scholarly journals The phosphoinositide coincidence detector Phafin2 promotes macropinocytosis by coordinating actin organisation at forming macropinosomes

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kay Oliver Schink ◽  
Kia Wee Tan ◽  
Hélène Spangenberg ◽  
Domenica Martorana ◽  
Marte Sneeggen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Extracellular Fluid ◽  
Coincidence Detection ◽  
Cancer Development ◽  
Actin Assembly ◽  
Actin Network ◽  
Ph Domain ◽  
Actin Reorganization ◽  
Domain Protein ◽  
Pass Through

AbstractUptake of large volumes of extracellular fluid by actin-dependent macropinocytosis has an important role in infection, immunity and cancer development. A key question is how actin assembly and disassembly are coordinated around macropinosomes to allow them to form and subsequently pass through the dense actin network underlying the plasma membrane to move towards the cell center for maturation. Here we show that the PH and FYVE domain protein Phafin2 is recruited transiently to newly-formed macropinosomes by a mechanism that involves coincidence detection of PtdIns3P and PtdIns4P. Phafin2 also interacts with actin via its PH domain, and recruitment of Phafin2 coincides with actin reorganization around nascent macropinosomes. Moreover, forced relocalization of Phafin2 to the plasma membrane causes rearrangement of the subcortical actin cytoskeleton. Depletion of Phafin2 inhibits macropinosome internalization and maturation and prevents KRAS-transformed cancer cells from utilizing extracellular protein as an amino acid source. We conclude that Phafin2 promotes macropinocytosis by controlling timely delamination of actin from nascent macropinosomes for their navigation through the dense subcortical actin network.

scholarly journals The PtdIns3P-binding protein Phafin2 escorts macropinosomes through the cortical actin cytoskeleton

2017 ◽  
Author(s):  
Kay Oliver Schink ◽  
Kia Wee Tan ◽  
Hélène Spangenberg ◽  
Domenica Martorana ◽  
Marte Sneeggen ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Cytoskeleton ◽  
Binding Protein ◽  
Extracellular Fluid ◽  
Cancer Development ◽  
Cross Linking ◽  
Filamin A ◽  
Actin Network ◽  
Cortical Actin ◽  
Cell Centre

AbstractUptake of large volumes of extracellular fluid by actin-dependent macropinocytosis plays important roles in infection, immunity and cancer development. A key question is how large macropinosomes are able to squeeze through the dense actin network underlying the plasma membrane in order to move towards the cell centre for maturation. Here we show that, immediately after macropinosomes have been sealed off from the plasma membrane, the PH-and FYVE domain-containing protein Phafin2 is recruited by a mechanism that involves binding to phosphatidylinositol 3-phosphate (PtdIns3P) generated in a non-canonical manner. Phafin2 in turn regulates the actin cross-linking protein Filamin A to promote entry of macropinosomes through the subcortical actin matrix and subsequent maturation. Depletion of Phafin2 inhibits macropinocytic internalization and maturation. We conclude that PtdIns3P and its effector Phafin2 are key components of a system that allows nascent macropinosomes to navigate through the dense subcortical actin network.

scholarly journals A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

2021 ◽  
Author(s):  
Timothy J HAWKINS ◽  
Michaela Kopischke ◽  
David Mentlak ◽  
Patrick Duckney ◽  
Johan Kroon ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Plasma Membrane ◽  
Guard Cell ◽  
Actin Filaments ◽  
Plant Immunity ◽  
Stomatal Closure ◽  
Actin Network ◽  
Actin Reorganization ◽  
Downstream Effectors ◽  
Membrane Bound

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cell's plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

scholarly journals Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

2020 ◽  
Author(s):  
Charlotte Kaplan ◽  
Sam J. Kenny ◽  
Shirley Chen ◽  
Johannes Schöneberg ◽  
Ewa Sitarska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Spatial Organization ◽  
Force Generation ◽  
List Type ◽  
Membrane Tension ◽  
Actin Assembly ◽  
Actin Network ◽  
Coated Pits ◽  
Actin Organization ◽  
Tightly Coupled

