scholarly journals The Monomeric Clathrin Assembly Protein, AP180, Regulates Contractile Vacuole Size in Dictyostelium discoideum

2006 ◽  
Vol 17 (12) ◽  
pp. 5381-5389 ◽  
Author(s):  
Irene Stavrou ◽  
Theresa J. O'Halloran
Keyword(s):  
Plasma Membrane ◽  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Adaptor Protein ◽  
Contractile Vacuole ◽  
Unique Contribution ◽  
Wild Type ◽  
Unique Role ◽  
Contractile Vacuoles ◽  
Assembly Proteins

AP180, one of many assembly proteins and adaptors for clathrin, stimulates the assembly of clathrin lattices on membranes, but its unique contribution to clathrin function remains elusive. In this study we identified the Dictyostelium discoideum ortholog of the adaptor protein AP180 and characterized a mutant strain carrying a deletion in this gene. Imaging GFP-labeled AP180 showed that it localized to punctae at the plasma membrane, the contractile vacuole, and the cytoplasm and associated with clathrin. AP180 null cells did not display defects characteristic of clathrin mutants and continued to localize clathrin punctae on their plasma membrane and within the cytoplasm. However, like clathrin mutants, AP180 mutants, were osmosensitive. When immersed in water, AP180 null cells formed abnormally large contractile vacuoles. Furthermore, the cycle of expansion and contraction for contractile vacuoles in AP80 null cells was twice as long as that of wild-type cells. Taken together, our results suggest that AP180 plays a unique role as a regulator of contractile vacuole morphology and activity in Dictyostelium.

Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281

1985 ◽  
Vol 78 (1) ◽  
pp. 49-65 ◽  
Author(s):  
N.J. Maihle ◽  
B.H. Satir
Keyword(s):  
Plasma Membrane ◽  
Mutant Strain ◽  
Protein Secretion ◽  
Tetrahymena Thermophila ◽  
Freeze Fracture ◽  
Size Class ◽  
Secretory Product ◽  
Wild Type ◽  
Intramembrane Particle ◽  
Mutant Cells

The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion. Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210. In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described. SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling. Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts. In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells. A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane. Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type.

scholarly journals Role of Clathrin Assembly Protein-2 Beta Subunit during White Spot Syndrome Virus Infection in Black Tiger Shrimp Penaeus monodon

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Thapanan Jatuyosporn ◽  
Pasunee Laohawutthichai ◽  
Premruethai Supungul ◽  
Rogerio R. Sotelo-Mundo ◽  
Adrian Ochoa-Leyva ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
White Spot Syndrome Virus ◽  
Penaeus Monodon ◽  
Adaptor Protein ◽  
Immunogold Labelling ◽  
Open Reading Frames ◽  
White Spot ◽  
Transmission Electron ◽  
White Spot Syndrome ◽  
Assembly Proteins

Abstract White spot syndrome virus (WSSV) is one of the most lethal viruses severely affecting shrimp industry. This disease can cause 100% mortality of farmed shrimp within a week. This work aims to characterize clathrin assembly proteins in Penaeus monodon and investigate their roles in WSSV entry. In general, clathrin assembly proteins form complexes with specific receptors and clathrins, leading to clathrin-mediated endocytosis. Adaptor protein 2 (AP-2), which is responsible for endocytosis at plasma membrane, consists of four subunits including α, β2, μ2 and σ2. Knockdown of clathrin coat AP17, or σ subunit of AP-2 dramatically reduced WSSV infectivity. Similar results were observed, when shrimp were pre-treated with chlorpromazine (CPZ), an inhibitor of clathrin-dependent endocytosis. The complete open reading frames of AP-2β and μ subunits of P. monodon are reported. PmAP-2 β was up-regulated about 4-fold at 6 and 36 h post-WSSV infection. Knockdown of PmAP-2β delayed shrimp mortality during WSSV infection, of which WSSV intermediate early 1 gene expression was also down-regulated. Immunogold-labelling and transmission electron microscopy revealed that PmAP-2β co-localized with WSSV particles at plasma membrane. In addition, PmAP-2β-silencing significantly affected the expression levels of PmSTAT, PmDOME, PmDorsal and ALFPm3 during WSSV infection. It is possible that PmAP-2β is associated with the JAK/STAT and the Toll pathway.

