scholarly journals Overproduction of Flotillin Influences Cell Differentiation and Shape in Bacillus subtilis

mBio ◽  
2013 ◽  
Vol 4 (6) ◽  
Author(s):  
Benjamin Mielich-Süss ◽  
Johannes Schneider ◽  
Daniel Lopez
Keyword(s):  
Signal Transduction ◽  
Bacillus Subtilis ◽  
Cell Differentiation ◽  
Lipid Rafts ◽  
Eukaryotic Cells ◽  
Membrane Microdomains ◽  
Functional Link ◽  
Working Model ◽  
Physiological Alterations

ABSTRACTBacteria organize many membrane-related signaling processes in functional microdomains that are structurally and functionally similar to the lipid rafts of eukaryotic cells. An important structural component of these microdomains is the protein flotillin, which seems to act as a chaperone in recruiting other proteins to lipid rafts to facilitate their interaction. In eukaryotic cells, the occurrence of severe diseases is often observed in combination with an overproduction of flotillin, but a functional link between these two phenomena is yet to be demonstrated. In this work, we used the bacterial modelBacillus subtilisas a tractable system to study the physiological alterations that occur in cells that overproduce flotillin. We discovered that an excess of flotillin altered specific signal transduction pathways that are associated with the membrane microdomains of bacteria. As a consequence of this, we detected significant defects in cell division and cell differentiation. These physiological alterations were in part caused by an unusual stabilization of the raft-associated protease FtsH. This report opens the possibility of using bacteria as a working model to better understand fundamental questions related to the functionality of lipid rafts.IMPORTANCEThe identification of signaling platforms in the membrane of bacteria that are functionally and structurally equivalent to eukaryotic lipid rafts reveals a level of sophistication in signal transduction and membrane organization unexpected in bacteria. It opens new and promising venues to address intricate questions related to the functionality of lipid rafts by using bacteria as a more tractable system. This is the first report that uses bacteria as a working model to investigate a fundamental question that was previously raised while studying the role of eukaryotic lipid rafts. It also provides evidence of the critical role of these signaling platforms in orchestrating diverse physiological processes in prokaryotic cells.

A Critical Role for Membrane Sulfatide in Platelet Aggregation.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 626-626
Author(s):  
Prasenjit Guchhait ◽  
Corie Shrimpton ◽  
Kochi Honke ◽  
Perumal Thiagarajan ◽  
Jose A. Lopez
Keyword(s):  
Platelet Aggregation ◽  
Lipid Rafts ◽  
Platelet Function ◽  
Critical Role ◽  
Membrane Microdomains ◽  
Systemic Lupus Erythematosis ◽  
Single Chain ◽  
Induce Platelet Aggregation ◽  
Adhesive Interactions

Abstract Sulfatide (galactocylceramide 3′-sulfate) is a sulfated glycosphingolipid expressed on the surfaces of erythrocytes, leukocytes, platelets and a variety other cells, that is known to interact with several cell adhesion molecules involved in hemostasis, including von Willebrand factor (VWF), laminin, thrombospondin, P-selectin and β2-glycoprotein I. Because these ligands are involved in many platelet adhesive interactions, we hypothesize that membrane sulfatide plays an important role in these processes. To examine this, we have cloned and purified a sulfatide-specific single-chain variable fragment (scFv) antibody from a phage-display library constructed from mRNA taken from the lymphocytes of patients with systemic lupus erythematosis. This scFv, PA38, specifically bound sulfatide, and did not react with the related sphingolipids cerebroside, ceramide, or sphingomyelin, or the phospholipids phosphatidylserine, phosphatidylcholine, or phosphatidylethanolamine. Using this tool, we examined the role of sulfatide in platelet function. We observed that PA38 dose-dependently (at 5 and 10 μg/ml) inhibited the aggregation of human platelets induced by either collagen or ADP. A control scFv produced in a similar manner had no effect. Furthermore, PA38 delayed platelet plug formation by 23 sec (with collagen-ADP agonist) and 46 sec (with collagen-epinephrine) in whole blood from normal human donors, as measured in a platelet function analyzer, PFA-100 (Dade Behring). Further, to verify that this was a sulfatide-specific effect, we compared collagen-induced platelet aggregation in normal mice to that of mice deficient in cerebroside sulfotransferase (CST)—a critical enzyme in the sulfatide synthetic pathway. The CST−/− mice fail to express sulfatide on the cell surface, and displayed defective platelet aggregation. Consistent with this, the PA38 also significantly inhibited collagen-induce platelet aggregation in wild-type mice. Given the importance of lipid rafts in signaling and adhesive processes, we looked for the localization of sulfatide in these membrane microdomains. Indeed, we found that sulfatide is enriched in lipid rafts suggesting a role for sulfatide in lipid-raft mediated events. Thus, we provide evidence for a key role of a membrane lipid, sulfatide in the adhesive interactions involved in platelet function. With one notable exception, the key adhesive roles in platelet-platelet interaction have all previously been assigned to proteins.