AbstractClathrin-mediated endocytosis (CME) remains robust despite variations in plasma membrane tension. Actin assembly-mediated force generation becomes essential for CME under high membrane tension, but the underlying mechanisms are not understood. We investigated actin network ultrastructure at each stage of CME by super-resolution imaging. Actin and N-WASP spatial organization indicate that polymerization initiates at the base of clathrin-coated pits and that the actin network then grows away from the plasma membrane. Actin network organization is not tightly coupled to endocytic clathrin coat growth and deformation. Membrane tension-dependent changes in actin organization explain this uncoupling. Under elevated membrane tension, CME dynamics slow down and the actin network grows higher, resulting in greater coverage of the clathrin coat. This adaptive mechanism is especially crucial during the initial membrane curvature-generating stages of CME. Our findings reveal that adaptive force generation by the actin network ensures robust CME progression despite changes in plasma membrane tension.Highlights-Clathrin coat surface area and actin ultra-structure adapt to elevated membrane tension.-The actin network is nucleated at the base of the clathrin-coated pit and grows upward.-Actin ultra-structural organization is not tightly coupled to CME progression.-Actin force generation is required earlier in CME progression under elevated membrane tension.SummaryKaplan et al. revealed that actin assembly compensates for changes in plasma membrane tension by an adaptive force generating mechanism to ensure robust endocytosis. Under elevated membrane tension the network grows deeper, even in early endocytic stages, from the base upward.

Immunocytochemical localization of p65 with one-nanometer gold particles following silver development

1989 ◽  
Vol 47 ◽  
pp. 1042-1043
Author(s):  
Richard W. Burry ◽  
Diane M. Hayes
Keyword(s):  
Plasma Membrane ◽  
Bovine Serum Albumin ◽  
Calf Serum ◽  
Specific Protein ◽  
Immunocytochemical Localization ◽  
Electron Microscopic ◽  
Gold Particles ◽  
Pass Through ◽  
Label Distribution ◽  
Phosphate Buffered Saline

Electron microscopic (EM) immunocytochemistry localization of the neuron specific protein p65 could show which organelles contain this antigen. Antibodies (Ab) labeled with horseradish peroxidase (HRP) followed by chromogen development show a broad diffuse label distribution within cells and restricting identification of organelles. Particulate label (e.g. 10 nm colloidal gold) is highly desirable but not practical because penetration into cells requires destroying the plasma membrane. We report pre-embedding immunocytochemistry with a particulate marker, 1 nm gold, that will pass through membranes treated with saponin, a mild detergent.Cell cultures of the rat cerebellum were fixed in buffered 4% paraformaldehyde and 0.1% glutaraldehyde (Glut.). The buffer for all incubations and rinses was phosphate buffered saline with: 1% calf serum, 0.2% saponin, 0.1% gelatin, 50 mM glycine 1 mg/ml bovine serum albumin, and (not in the HRP labeled cultures) 0.02% sodium azide. The monoclonal #48 to p65 was used with three label systems: HRP, 1 nm avidin gold with IntenSE M development, and 1 nm avidin gold with Danscher development.

scholarly journals Na delivery and ENaC mediate flow regulation of collecting duct endothelin-1 production

2012 ◽  
Vol 302 (10) ◽  
pp. F1325-F1330 ◽  
Author(s):  
Meghana M. Pandit ◽  
Kevin A. Strait ◽  
Toshio Matsuda ◽  
Donald E. Kohan
Keyword(s):  
Plasma Membrane ◽  
Collecting Duct ◽  
Extracellular Fluid ◽  
Na Channel ◽  
Endothelin 1 ◽  
Flow Response ◽  
Mrna Content ◽  
Static Conditions ◽  
Biologic System

