scholarly journals Mammalian inositol polyphosphate 5-phosphatase II can compensate for the absence of all three yeast Sac1-like-domain-containing 5-phosphatases

2001 ◽  
Vol 355 (3) ◽  
pp. 805-817 ◽  
Author(s):  
Cindy J. O'MALLEY ◽  
Brad K. McCOLL ◽  
Anne M. KONG ◽  
Sarah L. ELLIS ◽  
A. Primrose W. WIJAYARATNAM ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Inducible Promoter ◽  
Actin Binding ◽  
Inositol Polyphosphate ◽  
Null Mutant ◽  
Phosphatase Domain ◽  
Signalling Molecules ◽  
Yeast Strains ◽  
Bud Site

Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] plays a complex role in generating intracellular signalling molecules, and also in regulating actin-binding proteins, vesicular trafficking and vacuolar fusion. Four inositol polyphosphate 5-phosphatases (hereafter called 5-phosphatases) have been identified in Saccharomyces cerevisiae: Inp51p, Inp52p, Inp53p and Inp54p. Each enzyme contains a 5-phosphatase domain which hydrolyses PtdIns(4,5)P2, forming PtdIns4P, while Inp52p and Inp53p also express a polyphosphoinositide phosphatase domain within the Sac1-like domain. Disruption of any two yeast 5-phosphatases containing a Sac1-like domain results in abnormalities in actin polymerization, plasma membrane, vacuolar morphology and bud-site selection. Triple null mutant 5-phosphatase strains are non-viable. To investigate the role of PtdIns(4,5)P2 in mediating the phenotype of double and triple 5-phosphatase null mutant yeast, we determined whether a mammalian PtdIns(4,5)P2 5-phosphatase, 5-phosphatase II, which lacks polyphosphoinositide phosphatase activity, could correct the phenotype of triple 5-phosphatase null mutant yeast and restore cellular PtdIns(4,5)P2 levels to near basal values. Mammalian 5-phosphatase II expressed under an inducible promoter corrected the growth, cell wall, vacuolar and actin polymerization defects of the triple 5-phosphatase null mutant yeast strains. Cellular PtdIns(4,5)P2 levels in various 5-phosphatase double null mutant strains demonstrated significant accumulation (4.5-, 3- and 2-fold for ∆inp51∆inp53, ∆inp51∆inp52 and ∆inp52∆inp53 double null mutants respectively), which was corrected significantly following 5-phosphatase II expression. Collectively, these studies demonstrate the functional and cellular consequences of PtdIns(4,5)P2 accumulation and the evolutionary conservation of function between mammalian and yeast PtdIns(4,5)P2 5-phosphatases.

scholarly journals The WASP Homologue Las17 Activates the Novel Actin-regulatory Activity of Ysc84 to Promote Endocytosis in Yeast

2009 ◽  
Vol 20 (6) ◽  
pp. 1618-1628 ◽  
Author(s):  
Alastair S. Robertson ◽  
Ellen G. Allwood ◽  
Adam P.C. Smith ◽  
Fiona C. Gardiner ◽  
Rosaria Costa ◽  
...  
Keyword(s):  
Membrane Trafficking ◽  
Actin Polymerization ◽  
Actin Binding ◽  
The Novel ◽  
Cellular Processes ◽  
New Class ◽  
Actin Binding Domain ◽  
Kinetics Of ◽  
Actin Binding Site

Actin plays an essential role in many eukaryotic cellular processes, including motility, generation of polarity, and membrane trafficking. Actin function in these roles is regulated by association with proteins that affect its polymerization state, dynamics, and organization. Numerous proteins have been shown to localize with cortical patches of yeast actin during endocytosis, but the role of many of these proteins remains poorly understood. Here, we reveal that the yeast protein Ysc84 represents a new class of actin-binding proteins, conserved from yeast to humans. It contains a novel N-terminal actin-binding domain termed Ysc84 actin binding (YAB), which can bind and bundle actin filaments. Intriguingly, full-length Ysc84 alone does not bind to actin, but binding can be activated by a specific motif within the polyproline region of the yeast WASP homologue Las17. We also identify a new monomeric actin-binding site on Las17. Together, the polyproline region of Las17 and Ysc84 can promote actin polymerization. Using live cell imaging, kinetics of assembly and disassembly of proteins at the endocytic site were analyzed and reveal that loss of Ysc84 and its homologue Lsb3 decrease inward movement of vesicles consistent with a role in actin polymerization during endocytosis.

