scholarly journals Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: trigger analyses

1980 ◽  
Vol 46 (1) ◽  
pp. 41-60
Author(s):  
H. Matt ◽  
H. Plattner ◽  
K. Reichel ◽  
M. Lefort-Tran ◽  
J. Beisson
Keyword(s):  
Cell Membrane ◽  
Atpase Activity ◽  
Membrane Fusion ◽  
Dual Function ◽  
Genetic Dissection ◽  
Paramecium Tetraurelia ◽  
Attachment Sites ◽  
Ca2 Channels

A variety of trigger procedures were applied to analyse the exocytotic capability of different Paramecium tetraurelia strains. 7,S K 40I, kin 24I, and 9 (18 degrees C) are capable of exocytosis (permissive strains), in contrast to nd 6, nd 7, nd 9 (27 degrees C), tam 38 and ftb A, although all procedures used enhance [Ca2+]i in the cytoplasm of all strains tested and although strains nd 6, nd 7 and nd 9 (27 degrees C) contain a full set of morphologically normal trichocysts attended to the cell membrane. The results show that only those strains are permissive which were shown previously to contain a rosette of membrane-integrated particles and a Ca2+-ATPase activity in the cell membrane over the trichocyst attachment (exocytosis) sites. The results from trigger experiments with permissive and non-permissive strains would be compatible with a dual function of rosette particles as Ca2+ pumps and Ca2+ channels. Nevertheless, the latter aspect remains uncertain since we show that experiments along these lines published by others (introducing a Ca2+ ionophore from the outside) involve a solvent-induced artifact (pseudoexocytosis: matrix stretching in the absence of membrane fusion). In all strains, except for tam 38 and ftb A (which have abnormal trichocysts incapable of being attached to the cell membrane), the isolated trichocyst matrix can be transferred from the contracted to the expanded state in vitro with certain trigger procedures. Our data clearly show that an increase of [Ca2+]i in the cytoplasm is not sufficient for exocytosis to occur and that non-permissiveness is somehow due to an inability to perform membrane fusion. It remains open whether the lack of rosettes and Ca2+-ATPase activity at trichocyst attachment sites are primary cause of non-permissiveness.

scholarly journals Exocytosis induction in Paramecium tetraurelia cells by exogenous phosphoprotein phosphatase in vivo and in vitro: possible involvement of calcineurin in exocytotic membrane fusion.

1987 ◽  
Vol 105 (1) ◽  
pp. 181-189 ◽  
Author(s):  
M Momayezi ◽  
C J Lumpert ◽  
H Kersken ◽  
U Gras ◽  
H Plattner ◽  
...  
Keyword(s):  
Alkaline Phosphatase ◽  
Membrane Fusion ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Current Finding ◽  
Phosphoprotein Phosphatase ◽  
Negative Results ◽  
Molecular Components

Since it had been previously shown that in Paramecium cells exocytosis involves the dephosphorylation of a 65-kD phosphoprotein (PP), we tried to induce exocytotic membrane fusion by exogenous phosphatases (alkaline phosphatase or calcineurin [CaN]). The occurrence of calmodulin (CaM) at preformed exocytosis sites (Momayezi, M., H. Kersken, U. Gras, J. Vilmart-Seuwen, and H. Plattner, 1986, J. Histochem. Cytochem., 34:1621-1638) and the current finding of the presence of the 65-kD PP and of a CaN-like protein in cell surface fragments ("cortices") isolated from Paramecium cells led us to also test the effect of antibodies (Ab) against CaM or CaN on exocytosis performance. Microinjected anti-CaN Ab strongly inhibit exocytosis. (Negative results with microinjected anti-CaM Ab can easily be explained by the abundance of CaM.) Alternatively, microinjection of a Ca2+-CaM-CaN complex triggers exocytosis. The same occurs with alkaline phosphatase. All these effects can also be mimicked in vitro with isolated cortices. In vitro exocytosis triggered by adding Ca2+-CaM-CaN or alkaline phosphatase is paralleled by dephosphorylation of the 65-kD PP. Exocytosis can also be inhibited in cortices by anti-CaM Ab or anti-CaN Ab. In wild-type cells, compounds that inhibit phosphatase activity, but none that inhibit kinases or proteases, are able to inhibit exocytosis. Exocytosis cannot be induced by phosphatase injection in a membrane-fusion-deficient mutant strain (nd9-28 degrees C) characterized by a defective organization of exocytosis sites (Beisson, J., M. Lefort-Tran, M. Pouphile, M. Rossignol, and B. Satir, 1976, J. Cell Biol., 69:126-143). We conclude that exocytotic membrane fusion requires an adequate assembly of molecular components to allow for the dephosphorylation of a 65-kD PP and that this step is crucial for the induction of exocytotic membrane fusion in Paramecium cells. In vivo this probably involves a Ca2+-CaM-stimulated CaN-like PP phosphatase.