Modulation of Tissue Factor-Factor VIIa Signaling by Lipid Rafts and Caveolae.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1744-1744
Author(s):  
Vineet Awasthi ◽  
Samir Mandal ◽  
Veena Papanna ◽  
L. Vijaya Mohan Rao ◽  
Usha Pendurthi
Keyword(s):  
Cell Signaling ◽  
Lipid Rafts ◽  
G Proteins ◽  
Intracellular Signaling ◽  
Factor Viia ◽  
Membrane Microdomains ◽  
Caveolin 1 ◽  
Intracellular Signaling Pathways ◽  
Plasma Membrane Microdomains

Abstract Tissue factor (TF) is a cellular receptor for clotting factor VIIa (VIIa) and the formation of TF-VIIa complexes on cell surfaces not only triggers the coagulation cascade but also transduces cell signaling via activation of protease-activated receptors (PARs), particularly PAR2. Although a number of recent studies provide valuable information on intracellular signaling pathways that are activated by TF-VIIa, the role of various cell surface components in mediating the interaction of TF-VIIa with PARs, and the subsequent signal transmittance are unknown. Unlike thrombin and trypsin, VIIa has to bind to its cellular receptor (TF) to activate PARs. The inability of TF-VIIa to effectively activate Ca2+ signaling and failure to desensitize the signaling to subsequently added trypsin suggest that the TF-VIIa is a poor activator of PAR2. Despite this, a number of studies have shown that VIIa is as effective as trypsin or PAR2 agonist peptide in activating intracellular signaling pathways and gene expression in cells expressing TF. Although the potential mechanism for this phenomenon is unknown, compartmentalization of TF, PAR2, and G-proteins in plasma membrane microdomains could facilitate a robust TF-VIIa-induced PAR2-mediated cell signaling. Although certain G-protein coupled receptors and G-proteins are known to be segregated into specialized membrane microdomains, lipid rafts and caveolae, little is known whether PARs are segregated into lipid rafts and caveolae, and how such segregation might influence their activation by TF-VIIa and the subsequent coupling to G-proteins. To obtain answers to some of these questions, in the present study, we have characterized TF and PAR2 distribution on tumor cell surfaces and investigated the role of lipid raft/caveolae in modulating the TF-VIIa signaling in tumor cells. Detergent extraction of cells followed by fractionation on sucrose gradient centrifugation showed that TF and PAR2 were distributed both in lipid rafts (low-density) and soluble fractions. Immunofluorescence confocal microscopy revealed that TF at the cell surface is localized in discrete plasma membrane microdomains, and colocalized with caveolin-1, a structural integral protein of caveolae, indicating caveolar localization of TF. Similar to TF, PAR2 also displayed significant punctuate staining and colocalization with caveloin-1. Further, a substantial fraction of TF and PAR2 was colocalized in caveolae. Disruption of lipid rafts/caveolae by ß-methyl cyclodextrin or filipin treatments reduced TF association with PAR2 in lipid rafts and caveolar fractions and impaired the TF-VIIa-induced cell signaling (PI hydrolysis and IL-8 gene expression). Additional studies showed that both mßCD and filipin treatments specifically impaired TF-VIIa cleavage of PAR2 and but had no significant effect on trypsin cleavage of PAR2. Disruption of caveolae with caveolin-1 silencing had no effect on the TF-VIIa coagulant activity but inhibited the TF-VIIa-induced cell signaling. In summary, the data presented herein demonstrate that TF localization at the cell membrane could influence different functions of TF differently. While caveolar localization of TF had no influence in propagating the procoagulant activity of TF, it is essential in supporting the TF-VIIa-induced cell signaling.