scholarly journals AP180-Mediated Trafficking of Vamp7B Limits Homotypic Fusion of Dictyostelium Contractile Vacuoles

2009 ◽  
Vol 20 (20) ◽  
pp. 4278-4288 ◽  
Author(s):  
Yujia Wen ◽  
Irene Stavrou ◽  
Kirill Bersuker ◽  
Rebecca J. Brady ◽  
Arturo De Lozanne ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Coated Vesicles ◽  
Contractile Vacuole ◽  
Snare Proteins ◽  
In Vitro Assays ◽  
Accessory Proteins ◽  
Coated Pits ◽  
Contractile Vacuoles ◽  
Null Mutants

Clathrin-coated vesicles play an established role in endocytosis from the plasma membrane, but they are also found on internal organelles. We examined the composition of clathrin-coated vesicles on an internal organelle responsible for osmoregulation, the Dictyostelium discoideum contractile vacuole. Clathrin puncta on contractile vacuoles contained multiple accessory proteins typical of plasma membrane–coated pits, including AP2, AP180, and epsin, but not Hip1r. To examine how these clathrin accessory proteins influenced the contractile vacuole, we generated cell lines that carried single and double gene knockouts in the same genetic background. Single or double mutants that lacked AP180 or AP2 exhibited abnormally large contractile vacuoles. The enlarged contractile vacuoles in AP180-null mutants formed because of excessive homotypic fusion among contractile vacuoles. The SNARE protein Vamp7B was mislocalized and enriched on the contractile vacuoles of AP180-null mutants. In vitro assays revealed that AP180 interacted with the cytoplasmic domain of Vamp7B. We propose that AP180 directs Vamp7B into clathrin-coated vesicles on contractile vacuoles, creating an efficient mechanism for regulating the internal distribution of fusion-competent SNARE proteins and limiting homotypic fusions among contractile vacuoles. Dictyostelium contractile vacuoles offer a valuable system to study clathrin-coated vesicles on internal organelles within eukaryotic cells.

scholarly journals Impact of the Listeria monocytogenes Protein InlC on Infection in Mice

2013 ◽  
Vol 81 (4) ◽  
pp. 1334-1340 ◽  
Author(s):  
Nelly Leung ◽  
Antonella Gianfelice ◽  
Scott D. Gray-Owen ◽  
Keith Ireton
Keyword(s):  
Listeria Monocytogenes ◽  
Mutant Strain ◽  
Type Strain ◽  
Wild Type Strain ◽  
Bacterial Pathogen ◽  
Adaptor Protein ◽  
Single Amino Acid ◽  
Wild Type ◽  
Content Type

ABSTRACTThe bacterial pathogenListeria monocytogenescauses serious food-borne illnesses in pregnant women and the immunocompromised.L. monocytogenespromotes its internalization into host epithelial cells and then uses an F-actin-dependent motility process to spread from infected cells to surrounding healthy cells. In cultured enterocytes, efficient spread ofL. monocytogenesrequires the secreted bacterial protein InlC. InlC promotes dissemination by physically interacting with and antagonizing the function of the human adaptor protein Tuba. Here we examine the role of InlC and its interaction with host Tuba during infection in mice. The study took advantage of a single-amino-acid substitution (K173A) in InlC that impairs binding to human Tuba but does not affect InlC-mediated inhibition of the NF-κB pathway. Mice were inoculated intravenously with the wild-typeL. monocytogenesstrain EGD, an isogenic strain deleted for theinlCgene (ΔinlC), or a strain expressing K173A mutant InlC (inlC.K173A). The 50% lethal doses (LD50) for the ΔinlCorinlC.K173Amutant strain were approximately 4- or 6-fold greater than that for the wild-type strain, indicating a role forinlCin virulence. Compared to the wild-type strain, theinlC.K173Amutant strain exhibited lower bacterial loads in the liver. Histological analysis of livers indicated that the twoinlCmutant strains produced smaller foci of infection than did the wild-type strain. These smaller foci are consistent with a role for InlC in cell-to-cell spreadin vivo. Taken together, these results provide evidence that interaction of InlC with host Tuba is important for full virulence.

