scholarly journals Actin polymerization driven by WASH causes V-ATPase retrieval and vesicle neutralization before exocytosis

2011 ◽  
Vol 193 (5) ◽  
pp. 831-839 ◽  
Author(s):  
Michael Carnell ◽  
Tobias Zech ◽  
Simon D. Calaminus ◽  
Seiji Ura ◽  
Monica Hagedorn ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Dictyostelium Discoideum ◽  
Adenosine Triphosphatase ◽  
Actin Polymerization ◽  
Dominant Negative ◽  
Coated Vesicles ◽  
Filamentous Actin ◽  
Evolutionarily Conserved ◽  
Related Process

WASP and SCAR homologue (WASH) is a recently identified and evolutionarily conserved regulator of actin polymerization. In this paper, we show that WASH coats mature Dictyostelium discoideum lysosomes and is essential for exocytosis of indigestible material. A related process, the expulsion of the lethal endosomal pathogen Cryptococcus neoformans from mammalian macrophages, also uses WASH-coated vesicles, and cells expressing dominant negative WASH mutants inefficiently expel C. neoformans. D. discoideum WASH causes filamentous actin (F-actin) patches to form on lysosomes, leading to the removal of vacuolar adenosine triphosphatase (V-ATPase) and the neutralization of lysosomes to form postlysosomes. Without WASH, no patches or coats are formed, neutral postlysosomes are not seen, and indigestible material such as dextran is not exocytosed. Similar results occur when actin polymerization is blocked with latrunculin. V-ATPases are known to bind avidly to F-actin. Our data imply a new mechanism, actin-mediated sorting, in which WASH and the Arp2/3 complex polymerize actin on vesicles to drive the separation and recycling of proteins such as the V-ATPase.

scholarly journals Characterization of the megakaryocyte demarcation membrane system and its role in thrombopoiesis

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 3868-3875 ◽  
Author(s):  
Harald Schulze ◽  
Manav Korpal ◽  
Jonathan Hurov ◽  
Sang-We Kim ◽  
Jinghang Zhang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Plasma Membranes ◽  
Blood Platelets ◽  
Membrane Surface ◽  
Cell Mass ◽  
Membrane System ◽  
Dominant Negative ◽  
Filamentous Actin ◽  
Local Assembly ◽  
Actin Expression

To produce blood platelets, megakaryocytes elaborate proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. The invaginated demarcation membrane system (DMS), a hallmark of mature cells, has been proposed as the source of proplatelet membranes. By direct visualization of labeled DMS, we demonstrate that this is indeed the case. Late in megakaryocyte ontogeny, the DMS gets loaded with PI-4,5-P2, a phospholipid that is confined to plasma membranes in other cells. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinase α (PIP4Kα), and short hairpin (sh) RNA-mediated loss of PIP4Kα impairs both DMS development and expansion of megakaryocyte size. Thus, PI-4,5-P2 is a marker and possibly essential component of internal membranes. PI-4,5-P2 is known to promote actin polymerization by activating Rho-like GTPases and Wiskott-Aldrich syndrome (WASp) family proteins. Indeed, PI-4,5-P2 in the megakaryocyte DMS associates with filamentous actin. Expression of a dominant-negative N-WASp fragment or pharmacologic inhibition of actin polymerization causes similar arrests in proplatelet formation, acting at a step beyond expansion of the DMS and cell mass. These observations collectively suggest a signaling pathway wherein PI-4,5-P2 might facilitate DMS development and local assembly of actin fibers in preparation for platelet biogenesis.

Rho and Rac promote acinar morphological changes, actin reorganization, and amylase secretion

2005 ◽  
Vol 289 (3) ◽  
pp. G561-G570 ◽  
Author(s):  
Yan Bi ◽  
Sophie Le Page ◽  
John A. Williams
Keyword(s):  
Actin Polymerization ◽  
Morphological Changes ◽  
Dominant Negative ◽  
Amylase Secretion ◽  
Filamentous Actin ◽  
Pancreatic Acini ◽  
Actin Reorganization ◽  
Supramaximal Stimulation ◽  
Rho Family ◽  
High Concentrations

Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.