Aldolase-localization in cultured cells: Cell-type and substrate-specific regulation of cytoskeletal associations

2001 ◽  
Vol 79 (6) ◽  
pp. 719-728 ◽  
Author(s):  
Ralf Schindler ◽  
Elke Weichselsdorfer ◽  
Oliver Wagner ◽  
Jürgen Bereiter-Hahn
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filaments ◽  
Actin Polymerization ◽  
Cultured Cells ◽  
Actin Binding ◽  
Permeabilized Cells ◽  
Specific Regulation ◽  
Actin Fibrils ◽  
Fructose 1,6 Bisphosphate

The role of aldolase as a true F- and G-actin binding protein, including modulating actin polymerization, initiating bundling, and giving rise to supramolecular structures that emanate from actin fibrils, has been established using indirect immunofluorescence, permeabilization of XTH-2 cells and keratocytes, and microinjection of fluorescence-labeled aldolase. In addition, binding to intermediate filaments, vimentin, and cytokeratins has been demonstrated. In permeabilized cells in the presence of fructose-1,6-bisphosphate (20–2000 µM) aldolase shifts from association with actin fibres to intermediate filaments. Plenty of free binding sites on microtubules have been revealed by addition of fluorochromed aldolase derived from rabbit skeletal muscle. However, endogenous aldolase was never found associated with microtubules. Differences in actin polymerization in the presence of aldolase as revealed by pyrene-labeled actin fluorimetry and viscosimetry were explained by electron microscopy showing the formation of rod-like structures (10 nm wide, 20–60 nm in length) by association of aldolase with G-actin, which prevents further polymerization. Upon the addition of fructose-1,6-bisphosphate, G-actin–aldolase mixture polymerizes to a higher viscosity and forms stiffer filaments than pure actin of the same concentration.Key words: aldolase, cytoskeleton, electron microscopy, viscosimetry.

scholarly journals A33 THE ROLE OF A HOMOZYGOUS PROTEIN CODING VARIANT OF EPS8 IN THE PATHOGENESIS OF PEDIATRIC IBD

2020 ◽  
Vol 3 (Supplement_1) ◽  
pp. 40-41
Author(s):  
K Long ◽  
N Warner ◽  
J Pan ◽  
C Guo ◽  
A Muise
Keyword(s):  
Early Onset ◽  
Actin Polymerization ◽  
Cell Structure ◽  
Single Gene ◽  
Actin Binding ◽  
Western Blots ◽  
Very Early Onset Ibd ◽  
Normal Controls ◽  
Pediatric Ibd

