Involvement of the Ca2+-ATPase PAT1 and the contractile vacuole in calcium regulation in Dictyostelium discoideum

1999 ◽  
Vol 112 (3) ◽  
pp. 405-414 ◽  
Author(s):  
J. Moniakis ◽  
M.B. Coukell ◽  
A. Janiec
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Growth And Development ◽  
Molecular Mechanisms ◽  
Calcium Regulation ◽  
Contractile Vacuole ◽  
Rich Medium ◽  
Immunofluorescence Analysis

In Dictyostelium discoideum, the Ca2+-ATPase, PAT1, is localized to membranes of the contractile vacuole and its expression is upregulated substantially when the cells are grown in Ca2+-rich medium. In this study, we have analyzed the cellular/molecular mechanisms regulating PAT1 expression and examined the role of PAT1 and the contractile vacuole in Ca2+ regulation. During both growth and development, Dictyostelium cells respond to low millimolar concentrations of extracellular Ca2+ and upregulate PAT1 in a few hours. This process is dependent on protein synthesis and the serine/threonine phosphatase, calcineurin. Immunofluorescence analysis indicates that the upregulated PAT1 is associated mainly with the contractile vacuole, but it is also on the plasma membrane. This latter finding suggests that the contractile vacuole fuses with the plasma membrane to eliminate excess intracellular Ca2+. In support of this idea, it was observed that conditions which impair contractile vacuolar function reduce the rate of Ca2+ secretion. It was also found that cells deficient in PAT1, due to the expression of antisense patA RNA or to the presence of calcineurin antagonists, grow normally in low Ca2+ medium but poorly or not at all in high Ca2+ medium. Together, these results suggest that PAT1 and the contractile vacuole are components of a Ca2+ sequestration and excretion pathway, which functions to help maintain Ca2+ homeostasis, especially under conditions of Ca2+ stress.

Stimulation of calcium influx by platelet activating factor in Dictyostelium

1995 ◽  
Vol 108 (4) ◽  
pp. 1597-1603
Author(s):  
R. Schaloske ◽  
C. Sordano ◽  
S. Bozzaro ◽  
D. Malchow
Keyword(s):  
Plasma Membrane ◽  
G Protein ◽  
Dictyostelium Discoideum ◽  
Calcium Influx ◽  
Platelet Activating Factor ◽  
Inactive Compound ◽  
Time Period ◽  
Dependent Pathway ◽  
Stimulation Of

Platelet activating factor (PAF) induces Ca2+ influx in Dictyostelium discoideum. In this investigation we used this activity to analyze the mechanism of PAF action. We found that PAF activity was confined to the period of spike-shaped oscillations and suggest that the role of PAF is to augment cAMP relay. PAF seems to act only a few times during this time period of two hours, since Ca2+ entry adapted to a subsequent stimulus for about 30 minutes. PAF showed a reduced response in the G protein beta- strain LW14 and was unable to induce Ca2+ influx in the G alpha 2- strains HC85 and JM1. The latter expresses the cAMP receptors cAR1 constitutively, and exhibits cAMP-induced Ca2+ influx, albeit at a reduced level. In order to decide whether the inability of PAF to elicit a Ca2+ response in JM1 cells was due to the lack of differentiation and/or the lack of G alpha 2, we inhibited the IP3-dependent pathway with compound U73122 and found that Ca2+ entry was blocked, whereas a closely related inactive compound, U73343, did not alter the response. In agreement with this, NBD-Cl, an inhibitor of Ca2+ uptake into the IP3-sensitive store in Dictyostelium, also abolished PAF activity. The latter was not inhibited by the plasma membrane antagonists BN-52021 or WEB 2170. Therefore PAF seems to operate intracellularly via the IP3-signalling pathway at or upstream of the IP3-sensitive store.

scholarly journals ESCRT-dependent targeting of plasma membrane localized KCa3.1 to the lysosomes

2010 ◽  
Vol 299 (5) ◽  
pp. C1015-C1027 ◽  
Author(s):  
Corina M. Balut ◽  
Yajuan Gao ◽  
Sandra A. Murray ◽  
Patrick H. Thibodeau ◽  
Daniel C. Devor
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Close Association ◽  
Significant Inhibition ◽  
Dominant Negative ◽  
Human Embryonic Kidney Cells ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Interfering Rna