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.

scholarly journals A Dictyostelium discoideum mutant that missorts and oversecretes lysosomal enzyme precursors is defective in endocytosis.

1989 ◽  
Vol 109 (4) ◽  
pp. 1445-1456 ◽  
Author(s):  
D L Ebert ◽  
H H Freeze ◽  
J Richardson ◽  
R L Dimond ◽  
J A Cardelli
Keyword(s):  
Dictyostelium Discoideum ◽  
Mutant Strain ◽  
Lysosomal Enzyme ◽  
Fluid Phase ◽  
Wild Type ◽  
Lysosomal Hydrolases ◽  
Chase Period ◽  
Secondary Lysosomes ◽  
Beta Glucosidase ◽  
Enzyme Targeting

A mutant strain of Dictyostelium discoideum, HMW570, oversecretes several lysosomal enzyme activities during growth. Using a radiolabel pulse-chase protocol, we followed the synthesis and secretion of two of these enzymes, alpha-mannosidase and beta-glucosidase. A few hours into the chase period, HMW570 had secreted 95% of its radiolabeled alpha-mannosidase and 86% of its radiolabeled beta-glucosidase as precursor polypeptides compared to the secretion of less than 10% of these forms from wild-type cells. Neither alpha-mannosidase nor beta-glucosidase in HMW570 were ever found in the lysosomal fractions of sucrose gradients consistent with HMW570 being defective in lysosomal enzyme targeting. Also, both alpha-mannosidase and beta-glucosidase precursors in the mutant strain were membrane associated as previously observed for wild-type precursors, indicating membrane association is not sufficient for lysosomal enzyme targeting. Hypersecretion of the alpha-mannosidase precursor by HMW570 was not accompanied by major alterations in N-linked oligosaccharides such as size, charge, and ratio of sulfate and phosphate esters. However, HMW570 was defective in endocytosis. A fluid phase marker, [3H]dextran, accumulated in the mutant at one-half of the rate of wild-type cells and to only one-half the normal concentration. Fractionation of cellular organelles on self-forming Percoll gradients revealed that the majority of the fluid-phase marker resided in compartments in mutant cells with a density characteristic of endosomes. In contrast, in wild-type cells [3H]dextran was predominantly located in vesicles with a density identical to secondary lysosomes. Furthermore, the residual lysosomal enzyme activity in the mutant accumulated in endosomal-like vesicles. Thus, the mutation in HMW570 may be in a gene required for both the generation of dense secondary lysosomes and the sorting of lysosomal hydrolases.

Interaction between intracellular vacuoles and the cell surface analysed by finite aperture theory interference reflection microscopy

1982 ◽  
Vol 54 (1) ◽  
pp. 287-298
Author(s):  
D. Gingell ◽  
I. Todd ◽  
N. Owens
Keyword(s):  
Plasma Membrane ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Surface Membrane ◽  
Contractile Vacuole ◽  
Contractile Mechanism ◽  
Cell Surface Membrane ◽  
Minimal Thickness ◽  
Finite Aperture ◽  
Mechanical Consequence

Using finite aperture theory we have shown that localized very dark areas in the interference reflection images of Dictyostelium discoideum amoebae are due to the close intracellular approach of vesicles and tubular elements of the contractile vacuole system to the plasma membrane adjacent to the substratum. Vesicles interacting in this way become locally deformed to the planar contour of the substratum and are separated from the cell surface membrane by a constant approximately less than 0.1 micron of cytoplasm. Lamellar processes formed by these cells on very adhesive surfaces have identical dimensions. This minimal thickness may be a mechanical consequence of a contractile mechanism which pulls membranes together.