Role for lipid rafts in regulating interleukin-2 receptor signaling

Blood ◽  
2001 ◽  
Vol 98 (5) ◽  
pp. 1489-1497 ◽  
Author(s):  
Mina D. Marmor ◽  
Michael Julius
Keyword(s):  
Lipid Rafts ◽  
Interleukin 2 ◽  
Phosphatidyl Inositol ◽  
Membrane Microdomains ◽  
Lipid Microdomains ◽  
Interleukin 2 Receptor ◽  
Lipid Environment ◽  
Plasma Membrane Microdomains ◽  
Nonionic Detergents

Lipid rafts are plasma membrane microdomains characterized by a unique lipid environment enriched in gangliosides and cholesterol, leading to their insolubility in nonionic detergents. Many receptors are constitutively or inducibly localized in lipid rafts, which have been shown to function as platforms coordinating the induction of signaling pathways. In this report, the first evidence is provided for a role of these lipid microdomains in regulating interleukin-2 receptor (IL-2R) signaling. It is demonstrated that antibody- or ligand-mediated immobilization of components of lipid rafts, glycosyl-phosphatidyl-inositol–anchored proteins, and the GM1 ganglioside, respectively, inhibit IL-2–induced proliferation in T cells. IL-2Rα is shown to be constitutively enriched in rafts and further enriched in the presence of immobilized anti–Thy-1. In contrast, IL-2Rβ and IL-2Rγ, as well as JAK1 and JAK3, are found in soluble membrane fractions, and their localization is not altered by anti–Thy-1. IL-2–mediated heterotrimerization of IL-2R chains is shown to occur within soluble membrane fractions, exclusively, as is the activation of JAK1 and JAK3. As predicted by these results, the disruption of lipid raft integrity did not impair IL-2–induced signaling. Thus, the sequestration of IL-2Rα within lipid microdomains restricts its intermolecular interactions and regulates IL-2R signaling through impeding its association with IL-2Rβ and IL-2Rγ.

scholarly journals Genetic evidence for signal transduction within the Bacillus subtilis GerA germinant receptor

2021 ◽  
Author(s):  
Jeremy D. Amon ◽  
Lior Artzi ◽  
David Z. Rudner
Keyword(s):  
Signal Transduction ◽  
Bacillus Subtilis ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Transduction Pathway ◽  
Sense And Respond ◽  
Enrichment Strategy ◽  
Functional Receptor

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to L-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA , gerAB , and gerAC . The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA* ) that carries a mutation in gerAA and show it constitutively activates germination even in the presence of a wild-type copy of gerA . Using an enrichment strategy to screen for suppressors of gerA* , we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one ( gerAB[E105K]) reduces the GerA complex's ability to respond to L-alanine, while another ( gerAB[F259S] ) disrupts the germinant signal downstream of L-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway. Importance Endospore formers are a broad group of bacteria that can enter dormancy upon starvation and exit dormancy upon sensing the return of nutrients. How dormant spores sense and respond to these nutrients is poorly understood. Here, we identify a key step in the signal transduction pathway that is activated after spores detect the amino acid L-alanine. We present a model that provides a more complete picture of this process that is critical for allowing dormant spores to germinate and resume growth.

scholarly journals Genetic evidence for signal transduction within the Bacillus subtilis GerA germinant receptor

2021 ◽  
Author(s):  
Jeremy D. Amon ◽  
Lior Artzi ◽  
David Z. Rudner
Keyword(s):  
Signal Transduction ◽  
Bacillus Subtilis ◽  
Signal Transduction Pathway ◽  
Dominant Negative ◽  
Bacterial Spores ◽  
Wild Type ◽  
Enrichment Strategy ◽  
Functional Receptor

Bacterial spores can rapidly exit dormancy through the process of germination. This process begins with the activation of nutrient receptors embedded in the spore membrane. The prototypical germinant receptor in Bacillus subtilis responds to L-alanine and is thought to be a complex of proteins encoded by the genes in the gerA operon: gerAA, gerAB, and gerAC. The GerAB subunit has recently been shown to function as the nutrient sensor, but beyond contributing to complex stability, no additional functions have been attributed to the other two subunits. Here, we investigate the role of GerAA. We resurrect a previously characterized allele of gerA (termed gerA*) that carries a mutation in gerAA and show it constitutively activates germination even in the presence of a wild-type copy of gerA. Using an enrichment strategy to screen for suppressors of gerA*, we identified mutations in all three gerA genes that restore a functional receptor. Characterization of two distinct gerAB suppressors revealed that one (gerAB-E105K) reduces the GerA complex's ability to respond to L-alanine, while another (gerAB-F259S) disrupts the germinant signal downstream of L-alanine recognition. These data argue against models in which GerAA is directly or indirectly involved in germinant sensing. Rather, our data suggest that GerAA is responsible for transducing the nutrient signal sensed by GerAB. While the steps downstream of gerAA have yet to be uncovered, these results validate the use of a dominant-negative genetic approach in elucidating the gerA signal transduction pathway.

