scholarly journals Orai1α, but not Orai1β, co-localizes with TRPC1 and is required for its plasma membrane location and activation in HeLa cells

2022 ◽  
Vol 79 (1) ◽  
Author(s):  
Jose Sanchez-Collado ◽  
Jose J. Lopez ◽  
Isaac Jardin ◽  
Alejandro Berna-Erro ◽  
Pedro J. Camello ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
Dependent Inactivation ◽  
Crac Channel ◽  
Membrane Location

AbstractThe identification of two variants of the canonical pore-forming subunit of the Ca2+ release-activated Ca2+ (CRAC) channel Orai1, Orai1α and Orai1β, in mammalian cells arises the question whether they exhibit different functional characteristics. Orai1α and Orai1β differ in the N-terminal 63 amino acids, exclusive of Orai1α, and show different sensitivities to Ca2+-dependent inactivation, as well as distinct ability to form arachidonate-regulated channels. We have evaluated the role of both Orai1 variants in the activation of TRPC1 in HeLa cells. We found that Orai1α and Orai1β are required for the maintenance of regenerative Ca2+ oscillations, while TRPC1 plays a role in agonist-induced Ca2+ influx but is not essential for Ca2+ oscillations. Using APEX2 proximity labeling, co-immunoprecipitation and the fluorescence of G-GECO1.2 fused to Orai1α our results indicate that agonist stimulation and Ca2+ store depletion enhance Orai1α–TRPC1 interaction. Orai1α is essential for TRPC1 plasma membrane location and activation. Thus, TRPC1 function in HeLa cells depends on Ca2+ influx through Orai1α exclusively.

scholarly journals Mutational Analysis of the Human Immunodeficiency Virus Type 1 Vpu Transmembrane Domain That Promotes the Enhanced Release of Virus-Like Particles from the Plasma Membrane of Mammalian Cells

1998 ◽  
Vol 72 (2) ◽  
pp. 1270-1279 ◽  
Author(s):  
Mousumi Paul ◽  
Suparna Mazumder ◽  
Nicholas Raja ◽  
M. Abdul Jabbar
Keyword(s):  
Amino Acids ◽  
Human Immunodeficiency Virus ◽  
Plasma Membrane ◽  
Human Immunodeficiency Virus Type ◽  
Hela Cells ◽  
Mammalian Cells ◽  
Transmembrane Domain ◽  
Wild Type ◽  
Immunodeficiency Virus

ABSTRACT Human immunodeficiency virus type 1 Vpu is a multifunctional phosphoprotein composed of the N-terminal transmembrane (VpuTM) and C-terminal cytoplasmic domains. Each of these domains regulates a distinct function of the protein; the transmembrane domain is critical in virus release, and phosphorylation of the cytoplasmic domain is necessary for CD4 proteolysis. We carried our experiments to identify amino acids in the VpuTM domain that are important in the process of virus-like particle (VLP) release from HeLa cells. VLPs are released from the plasma membrane of HeLa cells at constitutive levels, and Vpu expression enhanced the release of VLPs by a factor of 10 to 15. Deletion of two to five amino acids from both N- and C-terminal ends or the middle of the VpuTM domain generated mutant Vpu proteins that have lost the ability to enhance VLP release. These deletion mutants have not lost the ability to associate with the wild-type or mutant Vpu proteins and formed complexes with equal efficiency. They were also transported normally to the Golgi complex. Furthermore, a Vpu protein having the CD4 transmembrane and Vpu cytoplasmic domains was completely inactive, and Vpu proteins harboring hybrid Vpu-CD4 TM domains were also defective in the ability to enhance the release of VLPs. When tested for functional complementation in cotransfected cells, two inactive proteins were not able to reconstitute Vpu activity that enhances the release of Gag particles. Coexpression of functional CD4/Vpu hybrids or wild-type Vpu with inactive mutant CD4/Vpu proteins revealed that mutations in the VpuTM domain could dominantly interfere with Vpu activity in Gag release. Taken together, these results demonstrated that the structural integrity of the VpuTM domain is critical for Vpu activity in the release of VLPs from the plasma membrane of mammalian cells.

scholarly journals Regulation of Interorganellar Ca2+ Transfer and NFAT Activation by the Mitochondrial Ca2+ Uniporter

2021 ◽  
Author(s):  
Ryan E. Yoast ◽  
Scott M. Emrich ◽  
Xuexin Zhang ◽  
Ping Xin ◽  
Vikas Arige ◽  
...  
Keyword(s):  
Nuclear Translocation ◽  
Channel Activity ◽  
Crac Channels ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
Dependent Inactivation ◽  
Nfat Activation ◽  
Crac Channel ◽  
Cellular Bioenergetics ◽  
Trisphosphate Receptor