Collecting duct (CD) endothelin-1 (ET-1) is an important autocrine inhibitor of Na and water transport. CD ET-1 production is stimulated by extracellular fluid volume expansion and tubule fluid flow, suggesting a mechanism coupling CD Na delivery and ET-1 synthesis. A mouse cortical CD cell line, mpkCCDc14, was subjected to static or flow conditions for 2 h at 2 dyn/cm2, followed by determination of ET-1 mRNA content. Flow with 300 mosmol/l NaCl increased ET-1 mRNA to 65% above that observed under static conditions. Increasing perfusate osmolarity to 450 mosmol/l with NaCl or Na acetate increased ET-1 mRNA to ∼184% compared with no flow, which was not observed when osmolarity was increased using mannitol or urea. Reducing Na concentration to 150 mosmol/l while maintaining total osmolarity at 300 mosmol/l with urea or mannitol decreased the flow response. Inhibition of epithelial Na channel (ENaC) with amiloride or benzamil abolished the flow response, suggesting involvement of ENaC in flow-regulated ET-1 synthesis. Aldosterone almost doubled the flow response. Since Ca2+ enhances CD ET-1 production, the involvement of plasma membrane and mitochondrial Na/Ca2+ exchangers (NCX) was assessed. SEA0400 and KB-R7943, plasma membrane NCX inhibitors, did not affect the flow response. However, CGP37157, a mitochondrial NCX inhibitor, abolished the response. In summary, the current study indicates that increased Na delivery, leading to ENaC-mediated Na entry and mitochondrial NCX activity, is involved in flow-stimulated CD ET-1 synthesis. This constitutes the first report of either ENaC or mitochondrial NCX regulation of an autocrine factor in any biologic system.

EGF-and NGF-stimulated translocation of cytohesin-1 to the plasma membrane of PC12 cells requires PI 3-kinase activation and a functional cytohesin-1 PH domain

1999 ◽  
Vol 112 (12) ◽  
pp. 1957-1965 ◽  
Author(s):  
K. Venkateswarlu ◽  
F. Gunn-Moore ◽  
J.M. Tavare ◽  
P.J. Cullen
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Laser Scanning ◽  
Time Course ◽  
Fluorescent Protein ◽  
Dominant Negative ◽  
Head Group ◽  
Nucleotide Exchange ◽  
Ph Domain

ADP-ribosylation factors (ARFs) are small GTP-binding proteins that function as regulators of eukaryotic vesicle trafficking. Cytohesin-1 is a member of a family of ARF guanine nucleotide-exchange factors that contain a C-terminal pleckstrin homology (PH) domain which has been proposed to bind the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate (PIP3). Here we demonstrate that in vitro, recombinant cytohesin-1 binds, via its PH domain, the inositol head group of PIP3, inositol 1,3,4, 5-tetrakisphosphate (IP4), with an affinity greater than 200-fold higher than the inositol head group of either phosphatidylinositol 4, 5-bisphosphate or phosphatidylinositol 3,4-bisphosphate. Moreover, addition of glycerol or diacetylglycerol to the 1-phosphate of IP4 does not alter the ability to interact with cytohesin-1, data which is entirely consistent with cytohesin-1 functioning as a putative PIP3 receptor. To address whether cytohesin-1 binds PIP3 in vivo, we have expressed a chimera of green fluorescent protein (GFP) fused to the N terminus of cytohesin-1 in PC12 cells. Using laser scanning confocal microscopy we demonstrate that either EGF- or NGF-stimulation of transiently transfected PC12 cells results in a rapid translocation of GFP-cytohesin-1 from the cytosol to the plasma membrane. This translocation is dependent on the cytohesin-1 PH domain and occurs with a time course that parallels the rate of plasma membrane PIP3 production. Furthermore, the translocation requires the ability of either agonist to activate PI 3-kinase, since it is inhibited by wortmannin (100 nM), LY294002 (50 microM) and by coexpression with a dominant negative p85. This data therefore suggests that in vivo cytohesin-1 can interact with PIP3 via its PH domain.

scholarly journals The PH Domain and the Polybasic c Domain of Cytohesin-1 Cooperate specifically in Plasma Membrane Association and Cellular Function

1998 ◽  
Vol 9 (8) ◽  
pp. 1981-1994 ◽  
Author(s):  
Wolfgang Nagel ◽  
Pierre Schilcher ◽  
Lutz Zeitlmann ◽  
Waldemar Kolanus
Keyword(s):  
Plasma Membrane ◽  
Cell Adhesion ◽  
Tyrosine Kinase ◽  
Biological Function ◽  
Common Mechanism ◽  
Membrane Association ◽  
Bruton’S Tyrosine Kinase ◽  
Ph Domain ◽  
Bruton's Tyrosine Kinase ◽  
Ph Domains

