Regulation of aurora B kinase by the lipid raft protein flotillin-1.
ABSTRACT: The lipid raft protein Flotillin-1 was previously shown to be required for cell proliferation. Here we show that it is critical for the maintenance of the levels of the mitotic regulator Aurora B. Knockdown of Flotillin-1 induced aberrant mitotic events similar to those produced by Aurora B depletion and led to a marked decline in Aurora B levels and activity. Transfection of wild-type full-length Flotillin-1 or forms directed to the nucleus increased Aurora B levels and activity. Flotillin-1 interacted with Aurora B directly through its SPFH domain in a complex distinct from the chromosomal passenger protein complex, and the two proteins co-purified in nuclear, non-raft fractions. These observations are the first evidence for a function of Flotillin-1 outside of lipid rafts and suggest its critical role in the maintenance of a pool of active Aurora B.
Project description:Lipid rafts are dynamic, nanoscale assemblies of specific proteins and lipids, distributed heterogeneously on eukaryotic membrane. Flotillin-1, a conserved eukaryotic raft marker protein (RMP) harbor SPFH (Stomatin, Prohibitin, Flotillin, and HflK/C) and oligomerization domains to regulate various cellular processes through its interactions with other signaling or transport proteins. Rafts were thought to be absent in prokaryotes hitherto, but recent report of its presence and significance in physiology of Bacillus subtilis prompted us to investigate the same in pathogenic bacteria (PB) also. In prokaryotes, proteins of SPFH2a subfamily show highest identity to SPFH domain of Flotillin-1. Moreover, bacterial genome organization revealed that Flotillin homolog harboring SPFH2a domain exists in an operon with an upstream gene containing NFeD domain. Here, presence of RMP in PB was initially investigated in silico by analyzing the presence of SPFH2a, oligomerization domains in the concerned gene and NfeD domain in the adjacent upstream gene. After investigating 300 PB, four were found to harbor RMP. Among them, domains of Bas0525 (FlotP) of Bacillus anthracis (BA) showed highest identity with characteristic domains of RMP. Considering the global threat of BA as the bioterror agent, it was selected as a model for further in vitro characterization of rafts in PB. In silico and in vitro analysis showed significant similarity of FlotP with numerous attributes of Flotillin-1. Its punctate distribution on membrane with exclusive localization in detergent resistant membrane fraction; strongly favors presence of raft with RMP FlotP in BA. Furthermore, significant effect of Zaragozic acid (ZA), a raft associated lipid biosynthesis inhibitor, on several patho-physiological attributes of BA such as growth, morphology, membrane rigidity etc., were also observed. Specifically, a considerable decrease in membrane rigidity, strongly recommended presence of an unknown raft associated lipid molecule on membrane of BA. In addition, treatment with ZA decreased secretion of anthrax toxins and FlotP expression, suggesting potential role of raft in pathogenesis and physiology of BA. Thus, the present study not only suggest the existence and role of raft like entity in pathophysiology of BA but also its possible use for the development of novel drugs or vaccines against anthrax.
Project description:Flotillin/reggie proteins are membrane-associated proteins present in all kinds of cells and belong to the family of proteins carrying the SPFH (stomatin, prohibitin, flotillin, and HflK/HflC) domain. In addition to this domain of unknown function, flotillin proteins are characterized by the flotillin domain, which is rich in heptad repeats. Bacterial flotillin orthologs have recently been shown to be part of lipid rafts, like their eukaryotic counterparts, and to be involved in signaling events. Double deletions of floT and the gene encoding the second flotillin-like protein in Bacillus subtilis, floA, show strong synthetic defects in cell morphology, motility, and transformation efficiency. The lack of FloT resulted in a marked defect in motility. Using total internal reflection fluorescence (TIRF) microscopy, we show that both proteins localize in characteristic focal structures within the cell membrane, which move in a highly dynamic and random manner but localize independently of each other. Thus, flotillin paralogs act in a spatially distinct manner. Flotillin domains in both FloA and FloT are essential for focal assemblies and for the proper function of flotillins. Both flotillin genes are situated next to genes encoding NfeD proteins. FloT dramatically affects the localization of NfeD2: FloT apparently recruits NfeD2 into the focal assemblies, documenting a close interaction between flotillins and NfeDs in bacteria. In contrast, the localization of NfeD1b is not affected by FloA, FloT, or NfeD2. FloA does not show a spatial connection with the upstream-encoded NfeD1b (YqeZ). Our work establishes that bacterial flotillin-like proteins have overlapping functions in a variety of membrane-associated processes and that flotillin domain-mediated assembly and NfeD proteins play important roles in setting up the flotillin raft-like structures in vivo.