Mitochondrial Ca2+ uptake is crucial for coupling receptor stimulation to cellular bioenergetics. Further, Ca2+ uptake by respiring mitochondria prevents Ca2+-dependent inactivation (CDI) of store-operated Ca2+ release-activated Ca2+ (CRAC) channels and inhibits Ca2+ extrusion to sustain cytosolic Ca2+ signaling. However, how Ca2+ uptake by the mitochondrial Ca2+ uniporter (MCU) shapes receptor-evoked interorganellar Ca2+ signaling is unknown. Here, we generated several cell lines with MCU-knockout (MCU-KO) as well as tissue-specific MCU-knockdown mice. We show that mitochondrial depolarization, but not MCU-KO, inhibits store-operated Ca2+ entry (SOCE). Paradoxically, despite enhancing Ca2+ extrusion and promoting CRAC channel CDI, MCU-KO increased cytosolic Ca2+ in response to store depletion. Further, physiological agonist stimulation in MCU-KO cells led to enhanced frequency of cytosolic Ca2+ oscillations, endoplasmic reticulum Ca2+ refilling, NFAT nuclear translocation and proliferation. However, MCU-KO did not affect inositol-1,4,5-trisphosphate receptor activity. Mathematical modeling supports that MCU-KO enhances cytosolic Ca2+, despite limiting CRAC channel activity.

scholarly journals Host Cell Plasma Membrane Phosphatidylserine Regulates the Assembly and Budding of Ebola Virus

2015 ◽  
Vol 89 (18) ◽  
pp. 9440-9453 ◽  
Author(s):  
Emmanuel Adu-Gyamfi ◽  
Kristen A. Johnson ◽  
Mark E. Fraser ◽  
Jordan L. Scott ◽  
Smita P. Soni ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Host Cell ◽  
Human Cell ◽  
Mammalian Cells ◽  
Matrix Protein ◽  
Ebola Virus ◽  
Cell Plasma Membrane ◽  
Replication Process ◽  
Cell Plasma

ABSTRACTLipid-enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. Ebola virus, which buds from the plasma membrane of the host cell, causes viral hemorrhagic fever and has a high fatality rate. To date, little has been known about how budding and egress of Ebola virus are mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of Ebola virus matrix protein VP40. VP40 binds PS-containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress, whereas PS is typically found only on the inner leaflet. Taking the data together, we present a model accounting for the role of plasma membrane PS in assembly of Ebola virus-like particles.IMPORTANCEThe lipid-enveloped Ebola virus causes severe infection with a high mortality rate and currently lacks FDA-approved therapeutics or vaccines. Ebola virus harbors just seven genes in its genome, and there is a critical requirement for acquisition of its lipid envelope from the plasma membrane of the human cell that it infects during the replication process. There is, however, a dearth of information available on the required contents of this envelope for egress and subsequent attachment and entry. Here we demonstrate that plasma membrane phosphatidylserine is critical for Ebola virus budding from the host cell plasma membrane. This report, to our knowledge, is the first to highlight the role of lipids in human cell membranes in the Ebola virus replication cycle and draws a clear link between selective binding and transport of a lipid across the membrane of the human cell and use of that lipid for subsequent viral entry.

scholarly journals The role of the yeast plasma membrane SPS nutrient sensor in the metabolic response to extracellular amino acids

2008 ◽  
Vol 42 (1) ◽  
pp. 215-228 ◽  
Author(s):  
Hanna Forsberg ◽  
C. Fredrik Gilstring ◽  
Arezou Zargari ◽  
Paula Martínez ◽  
Per O. Ljungdahl
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Metabolic Response ◽  
Yeast Plasma Membrane

scholarly journals THE ROLE OF METHIONINE DEFICIENCY IN POLIOVIRUS REPLICATION IN TISSUE CULTURES

1966 ◽  
Vol 123 (1) ◽  
pp. 17-24 ◽  
Author(s):  
Soussan Mohajer ◽  
Janis Gabliks
Keyword(s):  
Amino Acids ◽  
Hela Cells ◽  
Inhibitory Effect ◽  
Monkey Kidney ◽  
Kidney Cells ◽  
Tissue Cultures ◽  
Experimental Conditions ◽  
Methionine Deficiency ◽  
Intracellular Amino Acids

The role of methionine in poliovirus infection in HeLa and monkey kidney cells was investigated by using the methionine analogue l-ethionine. In the presence of 2.0 x 10–3 and 4.0 x 10–3 moles ethionine, the growth of HeLa and monkey kidney cells was significantly inhibited. Under the same experimental conditions, ethionine had no significant effect on the biosynthesis of two strains of poliovirus (Mahoney and Lansing) in HeLa cells, whereas in primary monkey kidney cells, it markedly inhibited the biosynthesis of the Lansing strain of poliovirus. HeLa cells partly depleted of their intracellular amino acids did not change the rate of viral biosynthesis. The inhibitory effect of ethionine on cell growth and viral biosynthesis was reversed by addition of an excess of l-methionine.