scholarly journals ATP keeps exocytosis sites in a primed state but is not required for membrane fusion: an analysis with Paramecium cells in vivo and in vitro.

1986 ◽  
Vol 103 (4) ◽  
pp. 1279-1288 ◽  
Author(s):  
J Vilmart-Seuwen ◽  
H Kersken ◽  
R Stürzl ◽  
H Plattner
Keyword(s):  
Membrane Fusion ◽  
Time Course ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Primed State ◽  
Different Strains ◽  
Exogenous Trigger ◽  
Free Media

We have tried to specify a widespread hypothesis on the requirement of ATP for exocytosis (membrane fusion). With Paramecium tetraurelia cells, synchronously (approximately 1 s) exocytosing trichocysts, ATP pools have been measured in different strains, including wild type cells, "non-discharge" (nd), "trichless" (tl), and other mutations. The occurrence of a considerable and rapid ATP consumption also in nd and tl mutations as well as its time course (with a maximum 3-5 s after exocytosis) in exocytosis-competent strains does not match the actual extent of exocytosis performance. However, from in vivo as well as from in vitro experiments, we came to the conclusion that ATP might be required to keep the system in a primed state and its removal might facilitate membrane fusion. (For the study of exocytosis in vitro we have developed a new system, consisting of isolated cortices). In vivo as well as in vitro exocytosis is inhibited by increased levels of ATP or by a nonhydrolyzable ATP analogue. In vitro exocytosis is facilitated in ATP-free media. In vivo-microinjected ATP retards exocytosis in response to chemical triggers, whereas microinjected apyrase triggers exocytosis without exogenous trigger. Experiments with this system also largely exclude any overlaps with other processes that normally accompany exocytosis. Our data also explain why it was frequently assumed that ATP would be required for exocytosis. We conclude that membrane fusion during exocytosis does not require the presence of ATP; the occurrence of membrane fusion might involve the elimination of ATP from primed fusogenic sites; most of the ATP consumption measured in the course of exocytosis may be due to other effects, probably to recovery phenomena.

scholarly journals The SARS-CoV-2 host cell membrane fusion protein TMPRSS2 is a tumor suppressor and its downregulation promotes antitumor immunity and immunotherapy response in lung adenocarcinoma

2021 ◽  
Author(s):  
Zhixian Liu ◽  
Zhilan Zhang ◽  
Qiushi Feng ◽  
Xiaosheng Wang
Keyword(s):  
Lung Cancer ◽  
Cell Membrane ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Antitumor Immunity ◽  
Host Cell Membrane ◽  
Membrane Fusion Protein ◽  
In Vivo Experiments

