scholarly journals Concentric zones of active RhoA and Cdc42 around single cell wounds

2005 ◽  
Vol 168 (3) ◽  
pp. 429-439 ◽  
Author(s):  
Hélène A. Benink ◽  
William M. Bement
Keyword(s):  
Single Cell ◽  
Rho Gtpases ◽  
Actin Filaments ◽  
Xenopus Oocytes ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Normal Zone ◽  
And Function ◽  
Distinct Features ◽  
Dependent Processes

Rho GTPases control many cytoskeleton-dependent processes, but how they regulate spatially distinct features of cytoskeletal function within a single cell is poorly understood. Here, we studied active RhoA and Cdc42 in wounded Xenopus oocytes, which assemble and close a dynamic ring of actin filaments (F-actin) and myosin-2 around wound sites. RhoA and Cdc42 are rapidly activated around wound sites in a calcium-dependent manner and segregate into distinct, concentric zones around the wound, with active Cdc42 in the approximate middle of the F-actin array and active RhoA on the interior of the array. These zones form before F-actin accumulation, and then move in concert with the closing array. Microtubules and F-actin are required for normal zone organization and dynamics, as is crosstalk between RhoA and Cdc42. Each of the zones makes distinct contributions to the organization and function of the actomyosin wound array. We propose that similar rho activity zones control related processes such as cytokinesis.

scholarly journals Modeling the roles of protein kinase Cβ and η in single-cell wound repair

2015 ◽  
Vol 26 (22) ◽  
pp. 4100-4108 ◽  
Author(s):  
William R. Holmes ◽  
Laura Liao ◽  
William Bement ◽  
Leah Edelstein-Keshet
Keyword(s):  
Mathematical Model ◽  
Protein Kinase ◽  
Single Cell ◽  
Protein Kinases ◽  
Rho Gtpases ◽  
Actin Filaments ◽  
Wound Repair ◽  
Xenopus Oocytes ◽  
Rho Gtpase ◽  
Characteristic Manner

Wounded cells such as Xenopus oocytes respond to damage by assembly and closure of an array of actin filaments and myosin-2 controlled by Rho GTPases, including Rho and Cdc42. Rho and Cdc42 are patterned around wounds in a characteristic manner, with active Rho concentrating in a ring-like zone inside a larger, ring-like zone of active Cdc42. How this patterning is achieved is unknown, but Rho and Cdc42 at wounds are subject to regulation by other proteins, including the protein kinases C. Specifically, Cdc42 and Rho activity are enhanced by PKCβ and inhibited by PKCη. We adapt a mathematical model of Simon and coworkers to probe the possible roles of these kinases. We show that PKCβ likely affects the magnitude of positive Rho–Abr feedback, whereas PKCη acts on Cdc42 inactivation. The model explains both qualitative and some overall quantitative features of PKC–Rho GTPase regulation. It also accounts for the previous, peculiar observation that ∼20% of cells overexpressing PKCη display zone inversions—that is, displacement of active Rho to the outside of the active Cdc42.

scholarly journals The expression of calpain 1 and calpain 2 in spermatogenic cells and spermatozoa of the mouse

Reproduction ◽  
2005 ◽  
Vol 129 (4) ◽  
pp. 435-442 ◽  
Author(s):  
Irit Ben-Aharon ◽  
Paula R Brown ◽  
Nir Etkovitz ◽  
Edward M Eddy ◽  
Ruth Shalgi
Keyword(s):  
Acrosome Reaction ◽  
Cysteine Proteases ◽  
Calpain Inhibitor ◽  
Dependent Manner ◽  
Spermatogenic Cells ◽  
Calcium Dependent ◽  
Protein Levels ◽  
Complex Process ◽  
And Function ◽  
Mitosis And Meiosis