Abstract Background The rate of onset for Inflammatory Bowel Disease (IBD) is rising worldwide—most significantly in the pediatric population. The pathogenesis of the disease involves a complicated interaction between the environment and genetics. Recent findings suggest that there is a broad range of rare, single-gene mutations that correlate with the IBD phenotype in children. Some of these single-gene defects can disrupt the epithelial barrier, which alters intestinal immune homeostasis. Epidermal Growth Factor Receptor Kinase Substrate 8 (EPS8) is crucial for the stabilization of F-actin by capping and bundling the barbed end. Previous literature shows that loss of expression of EPS8 disrupts the polymerization of F-actin in intestinal microvilli resulting in shortened brush borders. We identified a pediatric IBD patient with a homozygous missense EPS8 mutation(c.2099C>T, I700T) in the actin-binding domain. The patient presented with pancolitis, colonic strictures and other IBD-like symptoms at 6 weeks of age. Currently, the functional role of EPS8 in the pathogenesis of very early-onset IBD remains elusive. Aims 1)To investigate if the patient with EPS8 mutation has microvilli disorganization. 2)To determine EPS8 localization in patient samples and cell models. 3)To clarify the mechanism of how the EPS8 mutation affects F-actin in vitro. Ultimately, we want to clarify how the mutation of EPS8 might contribute to the onset of pediatric IBD. Methods A rare, damaging mutation in EPS8 was identified in a very early-onset IBD patient by Whole Exome Sequencing. From patient-derived colon biopsy samples, the localization of EPS8 and actin was visualized by immunofluorescence (IF) microscopy. The morphology of the patient’s intestinal microvilli was assessed using transmission electron microscopy (TEM). EPS8 expression was measured using western blot. An F-actin polymerization assay was performed comparing purified WT and mutant EPS8 protein to assess the rate of polymerization from monomeric G-actin to F-actin. Results IF microscopy of the colonic sections revealed lower co-localization of EPS8 and actin on the apical surfaces, compared to IBD and normal controls. Closer examination of the cell structure using TEM showed disruption of the microvilli in the EPS8 mutant, but not in IBD or normal controls. Western blots showed no differences in protein expression between wildtype and mutant EPS8 in HEK293T cells. F-actin polymerization assay revealed differences in the rate of G-actin to F-actin polymerization between wildtype and mutant EPS8. Conclusions The patient with EPS8 mutation had microvilli disorganization. EPS8 localized differently in the patient colon tissues compared to normal and IBD control samples. The EPS8 mutation may affect F-actin polymerization, which may lead to disruption of the microvilli. Funding Agencies CIHR

scholarly journals The Yeast Inositol Polyphosphate 5-Phosphatases Inp52p and Inp53p Translocate to Actin Patches following Hyperosmotic Stress: Mechanism for Regulating Phosphatidylinositol 4,5-Bisphosphate at Plasma Membrane Invaginations

2000 ◽  
Vol 20 (24) ◽  
pp. 9376-9390 ◽  
Author(s):  
Lisa M. Ooms ◽  
Brad K. McColl ◽  
Fenny Wiradjaja ◽  
A. P. W. Wijayaratnam ◽  
Paul Gleeson ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Actin Polymerization ◽  
Fluorescent Protein ◽  
Hyperosmotic Stress ◽  
Resting Cells ◽  
Inositol Polyphosphate ◽  
Rhodamine Phalloidin ◽  
Phosphatase Domain ◽  
Rich Domain ◽  
Green Fluorescent

ABSTRACT The Saccharomyces cerevisiae inositol polyphosphate 5-phosphatases (Inp51p, Inp52p, and Inp53p) each contain an N-terminal Sac1 domain, followed by a 5-phosphatase domain and a C-terminal proline-rich domain. Disruption of any two of these 5-phosphatases results in abnormal vacuolar and plasma membrane morphology. We have cloned and characterized the Sac1-containing 5-phosphatases Inp52p and Inp53p. Purified recombinant Inp52p lacking the Sac1 domain hydrolyzed phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] and PtdIns(3,5)P2. Inp52p and Inp53p were expressed in yeast as N-terminal fusion proteins with green fluorescent protein (GFP). In resting cells recombinant GFP-tagged 5-phosphatases were expressed diffusely throughout the cell but were excluded from the nucleus. Following hyperosmotic stress the GFP-tagged 5-phosphatases rapidly and transiently associated with actin patches, independent of actin, in both the mother and daughter cells of budding yeast as demonstrated by colocalization with rhodamine phalloidin. Both the Sac1 domain and proline-rich domains were able to independently mediate translocation of Inp52p to actin patches, following hyperosmotic stress, while the Inp53p proline-rich domain alone was sufficient for stress-mediated localization. Overexpression of Inp52p or Inp53p, but not catalytically inactive Inp52p, which lacked PtdIns(4,5)P2 5-phosphatase activity, resulted in a dramatic reduction in the repolarization time of actin patches following hyperosmotic stress. We propose that the osmotic-stress-induced translocation of Inp52p and Inp53p results in the localized regulation of PtdIns(3,5)P2 and PtdIns(4,5)P2 at actin patches and associated plasma membrane invaginations. This may provide a mechanism for regulating actin polymerization and cell growth as an acute adaptive response to hyperosmotic stress.