Abstract Background Cancer patients are susceptible to SARS-CoV-2 infection. An investigation into the association between the SARS-CoV-2 host cell membrane fusion protein TMPRSS2 and lung cancer is significant, considering that lung cancer is the leading cause of cancer death and that the lungs are the primary organ SARS-CoV-2 attacks. Methods Using five lung adenocarcinoma (LUAD) genomics datasets, we explored associations between TMPRSS2 expression and immune signatures, cancer-associated pathways, tumor progression phenotypes, and clinical prognosis in LUAD by the bioinformatics approach. We validated the findings from the bioinformatics analysis through in vitro and in vivo experiments and clinical samples we collected. Results TMPRSS2 expression levels were negatively correlated with the enrichment levels of both antitumor immune signatures and immunosuppressive signatures in LUAD. However, TMPRSS2 expression levels showed a significant positive correlation with the ratios of immune-stimulatory/immune-inhibitory signatures (CD8 + T cells/PD-L1) in LUAD. TMPRSS2 downregulation correlated with elevated activities of many oncogenic pathways in LUAD, including cell cycle, mismatch repair, p53, and extracellular matrix signaling. TMPRSS2 downregulation correlated with increased proliferation, stemness, genomic instability, tumor advancement, and worse survival in LUAD. In vitro and in vivo experiments validated the association of TMPRSS2 deficiency with increased tumor cell proliferation and invasion and antitumor immunity in LUAD. Moreover, in vivo experiments demonstrated that TMPRSS2-knockdown tumors were more sensitive to BMS-1, an inhibitor of PD-1/PD-L1. Conclusion TMPRSS2 is a tumor suppressor, while its downregulation is a positive biomarker of immunotherapy in LUAD. Our data provide a connection between lung cancer and pneumonia caused by SARS-CoV-2 infection.

Genetic dissection of the final exocytosis steps in Paramecium tetraurelia cells: cytochemical determination of Ca2+-ATPase activity over performed exocytosis sites

1980 ◽  
Vol 46 (1) ◽  
pp. 17-40 ◽  
Author(s):  
H. Plattner ◽  
K. Reichel ◽  
H. Matt ◽  
J. Beisson ◽  
M. Lefort-Tran ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Freeze Fracture ◽  
Secretory Vesicles ◽  
Genetic Dissection ◽  
Paramecium Tetraurelia ◽  
Electron Microscopic ◽  
X Ray ◽  
Electron Microscopic Cytochemistry ◽  
Function Correlation

In different Paramecium tetraurelia strains the occurrence of a Ca2+-ATPase (or p-nitro-phenylphosphatase) activity at the preformed attachment and exocytosis sites of the secretory vesicles (trichocysts) was analysed by electron-microscopic cytochemistry and X-ray microanalysis. In conjunction with freeze-fracture studies it was found that only those strains, which contain rosette particles, display this Ca2+-ATPase activity (7S, K 40I, nd 9 (18 degrees C), while other strains (nd 6, nd 9 (27 degrees C), tam 38) are devoid of both these characteristics. The presence (absence) of rosette particles and of Ca2+-ATPase activity at the preformed exocytosis sites is correlated with the capability (incapability) of performing exocytosis in these strains. We discuss several possible interpretations of this structure-function correlation.

scholarly journals The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro

1981 ◽  
Vol 193 (3) ◽  
pp. 781-792 ◽  
Author(s):  
E A H Baydoun ◽  
D H Northcote
Keyword(s):  
Zea Mays ◽  
Atpase Activity ◽  
Membrane Fusion ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Root Cells ◽  
Dependent Atpase

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.

scholarly journals Proton Leakage Is Sensed by IM30 and Activates IM30-Triggered Membrane Fusion

2020 ◽  
Vol 21 (12) ◽  
pp. 4530 ◽  
Author(s):  
Carmen Siebenaller ◽  
Benedikt Junglas ◽  
Annika Lehmann ◽  
Nadja Hellmann ◽  
Dirk Schneider
Keyword(s):  
Membrane Fusion ◽  
Thylakoid Membrane ◽  
Physiological Function ◽  
Membrane Lipids ◽  
Membrane System ◽  
Dual Function ◽  
Charged Membrane ◽  
Proton Leakage ◽  
Chloroplast Stroma