Recruitment of intracellular proteins to the plasma membrane is a commonly found requirement for the initiation of signal transduction events. The recently discovered pleckstrin homology (PH) domain, a structurally conserved element found in ∼100 signaling proteins, has been implicated in this function, because some PH domains have been described to be involved in plasma membrane association. Furthermore, several PH domains bind to the phosphoinositides phosphatidylinositol-(4,5)-bisphosphate and phosphatidylinositol-(3,4,5)-trisphosphate in vitro, however, mostly with low affinity. It is unclear how such weak interactions can be responsible for observed membrane binding in vivo as well as the resulting biological phenomena. Here, we investigate the structural and functional requirements for membrane association of cytohesin-1, a recently discovered regulatory protein of T cell adhesion. We demonstrate that both the PH domain and the adjacent carboxyl-terminal polybasic sequence of cytohesin-1 (c domain) are necessary for plasma membrane association and biological function, namely interference with Jurkat cell adhesion to intercellular adhesion molecule 1. Biosensor measurements revealed that phosphatidylinositol-(3,4,5)-trisphosphate binds to the PH domain and c domain together with high affinity (100 nM), whereas the isolated PH domain has a substantially lower affinity (2–3 μM). The cooperativity of both elements appears specific, because a chimeric protein, consisting of the c domain of cytohesin-1 and the PH domain of the β-adrenergic receptor kinase does not associate with membranes, nor does it inhibit adhesion. Moreover, replacement of the c domain of cytohesin-1 with a palmitoylation–isoprenylation motif partially restored the biological function, but the specific targeting to the plasma membrane was not retained. Thus we conclude that two elements of cytohesin-1, the PH domain and the c domain, are required and sufficient for membrane association. This appears to be a common mechanism for plasma membrane targeting of PH domains, because we observed a similar functional cooperativity of the PH domain of Bruton’s tyrosine kinase with the adjacent Bruton’s tyrosine kinase motif, a novel zinc-containing fold.

scholarly journals The CFTR trafficking mutation F508del inhibits the constitutive activity of SLC26A9

2017 ◽  
Vol 312 (6) ◽  
pp. L912-L925 ◽  
Author(s):  
Carol A. Bertrand ◽  
Shalini Mitra ◽  
Sanjay K. Mishra ◽  
Xiaohui Wang ◽  
Yu Zhao ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Family Member ◽  
Pdz Domain ◽  
Protein S ◽  
Constitutive Activity ◽  
Airway Epithelia ◽  
Anion Secretion ◽  
Anion Transporters ◽  
Bronchial Epithelia ◽  
Domain Protein

Several members of the SLC26A family of anion transporters associate with CFTR, forming complexes in which CFTR and SLC26A functions are reciprocally regulated. These associations are thought to be facilitated by PDZ scaffolding interactions. CFTR has been shown to be positively regulated by NHERF-1, and negatively regulated by CAL in airway epithelia. However, it is unclear which PDZ-domain protein(s) interact with SLC26A9, a SLC26A family member found in airway epithelia. We have previously shown that primary, human bronchial epithelia (HBE) from non-CF donors exhibit constitutive anion secretion attributable to SLC26A9. However, constitutive anion secretion is absent in HBE from CF donors. We examined whether changes in SLC26A9 constitutive activity could be attributed to a loss of CFTR trafficking, and what role PDZ interactions played. HEK293 coexpressing SLC26A9 with the trafficking mutant F508del CFTR exhibited a significant reduction in constitutive current compared with cells coexpressing SLC26A9 and wt CFTR. We found that SLC26A9 exhibits complex glycosylation when coexpressed with F508del CFTR, but its expression at the plasma membrane is decreased. SLC26A9 interacted with both NHERF-1 and CAL, and its interaction with both significantly increased with coexpression of wt CFTR. However, coexpression with F508del CFTR only increased SLC26A9’s interaction with CAL. Mutation of SLC26A9’s PDZ motif decreased this association with CAL, and restored its constitutive activity. Correcting aberrant F508del CFTR trafficking in CF HBE with corrector VX-809 also restored SLC26A9 activity. We conclude that when SLC26A9 is coexpressed with F508del CFTR, its trafficking defect leads to a PDZ motif-sensitive intracellular retention of SLC26A9.