The number of intermediate-conductance, Ca2+-activated K+ channels (KCa3.1) present at the plasma membrane is deterministic in any physiological response. However, the mechanisms by which KCa3.1 channels are removed from the plasma membrane and targeted for degradation are poorly understood. Recently, we demonstrated that KCa3.1 is rapidly internalized from the plasma membrane, having a short half-life in both human embryonic kidney cells (HEK293) and human microvascular endothelial cells (HMEC-1). In this study, we investigate the molecular mechanisms controlling the degradation of KCa3.1 heterologously expressed in HEK and HMEC-1 cells. Using immunofluorescence and electron microscopy, as well as quantitative biochemical analysis, we demonstrate that membrane KCa3.1 is targeted to the lysosomes for degradation. Furthermore, we demonstrate that either overexpressing a dominant negative Rab7 or short interfering RNA-mediated knockdown of Rab7 results in a significant inhibition of channel degradation rate. Coimmunoprecipitation confirmed a close association between Rab7 and KCa3.1. On the basis of these findings, we assessed the role of the ESCRT machinery in the degradation of heterologously expressed KCa3.1, including TSG101 [endosomal sorting complex required for transport (ESCRT)-I] and CHMP4 (ESCRT-III) as well as VPS4, a protein involved in the disassembly of the ESCRT machinery. We demonstrate that TSG101 is closely associated with KCa3.1 via coimmunoprecipitation and that a dominant negative TSG101 inhibits KCa3.1 degradation. In addition, both dominant negative CHMP4 and VPS4 significantly decrease the rate of membrane KCa3.1 degradation, compared with wild-type controls. These results are the first to demonstrate that plasma membrane-associated KCa3.1 is targeted for lysosomal degradation via a Rab7 and ESCRT-dependent pathway.

Mechanism of protein sorting during erythroblast enucleation: role of cytoskeletal connectivity

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1912-1919 ◽  
Author(s):  
James C.-M. Lee ◽  
J. Aura Gimm ◽  
Annie J. Lo ◽  
Mark J. Koury ◽  
Sharon W. Krauss ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Mechanisms ◽  
Immunofluorescence Microscopy ◽  
Protein Sorting ◽  
Membrane Cytoskeleton ◽  
Skeletal Protein ◽  
Regulate Protein ◽  
Membrane Components ◽  
Cytoskeletal Elements

AbstractDuring erythroblast enucleation, nuclei surrounded by plasma membrane separate from erythroblast cytoplasm. A key aspect of this process is sorting of erythroblast plasma membrane components to reticulocytes and expelled nuclei. Although it is known that cytoskeletal elements actin and spectrin partition to reticulocytes, little is understood about molecular mechanisms governing plasma membrane protein sorting. We chose glycophorin A (GPA) as a model integral protein to begin investigating protein-sorting mechanisms. Using immunofluorescence microscopy and Western blotting we found that GPA sorted predominantly to reticulocytes. We hypothesized that the degree of skeletal linkage might control the sorting pattern of transmembrane proteins. To explore this hypothesis, we quantified the extent of GPA association to the cytoskeleton in erythroblasts, young reticulocytes, and mature erythrocytes using fluorescence imaged microdeformation (FIMD) and observed that GPA underwent dramatic reorganization during terminal differentiation. We discovered that GPA was more connected to the membrane cytoskeleton, either directly or indirectly, in erythroblasts and young reticulocytes than in mature cells. We conclude that skeletal protein association can regulate protein sorting during enucleation. Further, we suggest that the enhanced rigidity of reticulocyte membranes observed in earlier investigations results, at least in part, from increased connectivity of GPA with the spectrin-based skeleton.

scholarly journals Silencing of Plasma Membrane Ca2+-ATPase Isoforms 2 and 3 Impairs Energy Metabolism in Differentiating PC12 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-13 ◽  
Author(s):  
Tomasz Boczek ◽  
Malwina Lisek ◽  
Bozena Ferenc ◽  
Antoni Kowalski ◽  
Magdalena Wiktorska ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Pc12 Cells ◽  
Molecular Mechanisms ◽  
Atp Synthesis ◽  
Mitochondrial Activity ◽  
Excitable Cells ◽  
Close Link ◽  
Atp Level ◽  
Atp Generation

A close link between Ca2+, ATP level, and neurogenesis is apparent; however, the molecular mechanisms of this relationship have not been completely elucidated. Transient elevations of cytosolic Ca2+may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+in cytosol. In differentiation process plasma membrane Ca2+ATPase (PMCA) is considered as one of the major players for Ca2+homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher[Ca2+]cresulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.