The inner membrane-associated protein of 30 kDa (IM30) is crucial for the development and maintenance of the thylakoid membrane system in chloroplasts and cyanobacteria. While its exact physiological function still is under debate, it has recently been suggested that IM30 has (at least) a dual function, and the protein is involved in stabilization of the thylakoid membrane as well as in Mg2+-dependent membrane fusion. IM30 binds to negatively charged membrane lipids, preferentially at stressed membrane regions where protons potentially leak out from the thylakoid lumen into the chloroplast stroma or the cyanobacterial cytoplasm, respectively. Here we show in vitro that IM30 membrane binding, as well as membrane fusion, is strongly increased in acidic environments. This enhanced activity involves a rearrangement of the protein structure. We suggest that this acid-induced transition is part of a mechanism that allows IM30 to (i) sense sites of proton leakage at the thylakoid membrane, to (ii) preferentially bind there, and to (iii) seal leaky membrane regions via membrane fusion processes.

Axial flow modulates proximal tubule NHE3 and H-ATPase activities by changing microvillus bending moments

2006 ◽  
Vol 290 (2) ◽  
pp. F289-F296 ◽  
Author(s):  
Zhaopeng Du ◽  
Qingshang Yan ◽  
Yi Duan ◽  
Sheldon Weinbaum ◽  
Alan M. Weinstein ◽  
...  
Keyword(s):  
Cell Membrane ◽  
Atpase Activity ◽  
Actin Cytoskeleton ◽  
Proximal Tubule ◽  
Axial Flow ◽  
Paracellular Permeability ◽  
Cytochalasin D ◽  
Proximal Tubules ◽  
Permeability Changes

We have previously demonstrated that mouse proximal tubules in vitro respond to changes in luminal flow with proportional changes in Na+ absorption (Du Z, Duan Y, Yan Q, Weinstein AM, Weinbaum S, and Wang T. Proc Natl Acad Sci USA 101: 13068–13073, 2004). It was hypothesized that brush-border microvilli function as a sensor to detect and amplify luminal hydrodynamic forces and transmit them to the actin cytoskeleton. In the present study we examine whether 1) flow-dependent HCO3− transport is proportional to flow-dependent variations in microvillous torque (bending moment); 2) both luminal membrane Na+/H+ exchange (NHE3) and H+-ATPase activity are modulated by axial flow; and 3) paracellular permeabilities contribute to the flux perturbations. HCO3− absorption is examined by microperfusion of mouse S2 proximal tubules in vitro, with varying perfusion rates, and in the presence of the Na/H-exchange inhibitor EIPA, the H+-ATPase inhibitor bafilomycin, and the actin cytoskeleton inhibitor cytochalasin D. Paracellular permeability changes are assessed with measurements of epithelial HCO3− permeability and transepithelial potential difference (PD). It is found that 1) an increase in perfusion rate enhances HCO3− absorption and microvillous torque, and the fractional changes of each are nearly identical; 2) inhibition of NHE3 by EIPA, or H+-ATPase by bafilomycin, produced only partial inhibition of flow-stimulated bicarbonate transport; 3) disruption of the actin cytoskeleton by cytochalasin D blocked the increment of HCO3− absorption by high flow; and 4) HCO3− permeability and transepithelial PD are not modulated by flow. We conclude that flow-dependent modulation of proximal tubule HCO3− reabsorption is due to changes in both NHE3 and H+-ATPase activity within the luminal cell membrane and this requires an intact actin cytoskeleton. Paracellular permeability changes do not contribute to this flow dependence. Perfusion-absorption balance in the proximal tubule is a direct effect of flow-induced torque on brush-border microvilli to regulate luminal cell membrane transporter activity.

scholarly journals Proteins Needed for Vesicle Budding from the Golgi Complex Are Also Required for the Docking Step of Homotypic Vacuole Fusion