The Actin Regulator Coronin-1A Modulates Platelet Shape Change and Consolidates Arterial Thrombosis

2018 ◽  
Vol 118 (12) ◽  
pp. 2098-2111 ◽  
Author(s):  
Thomas Stocker ◽  
Joachim Pircher ◽  
Artid Skenderi ◽  
Andreas Ehrlich ◽  
Clemens Eberle ◽  
...  
Keyword(s):  
Arterial Thrombosis ◽  
Actin Polymerization ◽  
Thrombus Formation ◽  
Shape Change ◽  
Actin Binding ◽  
Cellular Processes ◽  
Reduced Density ◽  
Induced Aggregation ◽  
Platelet Shape

AbstractCoronin-1A (Coro1A) belongs to a family of highly conserved actin-binding proteins that regulate cytoskeletal re-arrangement. In mammalians, Coro1A expression is most abundant in the haematopoietic lineage, where it regulates various cellular processes. The role of Coro1A in platelets has been previously unknown. Here, we identified Coro1A in human and mouse platelets. Genetic absence of Coro1A in mouse platelets inhibited agonist-induced actin polymerization and altered cofilin phosphoregulation, leading to a reduction in spreading and low-dose collagen induced aggregation. Furthermore, Coro1A-deficient mice displayed a defect in ferric chloride-induced arterial thrombosis with prolonged thrombus formation and reduced thrombus size. Immunofluorescence analysis revealed a less compact thrombus structure with reduced density of platelets and fibrinogen. In summary, Coro1A has a role in platelet biology with impact on spreading, aggregation and thrombosis.

scholarly journals The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

2001 ◽  
Vol 155 (6) ◽  
pp. 1065-1080 ◽  
Author(s):  
Jennifer M. Dyson ◽  
Cindy J. O'Malley ◽  
Jelena Becanovic ◽  
Adam D. Munday ◽  
Michael C. Berndt ◽  
...  
Keyword(s):  
Growth Factor ◽  
Striated Muscle ◽  
Leading Edge ◽  
Sh2 Domain ◽  
Actin Binding ◽  
Inositol Polyphosphate ◽  
Filamin A ◽  
Phosphatase Domain ◽  
Membrane Ruffling ◽  
Rich Domain

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton.

scholarly journals Adenylyl Cyclase G Is Activated by an Intramolecular Osmosensor

2004 ◽  
Vol 15 (3) ◽  
pp. 1479-1486 ◽  
Author(s):  
Shweta Saran ◽  
Pauline Schaap
Keyword(s):  
Adenylyl Cyclase ◽  
Catalytic Domain ◽  
Inducible Promoter ◽  
Null Mutant ◽  
Camp Production ◽  
Size Fractionation ◽  
Catalytic Domains ◽  
High Osmolality ◽  
Uv Induction