RIBOSOME-MEMBRANE INTERACTION AND PROTEIN SYNTHESIS

1973 ◽  
Vol 74 (Suppl) ◽  
pp. S192-S224 ◽  
Author(s):  
J. R. Tata
Keyword(s):  
Protein Synthesis ◽  
Growth And Development ◽  
Active Synthesis ◽  
Active Growth ◽  
Membrane Interaction ◽  
Major Function ◽  
Membrane Bound ◽  
Different Populations ◽  
Protein Secreting

ABSTRACT The role of secretion of proteins for the attachment of ribosomes to membranes has been well established. That another function must exist for membrane-ribosome interaction is suggested by observations on: (a) the active synthesis of proteins on membrane-bound ribosomes of predominantly non-protein secreting cells, and b) the massive proliferation of membrane-bound ribosomes during active growth and development of both secretory and non-secretory tissues. Literature on functional and compositional differences between membrane-bound and free ribosomes is reviewed and it is proposed that a major function of ribosome-membrane interaction is to effect a topological segregation of different populations of ribosomes synthesizing different classes of proteins.

scholarly journals MICROFILAMENTS AND CELL LOCOMOTION

1971 ◽  
Vol 49 (3) ◽  
pp. 595-613 ◽  
Author(s):  
Brian S. Spooner ◽  
Kenneth M. Yamada ◽  
Norman K. Wessells
Keyword(s):  
Protein Synthesis ◽  
Plasma Membrane ◽  
Cytochalasin B ◽  
Cell Movement ◽  
Leading Edge ◽  
Complete Recovery ◽  
Cell Locomotion ◽  
Cell Processes

The role of microfilaments in generating cell locomotion has been investigated in glial cells migrating in vitro. Such cells are found to contain two types of microfilament systems: First, a sheath of 50–70-A in diameter filaments is present in the cytoplasm at the base of the cells, just inside the plasma membrane, and in cell processes. Second, a network of 50-A in diameter filaments is found just beneath the plasma membrane at the leading edge (undulating membrane locomotory organelle) and along the sides of the cell. The drug, cytochalasin B, causes a rapid cessation of migration and a disruption of the microfilament network. Other organelles, including the microfilament sheath and microtubules, are unaltered by the drug, and protein synthesis is not inhibited. Removal of cytochalasin results in complete recovery of migratory capabilities, even in the absence of virtually all protein synthesis. Colchicine, at levels sufficient to disrupt all microtubules, has no effect on undulating membrane activity, on net cell movement, or on microfilament integrity. The microfilament network is, therefore, indispensable for locomotion.

scholarly journals The TUTase URT1 connects decapping activators and prevents the accumulation of excessively deadenylated mRNAs to avoid siRNA biogenesis

2020 ◽  
Author(s):  
Hélène Scheer ◽  
Caroline de Almeida ◽  
Emilie Ferrier ◽  
Quentin Simonnot ◽  
Laure Poirier ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
Growth And Development ◽  
Molecular Mechanisms ◽  
Tail Length ◽  
Mrna Degradation ◽  
In Planta ◽  
Rna Helicases ◽  
Eukaryotic Mrnas

AbstractUridylation is a widespread modification destabilizing eukaryotic mRNAs. Yet, molecular mechanisms underlying TUTase-mediated mRNA degradation remain mostly unresolved. Here, we report that the Arabidopsis TUTase URT1 participates in a molecular network connecting several translational repressors/decapping activators. URT1 directly interacts with DECAPPING 5 (DCP5), the Arabidopsis ortholog of human LSM14 and yeast Scd6, and this interaction connects URT1 to additional decay factors like DDX6/Dhh1-like RNA helicases. Nanopore direct RNA sequencing reveals a global role of URT1 in shaping poly(A) tail length, notably by preventing the accumulation of excessively deadenylated mRNAs. Based on in vitro and in planta data, we propose a model that explains how URT1 could reduce the accumulation of oligo(A)-tailed mRNAs both by favoring their degradation and because 3’ terminal uridines intrinsically hinder deadenylation. Importantly, preventing the accumulation of excessively deadenylated mRNAs avoids the biogenesis of illegitimate siRNAs that silence endogenous mRNAs and perturb Arabidopsis growth and development.

scholarly journals Cell motility through plasma membrane blebbing

2008 ◽  
Vol 181 (6) ◽  
pp. 879-884 ◽  
Author(s):  
Oliver T. Fackler ◽  
Robert Grosse
Keyword(s):  
Plasma Membrane ◽  
Cell Motility ◽  
Molecular Mechanisms ◽  
Cell Movement ◽  
Core Component ◽  
Membrane Blebbing ◽  
Membrane Blebs ◽  
Bleb Formation ◽  
Guanosine Triphosphatase

Plasma membrane blebs are dynamic cytoskeleton-regulated cell protrusions that have been implicated in apoptosis, cytokinesis, and cell movement. Influencing Rho–guanosine triphosphatase activities and subsequent actomyosin dynamics appears to constitute a core component for bleb formation. In this paper, we discuss recent evidence in support of a central role of nonapoptotic membrane blebbing for cell migration and cancer cell invasion as well as advances in our understanding of the underlying molecular mechanisms. Based on these studies, we propose that in a physiological context, bleb-associated cell motility reflects a cell's response to reduced substratum adhesion. The importance of blebbing as a functional protrusion is underscored by the existence of multiple molecular mechanisms that govern actin-mediated bleb retraction.

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