2000 ◽  
Vol 148 (6) ◽  
pp. 1223-1230 ◽  
Author(s):  
Albert Price ◽  
William Wickner ◽  
Christian Ungermann
Keyword(s):  
Membrane Fusion ◽  
Golgi Complex ◽  
Golgi Vesicle ◽  
Dual Function ◽  
Vesicle Budding ◽  
Cell Lysates ◽  
Vacuole Fusion ◽  
Transport Vesicles ◽  
In Trans

Vam2p/Vps41p is known to be required for transport vesicles with vacuolar cargo to bud from the Golgi. Like other VAM-encoded proteins, which are needed for homotypic vacuole fusion, we now report that Vam2p and its associated protein Vam6p/Vps39p are needed on each vacuole partner for homotypic fusion. In vitro vacuole fusion occurs in successive steps of priming, docking, and membrane fusion. While priming does not require Vam2p or Vam6p, the functions of these two proteins cannot be fulfilled until priming has occurred, and each is required for the docking reaction which culminates in trans-SNARE pairing. Consistent with their dual function in Golgi vesicle budding and homotypic fusion of vacuoles, approximately half of the Vam2p and Vam6p of the cell are recovered from cell lysates with purified vacuoles.

Trophoblast Cell Membrane Interactions at Implantation

1970 ◽  
Vol 28 ◽  
pp. 310-311
Author(s):  
A. C. Enders
Keyword(s):  
Epithelial Cells ◽  
Cell Membrane ◽  
Membrane Fusion ◽  
Trophoblast Cell ◽  
Membrane Interactions ◽  
Uterine Epithelial Cells ◽  
Functional Complex ◽  
Cytotrophoblast Cells ◽  
Complex Relationships ◽  
Syncytial Trophoblast

The alteration in membrane relationships seen at implantation include 1) interaction between cytotrophoblast cells to form syncytial trophoblast and addition to the syncytium by subsequent fusion of cytotrophoblast cells, 2) formation of a wide variety of functional complex relationships by trophoblast with uterine epithelial cells in the process of invasion of the endometrium, and 3) in the case of the rabbit, fusion of some uterine epithelial cells with the trophoblast.Formation of syncytium is apparently a membrane fusion phenomenon in which rapid confluence of cytoplasm often results in isolation of residual membrane within masses of syncytial trophoblast. Often the last areas of membrane to disappear are those including a desmosome where the cell membranes are apparently held apart from fusion.

Light Chain LC and TAT-EGFP-HCS of Botulinum Toxin Expression and Biological Function in vitro and in vivo

2019 ◽  
Vol 16 (3) ◽  
pp. 175-180
Author(s):  
Fengjin Hao ◽  
Yueqin Feng ◽  
Yifu Guan
Keyword(s):  
Biological Activity ◽  
Botulinum Toxin ◽  
Cell Membrane ◽  
Western Blot ◽  
Biological Function ◽  
C6 Cells ◽  
Green Fluorescence ◽  
Bv2 Cells

Objective: To verify whether the botulinum toxin heavy chain HCS has specific neuronal targeting function and to confirm whether TAT-EGFP-LC has hydrolyzable SNAP-25 and has transmembrane biological activity. Methods: We constructed the pET-28a-TAT-EGFP-HCS/LC plasmid. After the plasmid is expressed and purified, we co-cultured it with nerve cells or tumors. In addition, we used Western-Blot to identify whether protein LC and TAT-EGFP-LC can digest the protein SNAP-25. Results: Fluorescence imaging showed that PC12, BV2, C6 and HeLa cells all showed green fluorescence, and TAT-EGFP-HCS had the strongest fluorescence. Moreover, TAT-EGFP-LC can hydrolyze intracellular SNAP-25 in PC12 cells, C6 cells, BV2 cells and HeLa, whereas LC alone cannot. In addition, the in vivo protein TAT-EGFP-HCS can penetrate the blood-brain barrier and enter mouse brain tissue. Conclusion: TAT-EGFP-HSC expressed in vitro has neural guidance function and can carry large proteins across the cell membrane without influencing the biological activity.

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