, YFP) of apical cargo (GPI-anchored) and basolateral cargo (vesicular stomatitis virus glycoprotein, VSVG) were generated; see FIG 1. Fast dual color fluorescence video microscopy allowed visualization with high temporal and spatial resolution. Our studies revealed that apical and basolateral cargo progressively segregated into large domains in Golgi/TGN structures, excluded resident proteins, and exited in separate transport containers. These carries remained distinct and did not merge with endocytic structures en route to the plasma membrane. Interestingly, our data suggest that the primary sorting occurs by lateral segregation in the Golgi, prior to budding (FIG 2). Further characterization of morphological differences of apical versus basolateral transport carriers was achieved using a specialized microscopy technique called total internal reflection (TIR) microscopy. with this approach only the bottom of the cell (<100 nm) was illuminated by an exponentially decaying evanescent “wave” of light. A series of images, taken at ∼1 second intervals, shows a bright “flash” of fluorescence when the vesicle fuse with the plasma membrane and the fluorophore diffuses into the plasma membrane (FIG 3).

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