scholarly journals Analysis of the roles of phosphatidylinositol-4,5-bisphosphate and individual subunits in assembly, localization, and function of Saccharomyces cerevisiae target of rapamycin complex 2

2019 ◽  
Vol 30 (12) ◽  
pp. 1555-1574 ◽  
Author(s):  
Maria Nieves Martinez Marshall ◽  
Anita Emmerstorfer-Augustin ◽  
Kristin L. Leskoske ◽  
Lydia H. Zhang ◽  
Biyun Li ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Lipid Metabolism ◽  
Eukaryotic Cell ◽  
Target Of Rapamycin ◽  
Multiprotein Complex ◽  
Ph Domain ◽  
Evolutionarily Conserved ◽  
And Function

Eukaryotic cell survival requires maintenance of plasma membrane (PM) homeostasis in response to environmental insults and changes in lipid metabolism. In yeast, a key regulator of PM homeostasis is target of rapamycin (TOR) complex 2 (TORC2), a multiprotein complex containing the evolutionarily conserved TOR protein kinase isoform Tor2. PM localization is essential for TORC2 function. One core TORC2 subunit (Avo1) and two TORC2-­associated regulators (Slm1 and Slm2) contain pleckstrin homology (PH) domains that exhibit specificity for binding phosphatidylinositol-4,5- bisphosphate (PtdIns4,5P2). To investigate the roles of PtdIns4,5P2 and constituent subunits of TORC2, we used auxin-inducible degradation to systematically eliminate these factors and then examined localization, association, and function of the remaining TORC2 components. We found that PtdIns4,5P2 depletion significantly reduced TORC2 activity, yet did not prevent PM localization or cause disassembly of TORC2. Moreover, truncated Avo1 (lacking its C-terminal PH domain) was still recruited to the PM and supported growth. Even when all three PH-containing proteins were absent, the remaining TORC2 subunits were PM-bound. Revealingly, Avo3 localized to the PM independent of both Avo1 and Tor2, whereas both Tor2 and Avo1 required Avo3 for their PM anchoring. Our findings provide new mechanistic information about TORC2 and pinpoint Avo3 as pivotal for TORC2 PM localization and assembly in vivo.

scholarly journals Mechanisms of plasma membrane targeting of formin mDia2 through its amino terminal domains

2011 ◽  
Vol 22 (2) ◽  
pp. 189-201 ◽  
Author(s):  
Roman Gorelik ◽  
Changsong Yang ◽  
Vasumathi Kameswaran ◽  
Roberto Dominguez ◽  
Tatyana Svitkina
Keyword(s):  
Plasma Membrane ◽  
Electrostatic Interactions ◽  
Coiled Coil ◽  
Small Gtpases ◽  
Coincidence Detection ◽  
Membrane Targeting ◽  
Amino Terminal ◽  
N Terminus

The formin mDia2 mediates the formation of lamellipodia and filopodia during cell locomotion. The subcellular localization of activated mDia2 depends on interactions with actin filaments and the plasma membrane. We investigated the poorly understood mechanism of plasma membrane targeting of mDia2 and found that the entire N-terminal region of mDia2 preceding the actin-polymerizing formin homology domains 1 and 2 (FH1–FH2) module was potently targeted to the membrane. This localization was enhanced by Rif, but not by other tested small GTPases, and depended on a positively charged N-terminal basic domain (BD). The BD bound acidic phospholipids in vitro, suggesting that in vivo it may associate with the plasma membrane through electrostatic interactions. Unexpectedly, a fragment consisting of the GTPase-binding region and the diaphanous inhibitory domain (G-DID), thought to mediate the interaction with GTPases, was not targeted to the plasma membrane even in the presence of constitutively active Rif. Addition of the BD or dimerization/coiled coil domains to G-DID rescued plasma membrane targeting in cells. Direct binding of Rif to mDia2 N terminus required the presence of both G and DID. These results suggest that the entire N terminus of mDia2 serves as a coincidence detection module, directing mDia2 to the plasma membrane through interactions with phospholipids and activated Rif.

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