There is some evidence suggesting that Ca2+is involved in processes that occur during the development and function of spermatozoa. Calcium-dependent proteins, such as calmodulin, are expressed during mammalian spermatogenesis further suggesting that Ca2+takes part in its regulation. However, the precise roles of Ca2+in spermatogenesis remain to be elucidated. Calpains are a family of Ca2+-dependent cysteine proteases whose members are expressed ubiquitously or in a tissue-specific manner. Calpain has been demonstrated to mediate specific Ca2+-dependent processes including cell fusion, mitosis and meiosis. We herein followed the expression pattern of calpain’s ubiquitous isoforms, 1 and 2, throughout spermatogenesis at the RNA and protein levels by RT-PCR and Western blotting analysis. Both RNA and protein studies revealed that these isoforms are expressed in all spermatogenic cells. The expression of calpain 1 levels is slightly higher in spermatocytes entering the meiotic phase. Both calpain isoforms are also expressed in mouse spermatozoa and are localized to the acrosomal cap. Inducing capacitated spermatozoa to undergo the acrosome reaction in the presence of a selective calpain inhibitor significantly reduced the acrosome reaction rate in a dose-dependent manner. Thus, calpain, a pluripotential protease with numerous substrates, may serve as an effector in more than one pathway in the complex process of spermatogenesis and in the events preceding fertilization, such as the acrosome reaction.

scholarly journals Copine A Interacts with Actin Filaments and Plays a Role in Chemotaxis and Adhesion

Cells ◽  
2019 ◽  
Vol 8 (7) ◽  
pp. 758 ◽  
Author(s):  
Matthew J. Buccilli ◽  
April N. Ilacqua ◽  
Mingxi Han ◽  
Andrew A. Banas ◽  
Elise M. Wight ◽  
...  
Keyword(s):  
Protein Function ◽  
Actin Filaments ◽  
Model Organism ◽  
Dependent Manner ◽  
Developmental Defects ◽  
Calcium Dependent ◽  
Cellular Processes ◽  
Eukaryotic Organisms ◽  
Von Willebrand

Copines make up a family of calcium-dependent, phospholipid-binding proteins found in numerous eukaryotic organisms. Copine proteins consist of two C2 domains at the N-terminus followed by an A domain similar to the von Willebrand A domain found in integrins. We are studying copine protein function in the model organism, Dictyostelium discoideum, which has six copine genes, cpnA-cpnF. Previous research showed that cells lacking the cpnA gene exhibited a cytokinesis defect, a contractile vacuole defect, and developmental defects. To provide insight into the role of CpnA in these cellular processes, we used column chromatography and immunoprecipitation to isolate proteins that bind to CpnA. These proteins were identified by mass spectrometry. One of the proteins identified was actin. Purified CpnA was shown to bind to actin filaments in a calcium-dependent manner in vitro. cpnA− cells exhibited defects in three actin-based processes: chemotaxis, cell polarity, and adhesion. These results suggest that CpnA plays a role in chemotaxis and adhesion and may do so by interacting with actin filaments.

scholarly journals Influenza matrix protein 2 alters CFTR expression and function through its ion channel activity

2013 ◽  
Vol 304 (9) ◽  
pp. L582-L592 ◽  
Author(s):  
James D. Londino ◽  
Ahmed Lazrak ◽  
Asta Jurkuvenaite ◽  
James F. Collawn ◽  
James W. Noah ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Ion Channel ◽  
Xenopus Oocytes ◽  
Secretory Pathway ◽  
Matrix Protein ◽  
Protein A ◽  
Dependent Manner ◽  
Hek 293 ◽  
Concanamycin A ◽  
And Function

The human cystic fibrosis transmembrane conductance regulator (CFTR) is a cyclic AMP-activated chloride (Cl−) channel in the lung epithelium that helps regulate the thickness and composition of the lung epithelial lining fluid. We investigated whether influenza M2 protein, a pH-activated proton (H+) channel that traffics to the plasma membrane of infected cells, altered CFTR expression and function. M2 decreased CFTR activity in 1) Xenopus oocytes injected with human CFTR, 2) epithelial cells (HEK-293) stably transfected with CFTR, and 3) human bronchial epithelial cells (16HBE14o−) expressing native CFTR. This inhibition was partially reversed by an inhibitor of the ubiquitin-activating enzyme E1. Next we investigated whether the M2 inhibition of CFTR activity was due to an increase of secretory organelle pH by M2. Incubation of Xenopus oocytes expressing CFTR with ammonium chloride or concanamycin A, two agents that alkalinize the secretory pathway, inhibited CFTR activity in a dose-dependent manner. Treatment of M2- and CFTR-expressing oocytes with the M2 ion channel inhibitor amantadine prevented the loss in CFTR expression and activity; in addition, M2 mutants, lacking the ability to transport H+, did not alter CFTR activity in Xenopus oocytes and HEK cells. Expression of an M2 mutant retained in the endoplasmic reticulum also failed to alter CFTR activity. In summary, our data show that M2 decreases CFTR activity by increasing secretory organelle pH, which targets CFTR for destruction by the ubiquitin system. Alteration of CFTR activity has important consequences for fluid regulation and may potentially modify the immune response to viral infection.

scholarly journals Calmodulin triggers the resumption of meiosis in amphibian oocytes.