Phosphatidyl Inositol (4,5)P2 Marks Megakaryocyte Internal Membranes and Is Associated with Megakaryocyte Maturation and Platelet Release.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 732-732
Author(s):  
Harald Schulze ◽  
Manav Korpal ◽  
Jonathan Hurov ◽  
Sang-We Kim ◽  
Jinghang Zhang ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Blood Platelets ◽  
Membrane Surface ◽  
Small Gtpases ◽  
Dominant Negative ◽  
Pleckstrin Homology Domain ◽  
Acceptor Site ◽  
Cell Periphery ◽  
Filamentous Actin ◽  
Fluorescence Protein

Abstract To produce blood platelets, the megakaryocyte (MK) cytoplasm elaborates proplatelets, accompanied by expansion of membrane surface area and dramatic cytoskeletal rearrangements. Invaginated demarcation membranes (DMS) are thought to be the source for the proplatelet and platelet membranes, however, they have THUS far BEEN INSUFFICIENTLY characterized. We first used a mouse model where the cDNA encoding enhanced yellow fluorescence protein (EYFP) with a C-terminally introduced myristoyl acceptor site has been introduced into the GPIIb locus. Heterozygous knock-in mice reveal yellow fluroescent MKs with an internal staining pattern that resembles the reticiulated pattern of the DMS as found in micrographs. Proplatelet-forming MKs reveal contiguous membrane connection between the internally stained membranes and the outlines of the proplatelet shaft resulting in production of fluorescent platelets. We next sought to characterize the internal membranes biochemically and retrovirally infected MKs to express the green fluorescence protein (EGFP) tagged with the pleckstrin homology domain of phospholipase Cδ1 (PLCδ1) which binds with high specificity to phosphatidylinositol(4,5)P2 (PIP2). Young MKs stain the cell periphery as described for most other cell types. Mature MKs, however, stain the internal membranes, whereas the plasma membrane becomes PIP2-negative as shown by co-staining with CD41. Proplatelet membranes emanate from these internal PIP2-positive membranes, proving that the DMS is indeed the membrane reservoir during platelet biogenesis. Appearance of PI-4,5-P2 in the DMS occurs in proximity to PI-5-P-4-kinaseα (PI4Kα), a protein highly expressed in MKs and platelets, as shown by overexpressing EGFP-tagged kinase in primary MKs. In addition, shRNA-mediated loss of PIP4Kα or depletion of its presumptive substrate block DMS development and expansion of MK size. Thus, PI-4,5-P2 is a marker and essential component of internal membranes and is most likely introduced about the non-canonical pathway using PI5P as the substrate. PI-4,5-P2 promotes actin polymerization by activating small GTPases from the Rac/Rho superfamily as well as Wiskott-Aldrich Syndrome (WASp) family proteins. Indeed, PIP2 is associated with filamentous actin when MKs are co-stained with phalloidin. Expression of a dominant-negative N-WASp C-terminal fragment (CA-domain) that inactivats all WASp/WAVE family members leads to Arp3 binding without assembling the complete Arp2/3 complex, thus inhibiting actin filament nucleation. F-Actin staining in the infected MKs reveals a pattern similar to that of MKs treated with pharmacologic dosage of actin polymerization-antagonists like cytochalasin D, which disrupts actin filaments and inhibits proplatelet formation when administered early in MK culture. Dominant-negative WASp impairs proplatelet elaboration similarly, acting at a step past expansion of the cell volume. These observations implicate a signaling pathway wherein PI-4,5-P2 facilitates DMS development and suggests a pathway that links a DMS lipid marker with local assembly of actin fibers as a requirement for platelet biogenesis.

scholarly journals HEM1 deficiency disrupts mTORC2 and F-actin control in inherited immunodysregulatory disease

Science ◽  
2020 ◽  
Vol 369 (6500) ◽  
pp. 202-207 ◽  
Author(s):  
Sarah A. Cook ◽  
William A. Comrie ◽  
M. Cecilia Poli ◽  
Morgan Similuk ◽  
Andrew J. Oler ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Synapse Formation ◽  
Immune Cell ◽  
Filamentous Actin ◽  
Cortical Actin ◽  
Akt Phosphorylation ◽  
Actin Networks ◽  
Evolutionarily Conserved ◽  
Regulatory Complex ◽  
Immune Cell Migration