Examination of the Role of Lipid Rafts in GPCR Signal Transduction Using Plasmon Waveguide Resonance (PWR) Spectroscopy

2010 ◽  
pp. 750-753
Author(s):  
Victor J. Hruby ◽  
Isabel D. Alves ◽  
Savitha Devanathan ◽  
Zdzislaw Salamon ◽  
Gordon Tollin
Keyword(s):  
Signal Transduction ◽  
Lipid Rafts ◽  
Gpcr Signal ◽  
Waveguide Resonance

A critical role of lipid rafts in the organization of a key FcγRIIa-mediated signaling pathway in human platelets

2003 ◽  
Vol 89 (02) ◽  
pp. 318-330 ◽  
Author(s):  
Stéphane Bodin ◽  
Cécile Viala ◽  
Ashraf Ragab ◽  
Bernard Payrastre
Keyword(s):  
Lipid Rafts ◽  
Critical Role ◽  
Adenosine Diphosphate ◽  
Human Platelets ◽  
Temporal Organization ◽  
Membrane Microdomains ◽  
Cross Linking ◽  
Dependent Manner ◽  
Signaling Complex

SummaryThe involvement of platelet FcγRIIa in heparin-associated thrombocytopenia (HIT) is now well established. However, the precise sequence of molecular events initiated by FcγRIIa cross-linking in platelets remains partly characterized. We investigated here the role of lipid rafts in the spatio-temporal organization of the FcγRIIa-dependent signaling events. Upon cross-linking, FcγRIIa relocated in rafts where the kinase Lyn and the adapter LAT were among the major phosphotyrosyl proteins. Upon stimulation by HIT sera, the second messenger phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) accumulated in rafts in a P2Y12 adenosine diphosphate (ADP) recep- tor-dependent manner. PtdIns(3,4,5)P3 was then essential to specifically recruit phospholipase Cγ2 (PLCγ2) to these membrane microdomains. Controlled disruption of rafts by methyl γ-cyclodextrin reversibly abolished PtdIns(3,4,5)P3 production, PLC activation and platelet responses induced by FcγRIIa cross-linking without affecting the tyrosine phosphorylation events. This work demonstrates that platelet rafts are essential for the integration of a key signaling complex leading to the rapid production of PtdIns(3,4,5)P3 and in turn PLCγ2 activation during HIT.

CtaG is required for formation of active cytochrome c oxidase in Bacillus subtilis

Microbiology ◽  
2004 ◽  
Vol 150 (2) ◽  
pp. 415-425 ◽  
Author(s):  
Jenny Bengtsson ◽  
Claes von Wachenfeldt ◽  
Lena Winstedt ◽  
Per Nygaard ◽  
Lars Hederstedt
Keyword(s):  
Bacillus Subtilis ◽  
Copper Ions ◽  
Eukaryotic Cells ◽  
Gram Positive ◽  
Gram Positive Bacteria ◽  
Assembly Factor ◽  
Membrane Bound ◽  
Respiratory Oxidases ◽  
Copper Centre

The Gram-positive bacterium Bacillus subtilis contains two respiratory oxidases of the haem-copper superfamily: cytochrome aa 3, which is a quinol oxidase, and cytochrome caa 3, which is a cytochrome c oxidase. Cytochrome c oxidase uniquely contains a di-copper centre, CuA. B. subtilis CtaG is a membrane protein encoded by the same gene cluster as that which encodes the subunits of cytochrome c oxidase. The role of B. subtilis CtaG and orthologous proteins present in many other Gram-positive bacteria has remained unexplored. The sequence of CtaG is unrelated to that of CtaG/Cox11p of proteobacteria and eukaryotic cells. This study shows that B. subtilis CtaG is essential for the formation of active cytochrome caa 3 but is not required for assembly of the core subunits I and II with haem in the membrane and it has no role in the synthesis of active cytochrome aa 3. B. subtilis YpmQ, a homologue to Sco1p of eukaryotic cells, is also a membrane-bound cytochrome c oxidase-specific assembly factor. Properties of CtaG- and YpmQ-deficient mutants were compared. Cells lacking YpmQ showed a low cytochrome c oxidase activity and this defect was suppressed by the supplementation of the growth medium with copper ions. It has previously been proposed that YpmQ/Sco1p is involved in synthesis of the CuA centre. The results of this study are consistent with this proposal but the exact role of YpmQ in assembly of cytochrome c oxidase remains to be elucidated.

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