1981 ◽  
Vol 89 (3) ◽  
pp. 389-394 ◽  
Author(s):  
W J Wasserman ◽  
L D Smith
Keyword(s):  
Calcium Binding ◽  
Xenopus Oocytes ◽  
Bovine Brain ◽  
Affinity Column ◽  
Xenopus Laevis Oocytes ◽  
Dependent Manner ◽  
Calcium Dependent ◽  
Calcium Calmodulin Dependent ◽  
Dependent Protein ◽  
Sepharose 4B

The calcium-binding protein, calmodulin, has been purified from Xenopus laevis oocytes. This 18,500-dalton protein, pl 4.3, has two high-affinity calcium-binding sites per mole protein having a dissociation constant of 2.8 x 10(-6) M. Full-grown Xenopus oocytes, arrested in late G2 of the meiotic cell cycle, resumed meiosis when microinjected with 60-80 ng (3-4 pmol) of calmodulin in the form of a calcium-calmodulin complex. The timing of the meiotic events in these recipient oocytes was the same as that normally induced by progesterone. Xenopus ovarian calmodulin stimulated bovine brain phosphodiesterase (PDE) 3- to 10-fold in a calcium-dependent manner, but it had no apparent effect on ovarian PDE activity. A calcium-calmodulin-dependent protein kinase has been isolated from Xenopus oocytes using a calmodulin-Sepharose 4B affinity column. The possible role for this kinase in regulating the G2-M transition in oocytes has been discussed.

scholarly journals Toxoplasma gondii establishes an extensive filamentous network consisting of stable F-actin during replication

2016 ◽  
Author(s):  
Javier Periz ◽  
Jamie Whitelaw ◽  
Clare Harding ◽  
Leandro Lemgruber ◽  
Simon Gras ◽  
...  
Keyword(s):  
Life Cycle ◽  
Actin Filaments ◽  
Critical Concentration ◽  
Physiological Role ◽  
Dependent Manner ◽  
Material Transport ◽  
Calcium Dependent ◽  
Functional Models ◽  
Parasite Replication

AbstractApicomplexan actin is well conserved and clearly important during the parasite's life cycle. Several studies assert that its polymerization kinetics are unusual, permitting only short, unstable F-actin filaments. However, it has not been possible to study actin in vivo, so its physiological role has remained obscure. This has led to functional models which are mutually conflicting, incompatible with actin behavior from other eukaryotes, and cannot explain actin's importance during basic processes such as parasite replication and egress. Here we use a chromobody that specifically binds F-actin to demonstrate that Toxoplasma forms stable actin filaments in vivo. F-actin is not only important for parasite replication, but also forms an extensive network that connects individuals both within and between parasitophorous vacuoles, and allows vesicles to be exchanged between parasites within a vacuole. During host cell egress, prior to motility, this network collapses in a calcium-dependent manner. This study demonstrates unexpected roles of Toxoplasma actin during the asexual life cycle, and proves that formation of F-actin depends on a critical concentration of G-actin, implying a polymerization mechanism similar to mammalian actin.One Sentence SummaryToxoplasma establishes a stable F-actin network that is essential for replication and material transport between individual parasites.

scholarly journals Gradients of phosphatidylserine contribute to plasma membrane charge localization and cell polarity in fission yeast

2017 ◽  
Vol 28 (1) ◽  
pp. 210-220 ◽  
Author(s):  
Armin Haupt ◽  
Nicolas Minc
Keyword(s):  
Plasma Membrane ◽  
Fission Yeast ◽  
Rho Gtpases ◽  
Cell Types ◽  
Dependent Manner ◽  
Surface Charges ◽  
Charge Localization ◽  
Membrane Charge ◽  
Altered Cell ◽  
And Function

Surface charges at the inner leaflet of the plasma membrane may contribute to regulate the surface recruitment of key signaling factors. Phosphatidylserine (PS) is an abundant charged lipid that may regulate charge distribution in different cell types. Here we characterize the subcellular distribution and function of PS in the rod-shaped, polarized fission yeast. We find that PS preferably accumulates at cell tips and defines a gradient of negative charges along the cell surface. This polarization depends on actin-mediated endocytosis and contributes to the subcellular partitioning of charged polarity-regulating Rho GTPases like Rho1 or Cdc42 in a protein charge–dependent manner. Cells depleted of PS have altered cell dimensions and fail to properly regulate growth from the second end, suggesting a role for PS and membrane charge in polarized cell growth.

scholarly journals Actin-membrane interaction in fibroblasts: what proteins are involved in this association?