Interaction of vaginal Lactobacillus strains with HeLa cells plasma membrane

2017 ◽  
Vol 8 (4) ◽  
pp. 625-633 ◽  
Author(s):  
N. Calonghi ◽  
C. Parolin ◽  
G. Sartor ◽  
L. Verardi ◽  
B. Giordani ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Physical Properties ◽  
Hela Cells ◽  
Protective Role ◽  
Vaginal Infections ◽  
Vaginal Lactobacilli ◽  
Cell Line Hela ◽  
Cervical Cells ◽  
Host Epithelium

Vaginal lactobacilli offer protection against recurrent urinary and vaginal infections. The precise mechanisms underlying the interaction between lactobacilli and the host epithelium remain poorly understood at the molecular level. Deciphering such events can provide valuable information on the mode of action of commensal and probiotic bacteria in the vaginal environment. We investigated the effects exerted by five Lactobacillus strains of vaginal origin (Lactobacillus crispatus BC1 and BC2, Lactobacillus gasseri BC9 and BC11 and Lactobacillus vaginalis BC15) on the physical properties of the plasma membrane in a cervical cell line (HeLa). The interaction of the vaginal lactobacilli with the cervical cells determined two kinds of effects on plasma membrane: (1) modification of the membrane polar lipid organisation and the physical properties (L. crispatus BC1 and L. gasseri BC9); (2) modification of α5β1 integrin organisation (L. crispatus BC2, L. gasseri BC11 and L. vaginalis BC15). These two mechanisms can be at the basis of the protective role of lactobacilli against Candida albicans adhesion. Upon stimulation with all Lactobacillus strains, we observed a reduction of the basal oxidative stress in HeLa cells that could be related to modifications in physical properties and organisation of the plasma membrane. These results confirm the strictly strain-specific peculiarities of Lactobacillus and deepen the understanding of the mechanisms underlying the health-promoting role of this genus within the vaginal ecosystem.

scholarly journals Different consequences of cataract-associated mutations at adjacent positions in the first extracellular boundary of connexin50

2011 ◽  
Vol 300 (5) ◽  
pp. C1055-C1064 ◽  
Author(s):  
Jun-Jie Tong ◽  
Peter J. Minogue ◽  
Wenji Guo ◽  
Tung-Ling Chen ◽  
Eric C. Beyer ◽  
...  
Keyword(s):  
Amino Acids ◽  
Plasma Membrane ◽  
Hela Cells ◽  
Xenopus Oocytes ◽  
Dominant Negative ◽  
Wild Type ◽  
Gap Junction Channels ◽  
Dominant Negative Inhibitor ◽  
Intercellular Channels ◽  
Gap Junctional

Gap junction channels, which are made of connexins, are critical for intercellular communication, a function that may be disrupted in a variety of diseases. We studied the consequences of two cataract-associated mutations at adjacent positions at the first extracellular boundary in human connexin50 (Cx50), W45S and G46V. Both of these mutants formed gap junctional plaques when they were expressed in HeLa cells, suggesting that they trafficked to the plasma membrane properly. However, their functional properties differed. Dual two-microelectrode voltage-clamp studies showed that W45S did not form functional intercellular channels in paired Xenopus oocytes or hemichannel currents in single oocytes. When W45S was coexpressed with wild-type Cx50, the mutant acted as a dominant negative inhibitor of wild-type function. In contrast, G46V formed both functional gap junctional channels and hemichannels. G46V exhibited greatly enhanced currents compared with wild-type Cx50 in the presence of physiological calcium concentrations. This increase in hemichannel activity persisted when G46V was coexpressed with wild-type lens connexins, consistent with a dominant gain of hemichannel function for G46V. These data suggest that although these two mutations are in adjacent amino acids, they have very different effects on connexin function and cause disease by different mechanisms: W45S inhibits gap junctional channel function; G46V reduces cell viability by forming open hemichannels.

scholarly journals Critical Role of Dispensable Genes in Mycoplasma agalactiae Interaction with Mammalian Cells