Project description:Venosomes are extracellular vesicles found in the venom of Leptopilina endoparasitoids wasps, which transport and target virulence factors to impair the parasitoid egg encapsulation by the lamellocytes of their Drosophila melanogaster host larva. Using the co-immunolocalization of fluorescent L. boulardi venosomes and one of the putative-transported virulence factors, LbGAP, with known markers of cellular endocytosis, we show that venosomes endocytosis by lamellocytes is not a process dependent on clathrin or macropinocytosis and internalization seems to bypass the early endosomal compartment Rab5. After internalization, LbGAP colocalizes strongly with flotillin-1 and the GPI-anchored protein Atilla/L1 (a lamellocyte surface marker) suggesting that entry occurs via a flotillin/lipid raft-dependent pathway. Once internalized, venosomes reach all intracellular compartments, including late and recycling endosomes, lysosomes, and the endoplasmic reticulum network. Venosomes therefore enter their target cells by a specific mechanism and the virulence factors are widely distributed in the lamellocytes' compartments to impair their functions.
Project description:Plasma membranes of lens fiber cells have high levels of long-chain saturated fatty acids, cholesterol, and sphingolipids-key components of lipid rafts. Thus, lipid rafts are expected to constitute a significant portion of fiber cell membranes and play important roles in lens biology. The purpose of this study was to characterize the lens lipid raft proteome.Quantitative proteomics, both label-free and iTRAQ methods, were used to characterize lens fiber cell lipid raft proteins. Detergent-resistant, lipid raft membrane (DRM) fractions were isolated by sucrose gradient centrifugation. To confirm protein localization to lipid rafts, protein sensitivity to cholesterol removal by methyl-?-cyclodextrin was quantified by iTRAQ analysis.A total of 506 proteins were identified in raft-like detergent-resistant membranes. Proteins identified support important functions of raft domains in fiber cells, including trafficking, signal transduction, and cytoskeletal organization. In cholesterol-sensitivity studies, 200 proteins were quantified and 71 proteins were strongly affected by cholesterol removal. Lipid raft markers flotillin-1 and flotillin-2 and a significant fraction of AQP0, MP20, and AQP5 were found in the DRM fraction and were highly sensitive to cholesterol removal. Connexins 46 and 50 were more abundant in nonraft fractions, but a small fraction of each was found in the DRM fraction and was strongly affected by cholesterol removal. Quantification of modified AQP0 confirmed that fatty acylation targeted this protein to membrane raft domains.These data represent the first comprehensive profile of the lipid raft proteome of lens fiber cells and provide information on membrane protein organization in these cells.
Project description:The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen.
Project description:Flotillin-1 (Flot1) is an evolutionary conserved, ubiquitously expressed lipid raft-associated scaffolding protein. Migration of Flot1-deficient neutrophils is impaired because of a decrease in myosin II-mediated contractility. Flot1 also accumulates in the uropod of polarized T cells, suggesting an analogous role in T cell migration. In this study, we analyzed morphology and migration parameters of murine wild-type and Flot1-/- CD8+ T cells using in vitro assays and intravital two-photon microscopy of lymphoid and nonlymphoid tissues. Flot1-/- CD8+ T cells displayed significant alterations in cell shape and motility parameters in vivo but showed comparable homing to lymphoid organs and intact in vitro migration to chemokines. Furthermore, their clonal expansion and infiltration into nonlymphoid tissues during primary and secondary antiviral immune responses was comparable to wild-type CD8+ T cells. Taken together, Flot1 plays a detectable but unexpectedly minor role for CD8+ T cell behavior under physiological conditions.