1984 ◽  
Vol 99 (1) ◽  
pp. 95s-103s ◽  
Author(s):  
P Mangeat ◽  
K Burridge
Keyword(s):  
Plasma Membrane ◽  
Actin Filaments ◽  
Stress Fiber ◽  
Membrane Interaction ◽  
Microfilament Bundles ◽  
The Family ◽  
Form Part ◽  
Membrane Attachment ◽  
A Chain ◽  
And Function

In this review we discuss some of the proteins for which a role in linking actin to the fibroblast plasma membrane has been suggested. We focus on the family of proteins related to erythrocyte spectrin, proteins that have generally been viewed as having an organization and a function in actin-membrane attachment similar to those of erythrocyte spectrin. Experiments in which we precipitated the nonerythrocyte spectrin within living fibroblasts have led us to question this supposed similarity of organization and function of the nonerythrocyte and erythrocyte spectrins. Intracellular precipitation of fibroblast spectrin does not affect the integrity of the major actin-containing structures, the stress fiber microfilament bundles. Unexpectedly, however, we found that the precipitation of spectrin results in a condensation and altered distribution of the vimentin class of intermediate filaments in most cells examined. Although fibroblast spectrin may have a role in the attachment of some of the cortical, submembranous actin, it is surprising how little the intracellular immunoprecipitation of the spectrin affects the cells. Several proteins have been found concentrated at the ends of stress fibers, where the actin filaments terminate at focal contacts. Two of these proteins, alpha-actinin and fimbrin, have properties that suggest that they are not involved in the attachment of the ends of the bundles to the membrane but are more probably involved in the organization and cross-linking of the filaments within the bundles. On the other hand, vinculin and talin are two proteins that interact with each other and may form part of a chain of attachments between the ends of the microfilament bundles and the focal contact membrane. Their role in this attachment, however, has not been established and further work is needed to examine their interaction with actin and to identify any other components with which they may interact, particularly in the plasma membrane.

scholarly journals Ca2+-Dependent Protein Kinase 6 Enhances KAT2 Shaker Channel Activity in Arabidopsis thaliana

2021 ◽  
Vol 22 (4) ◽  
pp. 1596
Author(s):  
Elsa Ronzier ◽  
Claire Corratgé-Faillie ◽  
Frédéric Sanchez ◽  
Christian Brière ◽  
Tou Cheu Xiong
Keyword(s):  
Arabidopsis Thaliana ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Physical Interaction ◽  
Fluorescence Lifetime Imaging ◽  
Dependent Manner ◽  
In Planta ◽  
Knock Out ◽  
Calcium Dependent ◽  
Dependent Protein

Post-translational regulations of Shaker-like voltage-gated K+ channels were reported to be essential for rapid responses to environmental stresses in plants. In particular, it has been shown that calcium-dependent protein kinases (CPKs) regulate Shaker channels in plants. Here, the focus was on KAT2, a Shaker channel cloned in the model plant Arabidopsis thaliana, where is it expressed namely in the vascular tissues of leaves. After co-expression of KAT2 with AtCPK6 in Xenopuslaevis oocytes, voltage-clamp recordings demonstrated that AtCPK6 stimulates the activity of KAT2 in a calcium-dependent manner. A physical interaction between these two proteins has also been shown by Förster resonance energy transfer by fluorescence lifetime imaging (FRET-FLIM). Peptide array assays support that AtCPK6 phosphorylates KAT2 at several positions, also in a calcium-dependent manner. Finally, K+ fluorescence imaging in planta suggests that K+ distribution is impaired in kat2 knock-out mutant leaves. We propose that the AtCPK6/KAT2 couple plays a role in the homeostasis of K+ distribution in leaves.

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