2010 ◽  
Vol 78 (4) ◽  
pp. 1542-1551 ◽  
Author(s):  
Eric Baranowski ◽  
Sébastien Guiral ◽  
Eveline Sagné ◽  
Agnès Skapski ◽  
Christine Citti
Keyword(s):  
Hela Cells ◽  
Mammalian Cells ◽  
Critical Role ◽  
Genomic Region ◽  
Mycoplasma Agalactiae ◽  
Transposon Insertion ◽  
Mammalian Cell Lines ◽  
Fold Reduction ◽  
Genomic Regions

ABSTRACT Mycoplasmas are minimal bacteria whose genomes barely exceed the smallest amount of information required to sustain autonomous life. Despite this apparent simplicity, several mycoplasmas are successful pathogens of humans and animals, in which they establish intimate interactions with epithelial cells at mucosal surfaces. To identify biological functions mediating mycoplasma interactions with mammalian cells, we produced a library of transposon knockout mutants in the ruminant pathogen Mycoplasma agalactiae and used this library to identify mutants displaying a growth-deficient pheonotype in cell culture. M. agalactiae mutants displaying a 3-fold reduction in CFU titers to nearly complete extinction in coculture with HeLa cells were identified. Mapping of transposon insertion sites revealed 18 genomic regions putatively involved in the interaction of M. agalactiae with HeLa cells. Several of these regions encode proteins with features of membrane lipoproteins and/or were involved in horizontal gene transfer with phylogenetically distant pathogenic mycoplasmas of ruminants. Two mutants with the most extreme phenotype carry a transposon in a genomic region designated the NIF locus which encodes homologues of SufS and SufU, two proteins presumably involved in [Fe-S] cluster biosynthesis in Gram-positive bacteria. Complementation studies confirmed the conditional essentiality of the NIF locus, which was found to be critical for proliferation in the presence of HeLa cells and several other mammalian cell lines but dispensable for axenic growth. While our results raised questions regarding essential functions in mycoplasmas, they also provide a means for studying the role of mycoplasmas as minimal pathogens.

scholarly journals Omnitemporal choreographies of IP3R and all five STIM/Orai underlie the complexity of mammalian Ca2+ signaling

2020 ◽  
Author(s):  
Scott M. Emrich ◽  
Ryan E. Yoast ◽  
Ping Xin ◽  
Vikas Arige ◽  
Larry E. Wagner ◽  
...  
Keyword(s):  
Gradual Increase ◽  
Receptor Activation ◽  
List Type ◽  
Activated T Cells ◽  
Full Spectrum ◽  
Store Depletion ◽  
Agonist Stimulation ◽  
Evolutionarily Conserved ◽  
Redundant Functions

SummaryInvertebrates express one endoplasmic reticulum (ER)-resident Ca2+-sensing stromal-interaction molecule (Stim) and one Orai plasma membrane channel protein. Stim conveys store depletion to Orai, mediating the evolutionarily conserved Ca2+ release-activated Ca2+ (CRAC) current. The crucial role of their vertebrate homologues, STIM1 and Orai1 in mediating CRAC activity in mammals is well-established. However, mammals possess two STIM and three Orai isoforms and the choreography of their interactions under physiological receptor activation is unknown. We show that the five mammalian STIM1/2 and Orai1/2/3 isoforms have non-redundant functions. Yet, all five isoforms are always required together to ensure the graded diversity of mammalian Ca2+ signaling events in response to the full spectrum of agonist strengths. Receptor-activated Ca2+ signaling across the range of stimulus intensities requires functional interactions between not only STIM1/2 and Orai1/2/3, but also IP3R, ensuring that receptor-mediated Ca2+ release is precisely tailored to Ca2+ entry and activation of nuclear factor of activated T-cells (NFAT). This is orchestrated by two interdependent and counterbalancing paradigms: the N-termini Ca2+-binding ER-luminal domains of unactivated STIM1/2 inhibit IP3R-evoked Ca2+ release. Gradual increase in agonist intensity leads to gradual STIM1/2 activation and relief of IP3R inhibition. Concomitantly, the cytosolic C-termini of activated STIM1/2 differentially interact with Orai1/2/3 proteins as agonist intensity increases. Thus, coordinated and omnitemporal functions of all five STIM/Orai proteins and IP3Rs at the ER-lumen and cytosol translate the strength of agonist stimulation to precise levels of Ca2+ release, Ca2+ entry and NFAT induction, ensuring the diversity and fidelity of complex mammalian Ca2+ signaling.HighlightsAll five STIM/Orai and IP3R are always required together in mammalian Ca2+ signallingUnactivated STIM1/2 inhibit IP3R and activated STIM1/2 cooperatively activate Orai1/2/3STIM1 contribution increases and that of STIM2 decreases as agonist intensifiesGraded IP3R disinhibition and Orai activation tailor receptor activity to NFAT induction

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