Adenylyl cyclase G (ACG) is activated by high osmolality and mediates inhibition of spore germination by this stress factor. The catalytic domains of all eukaryote cyclases are active as dimers and dimerization often mediates activation. To investigate the role of dimerization in ACG activation, we coexpressed ACG with an ACG construct that lacked the catalytic domain (ACGΔcat) and was driven by a UV-inducible promoter. After UV induction of ACGΔcat, cAMP production by ACG was strongly inhibited, but osmostimulation was not reduced. Size fractionation of native ACG showed that dimers were formed between ACG molecules and between ACG and ACGΔcat. However, high osmolality did not alter the dimer/monomer ratio. This indicates that ACG activity requires dimerization via a region outside the catalytic domain but that dimer formation does not mediate activation by high osmolality. To establish whether ACG required auxiliary sensors for osmostimulation, we expressed ACG cDNA in a yeast adenylyl cyclase null mutant. In yeast, cAMP production by ACG was similarly activated by high osmolality as in Dictyostelium. This strongly suggests that the ACG osmosensor is intramolecular, which would define ACG as the first characterized primary osmosensor in eukaryotes.

scholarly journals The role of the Arp2/3 complex in shaping the dynamics and structures of branched actomyosin networks

2020 ◽  
Vol 117 (20) ◽  
pp. 10825-10831 ◽  
Author(s):  
James Liman ◽  
Carlos Bueno ◽  
Yossi Eliaz ◽  
Nicholas P. Schafer ◽  
M. Neal Waxham ◽  
...  
Keyword(s):  
Actin Polymerization ◽  
Active Networks ◽  
Actin Dynamics ◽  
Actin Binding ◽  
Marginal Stability ◽  
Actin Assembly ◽  
Heterogeneous Distribution ◽  
Energy Consuming ◽  
Far From Equilibrium

Actomyosin networks give cells the ability to move and divide. These networks contract and expand while being driven by active energy-consuming processes such as motor protein walking and actin polymerization. Actin dynamics is also regulated by actin-binding proteins, such as the actin-related protein 2/3 (Arp2/3) complex. This complex generates branched filaments, thereby changing the overall organization of the network. In this work, the spatiotemporal patterns of dynamical actin assembly accompanying the branching-induced reorganization caused by Arp2/3 were studied using a computational model (mechanochemical dynamics of active networks [MEDYAN]); this model simulates actomyosin network dynamics as a result of chemical reactions whose rates are modulated by rapid mechanical equilibration. We show that branched actomyosin networks relax significantly more slowly than do unbranched networks. Also, branched networks undergo rare convulsive movements, “avalanches,” that release strain in the network. These avalanches are associated with the more heterogeneous distribution of mechanically linked filaments displayed by branched networks. These far-from-equilibrium events arising from the marginal stability of growing actomyosin networks provide a possible mechanism of the “cytoquakes” recently seen in experiments.

Current Study of RhoA and Associated Signaling Pathways in Gastric Cancer

2020 ◽  
Vol 15 (7) ◽  
pp. 607-613 ◽  
Author(s):  
Haiping Liu ◽  
Yiqian Liu ◽  
Xiaochuan Zhang ◽  
Xiaodong Wang
Keyword(s):  
Gastric Cancer ◽  
Survival Rate ◽  
Signaling Pathways ◽  
Tumor Cells ◽  
Actin Polymerization ◽  
Cell Transformation ◽  
Cell Movement ◽  
Invasion And Metastasis ◽  
Common Cancer

Gastric cancer (GC) is the fourth-most common cancer in the world, with an estimated 1.034 million new cases in 2015, and the third-highest cause of cancer deaths, estimated at 785,558, in 2014. Early diagnosis and treatment greatly affect the survival rate in patients with GC: the 5‐year survival rate of early GC reaches 90%‐95%, while the mortality rate significantly increases if GC develops to the late stage. Recently, studies for the role of RhoA in the diseases have become a hot topic, especially in the development of tumors. A study found that RhoA can regulate actin polymerization, cell adhesion, motor-myosin, cell transformation, and the ability to participate in the activities of cell movement, proliferation, migration, which are closely related to the invasion and metastasis of tumor cells. However, the specific role of RhoA in tumor cells remains to be studied. Therefore, our current study aimed to briefly review the role of RhoA in GC, especially for its associated signaling pathways involved in the GC progression.

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