Immunodeficiency often coincides with hyperactive immune disorders such as autoimmunity, lymphoproliferation, or atopy, but this coincidence is rarely understood on a molecular level. We describe five patients from four families with immunodeficiency coupled with atopy, lymphoproliferation, and cytokine overproduction harboring mutations in NCKAP1L, which encodes the hematopoietic-specific HEM1 protein. These mutations cause the loss of the HEM1 protein and the WAVE regulatory complex (WRC) or disrupt binding to the WRC regulator, Arf1, thereby impairing actin polymerization, synapse formation, and immune cell migration. Diminished cortical actin networks caused by WRC loss led to uncontrolled cytokine release and immune hyperresponsiveness. HEM1 loss also blocked mechanistic target of rapamycin complex 2 (mTORC2)–dependent AKT phosphorylation, T cell proliferation, and selected effector functions, leading to immunodeficiency. Thus, the evolutionarily conserved HEM1 protein simultaneously regulates filamentous actin (F-actin) and mTORC2 signaling to achieve equipoise in immune responses.

scholarly journals A developmentally regulated Na-H exchanger in Dictyostelium discoideum is necessary for cell polarity during chemotaxis

2005 ◽  
Vol 169 (2) ◽  
pp. 321-329 ◽  
Author(s):  
Hitesh Patel ◽  
Diane L. Barber
Keyword(s):  
Dictyostelium Discoideum ◽  
Intracellular Ph ◽  
Actin Polymerization ◽  
De Novo ◽  
Cell Movement ◽  
Leading Edge ◽  
Ph Homeostasis ◽  
Developmentally Regulated ◽  
Evolutionarily Conserved ◽  
Morphological Polarity

Increased intracellular H+ efflux is speculated to be an evolutionarily conserved mechanism necessary for rapid assembly of cytoskeletal filaments and for morphological polarity during cell motility. In Dictyostelium discoideum, increased intracellular pH through undefined transport mechanisms plays a key role in directed cell movement. We report that a developmentally regulated Na-H exchanger in Dictyostelium discoideum (DdNHE1) localizes to the leading edge of polarized cells and is necessary for intracellular pH homeostasis and for efficient chemotaxis. Starved DdNHE1-null cells (Ddnhe1−) differentiate, and in response to the chemoattractant cAMP they retain directional sensing; however, they cannot attain a polarized morphology, but instead extend mislocalized pseudopodia around the cell and exhibit decreased velocity. Consistent with impaired polarity, in response to chemoattractant, Ddnhe1− cells lack a leading edge localization of F-actin and have significantly attenuated de novo F-actin polymerization but increased abundance of membrane-associated phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3). These findings indicate that during chemotaxis DdNHE1 is necessary for establishing the kinetics of actin polymerization and PI(3,4,5)P3 production and for attaining a polarized phenotype.

scholarly journals Dynamic cortical actin remodeling by ERM proteins controls BCR microcluster organization and integrity

2011 ◽  
Vol 208 (5) ◽  
pp. 1055-1068 ◽  
Author(s):  
Bebhinn Treanor ◽  
David Depoil ◽  
Andreas Bruckbauer ◽  
Facundo D. Batista
Keyword(s):  
Actin Cytoskeleton ◽  
B Cell ◽  
Protein Function ◽  
Actin Polymerization ◽  
Lymphocyte Activation ◽  
Dominant Negative ◽  
Temporary Increase ◽  
Cortical Actin ◽  
Erm Proteins ◽  
Diffusion Dynamics

Signaling microclusters are a common feature of lymphocyte activation. However, the mechanisms controlling the size and organization of these discrete structures are poorly understood. The Ezrin-Radixin-Moesin (ERM) proteins, which link plasma membrane proteins with the actin cytoskeleton and regulate the steady-state diffusion dynamics of the B cell receptor (BCR), are transiently dephosphorylated upon antigen receptor stimulation. In this study, we show that the ERM proteins ezrin and moesin influence the organization and integrity of BCR microclusters. BCR-driven inactivation of ERM proteins is accompanied by a temporary increase in BCR diffusion, followed by BCR immobilization. Disruption of ERM protein function using dominant-negative or constitutively active ezrin constructs or knockdown of ezrin and moesin expression quantitatively and qualitatively alters BCR microcluster formation, antigen aggregation, and downstream BCR signal transduction. Chemical inhibition of actin polymerization also altered the structure and integrity of BCR microclusters. Together, these findings highlight a crucial role for the cortical actin cytoskeleton during B cell spreading and microcluster formation and function.

scholarly journals Disruption of inositol biosynthesis through targeted mutagenesis in Dictyostelium discoideum: generation and characterization of inositol-auxotrophic mutants