scholarly journals The role of Zur-regulated lipoprotein A in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles in Acinetobacter baumannii

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Nayeong Kim ◽  
Hyo Jeong Kim ◽  
Man Hwan Oh ◽  
Se Yeon Kim ◽  
Mi Hyun Kim ◽  
...  
Keyword(s):  
Acinetobacter Baumannii ◽  
Outer Membrane ◽  
Mutant Strain ◽  
Antimicrobial Susceptibility ◽  
Type Strain ◽  
Wild Type Strain ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Wild Type ◽  
Bacterial Morphology

Abstract Background Zinc uptake-regulator (Zur)-regulated lipoprotein A (ZrlA) plays a role in bacterial fitness and overcoming antimicrobial exposure in Acinetobacter baumannii. This study further characterized the zrlA gene and its encoded protein and investigated the roles of the zrlA gene in bacterial morphology, antimicrobial susceptibility, and production of outer membrane vesicles (OMVs) in A. baumannii ATCC 17978. Results In silico and polymerase chain reaction analyses showed that the zrlA gene was conserved among A. baumannii strains with 97–100% sequence homology. Recombinant ZrlA protein exhibited a specific enzymatic activity of D-alanine-D-alanine carboxypeptidase. Wild-type A. baumannii exhibited more morphological heterogeneity than a ΔzrlA mutant strain during stationary phase. The ΔzrlA mutant strain was more susceptible to gentamicin than the wild-type strain. Sizes and protein profiles of OMVs were similar between the wild-type and ΔzrlA mutant strains, but the ΔzrlA mutant strain produced 9.7 times more OMV particles than the wild-type strain. OMVs from the ΔzrlA mutant were more cytotoxic in cultured epithelial cells than OMVs from the wild-type strain. Conclusions The present study demonstrated that A. baumannii ZrlA contributes to bacterial morphogenesis and antimicrobial resistance, but its deletion increases OMV production and OMV-mediated host cell cytotoxicity.

scholarly journals Low ABA concentration promotes root growth and hydrotropism through relief of ABA INSENSITIVE 1-mediated inhibition of plasma membrane H+-ATPase 2

2021 ◽  
Vol 7 (12) ◽  
pp. eabd4113
Author(s):  
Rui Miao ◽  
Wei Yuan ◽  
Yue Wang ◽  
Irene Garcia-Maquilon ◽  
Xiaolin Dang ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Root Growth ◽  
Experimental System ◽  
Aba Signaling ◽  
Wild Type ◽  
Normal Medium ◽  
C Terminus ◽  
Quadruple Mutant ◽  
Aba Insensitive ◽  
Aba Receptors

The hab1-1abi1-2abi2-2pp2ca-1 quadruple mutant (Qabi2-2) seedlings lacking key negative regulators of ABA signaling, namely, clade A protein phosphatases type 2C (PP2Cs), show more apoplastic H+ efflux in roots and display an enhanced root growth under normal medium or water stress medium compared to the wild type. The presence of low ABA concentration (0.1 micromolar), inhibiting PP2C activity via monomeric ABA receptors, enhances root apoplastic H+ efflux and growth of the wild type, resembling the Qabi2-2 phenotype in normal medium. Qabi2-2 seedlings also demonstrate increased hydrotropism compared to the wild type in obliquely-oriented hydrotropic experimental system, and asymmetric H+ efflux in root elongation zone is crucial for root hydrotropism. Moreover, we reveal that Arabidopsis ABA-insensitive 1, a key PP2C in ABA signaling, interacts directly with the C terminus of Arabidopsis plasma membrane H+-dependent adenosine triphosphatase 2 (AHA2) and dephosphorylates its penultimate threonine residue (Thr947), whose dephosphorylation negatively regulates AHA2.

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