Project description:Stomatin, prohibitin, flotillin, and HflK/C (SPFH) domain proteins are membrane proteins that are widely conserved from bacteria to mammals. The molecular functions of these proteins have not been established. In mammals, the domain is often found in raft-associated proteins such as flotillin and podocin. We determined the structure of the SPFH domain of PH0470 derived from Pyrococcus horikoshii using NMR. The structure closely resembles that of the SPFH domain of the paralog PH1511, except for two C-terminal helices. The results show that the SPFH domain forms stable dimers, trimers, tetramers, and multimers, although it lacks the coiled-coil region for oligomerization, which is a highly conserved region in this protein family. The oligomers exhibited unusual thermodynamic behavior, as determined by circular dichroism, NMR, gel filtration, chemical cross-linking, and analytical ultracentrifugation. The oligomers were converted into monomers when they were heated once and then cooled. This transition was one-way and irreversible. We propose a mechanism of domain swapping for forming dimers as well as successive oligomers. The results of this study provide what to our knowledge are new insights into the common molecular function of the SPFH domain, which may act as a membrane skeleton through oligomerization by domain swapping.
Project description:We show that highly efficient depletion of sphingolipids in two different cell lines does not abrogate the ability to isolate Lubrol-based DRMs (detergent-resistant membranes) or detergent-free lipid rafts from these cells. Compared with control, DRM/detergent-free lipid raft fractions contain equal amounts of protein, cholesterol and phospholipid, whereas the classical DRM/lipid raft markers Src, caveolin-1 and flotillin display the same gradient distribution. DRMs/detergent-free lipid rafts themselves are severely depleted of sphingolipids. The fatty acid profile of the remaining sphingolipids as well as that of the glycerophospholipids shows several differences compared with control, most prominently an increase in highly saturated C(16) species. The glycerophospholipid headgroup composition is unchanged in sphingolipid-depleted cells and cell-derived detergent-free lipid rafts. Sphingolipid depletion does not alter the localization of MRP1 (multidrug-resistance-related protein 1) in DRMs/detergent-free lipid rafts or MRP1-mediated efflux of carboxyfluorescein. We conclude that extensive sphingolipid depletion does not affect lipid raft integrity in two cell lines and does not affect the function of the lipid-raft-associated protein MRP1.
Project description:Studies of detergent-resistant membrane (DRM) rafts in mature erythrocytes have facilitated identification of proteins that regulate formation of endovacuolar structures such as the parasitophorous vacuolar membrane (PVM) induced by the malaria parasite Plasmodium falciparum. However, analyses of raft lipids have remained elusive because detergents interfere with lipid detection. Here, we use primaquine to perturb the erythrocyte membrane and induce detergent-free buoyant vesicles, which are enriched in cholesterol and major raft proteins flotillin and stomatin and contain low levels of cytoskeleton, all characteristics of raft microdomains. Lipid mass spectrometry revealed that phosphatidylethanolamine and phosphatidylglycerol are depleted in endovesicles while phosphoinositides are highly enriched, suggesting raft-based endovesiculation can be achieved by simple (non-receptor-mediated) mechanical perturbation of the erythrocyte plasma membrane and results in sorting of inner leaflet phospholipids. Live-cell imaging of lipid-specific protein probes showed that phosphatidylinositol (4,5) bisphosphate (PIP(2)) is highly concentrated in primaquine-induced vesicles, confirming that it is an erythrocyte raft lipid. However, the malarial PVM lacks PIP(2), although another raft lipid, phosphatidylserine, is readily detected. Thus, different remodeling/sorting of cytoplasmic raft phospholipids may occur in distinct endovacuoles. Importantly, erythrocyte raft lipids recruited to the invasion junction by mechanical stimulation may be remodeled by the malaria parasite to establish blood-stage infection.
Project description:Bacteria 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 model Bacillus subtilis as 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.The 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.