2006 ◽  
Vol 397 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Andreas Fischbach ◽  
Stephan Adelt ◽  
Alexander Müller ◽  
Günter Vogel
Keyword(s):  
Dictyostelium Discoideum ◽  
Genetic Manipulation ◽  
Inositol Phosphate ◽  
Targeted Mutagenesis ◽  
Inositol Polyphosphate ◽  
Fluid Medium ◽  
Physiological Processes ◽  
Evolutionarily Conserved ◽  
Cellular Slime

myo-Inositol and its downstream metabolites participate in diverse physiological processes. Nevertheless, considering their variety, it is likely that additional roles are yet to be uncovered. Biosynthesis of myo-inositol takes place via an evolutionarily conserved metabolic pathway and is strictly dependent on inositol-3-phosphate synthase (EC 5.5.1.4). Genetic manipulation of this enzyme will disrupt the cellular inositol supply. Two methods, based on gene deletion and antisense strategy, were used to generate mutants of the cellular slime mould Dictyostelium discoideum. These mutants are inositol-auxotrophic and show phenotypic changes under inositol starvation. One remarkable attribute is their inability to live by phagocytosis of bacteria, which is the exclusive nutrient source in their natural environment. Cultivated on fluid medium, the mutants lose their viability when deprived of inositol for longer than 24 h. Here, we report a study of the alterations in the first 24 h in cellular inositol, inositol phosphate and phosphoinositide concentrations, whereby a rapidly accumulating phosphorylated compound was detected. After its identification as 2,3-BPG (2,3-bisphosphoglycerate), evidence could be found that the internal disturbances of inositol homoeostasis trigger the accumulation. In a first attempt to characterize this as a physiologically relevant response, the efficient in vitro inhibition of a D. discoideum inositol-polyphosphate 5-phosphatase (EC 3.1.3.56) by 2,3-BPG is presented.

scholarly journals Evidence that coated vesicles isolated from brain are calcium-sequestering organelles resembling sarcoplasmic reticulum.

1977 ◽  
Vol 75 (1) ◽  
pp. 135-147 ◽  
Author(s):  
A L Blitz ◽  
R E Fine ◽  
P A Toselli
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Calcium Ions ◽  
Adenosine Triphosphatase ◽  
Sodium Dodecyl ◽  
Maximal Activity ◽  
Coated Vesicles ◽  
Potassium Oxalate ◽  
Triton X ◽  
Trapping Agent ◽  
The Brain

Coated vesicles from the brain have been purified to near morphological homogeneity by a modification of the method of Pearse. These vesicles resemble sarcoplasmic reticulum fragments isolated from skeletal muscle. They contain proteins with 100,000- and 55,000-dalton mol wt which co-migrate on polyacrylamide gels, in the presence of sodium dodecyl sulfate, with the two major proteins of the sarcoplasmic reticulum fragment. These vesicles contain adenosine triphosphatase (ATPase) activity which is stimulated by calcium ions in the presence of Triton X-100 (Rohm & Haas Co., Philadelphia, Pa.), displaying maximal activity at 8 x 10(-7) M Ca ++. They take up calcium ions from the medium, and this uptake is stimulated by ATP and by potassium oxalate, a calcium-trapping agent. The 100,000-dalton protein of the coated vesicles displays immunological reactivity with an antiserum directed against the 100,000-dalton, calcium-stimulated ATPase of the sarcoplasmic reticulum. As with the sarcoplasmic reticulum fragment, this protein becomes radiolabeled when coated vesicles are briefly incubated with gamma-labeled [32P]ATP. The possible functions of coated vesicles as calcium-sequestering organelles are discussed.

scholarly journals Atypical Protein Kinase C Is Involved in the Evolutionarily Conserved Par Protein Complex and Plays a Critical Role in Establishing Epithelia-Specific Junctional Structures

2001 ◽  
Vol 152 (6) ◽  
pp. 1183-1196 ◽  
Author(s):  
Atsushi Suzuki ◽  
Tomoyuki Yamanaka ◽  
Tomonori Hirose ◽  
Naoyuki Manabe ◽  
Keiko Mizuno ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Cell Polarity ◽  
Protein Complex ◽  
Mdck Cells ◽  
Critical Role ◽  
Dominant Negative ◽  
Dominant Negative Mutant ◽  
Surface Polarity ◽  
C Elegans ◽  
Evolutionarily Conserved

We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607–3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S. Ohno. 1998. J. Cell Biol. 143:95–106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here, we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or nonionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+,K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6), and thereby mediates the formation of an aPKC-ASIP/PAR-3–PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

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