Dissecting the Molecular Properties of Prokaryotic Flotillins

Flotillins are universally conserved proteins that are present in all kingdoms of life. Recently it was demonstrated that the B. subtilis flotillin YuaG (FloT) has a direct influence on membrane domain formation by orchestrating lipid domains. Thereby it allocates a proper environment for diverse cellular machineries. YuaG creates platforms for signal transduction, processes crucial for biofilm formation, sporulation, competence, secretion, and others. Even though, flotillins are an emerging topic of research in the field of microbiology little is known about the molecular architecture of prokaryotic flotillins. All flotillins share common structural elements and are tethered to the membrane N’- terminally, followed by a so called PHB domain and a flotillin domain. We show here that prokaryotic flotillins are, similarly to eukaryotic flotillins, tethered to the membrane via a hairpin loop. Further it is demonstrated by sedimentation assays that B. subtilis flotillins do not bind to the membrane via their PHB domain contrary to eukaryotic flotillins. Size exclusion chromatography experiments, blue native PAGE and cross linking experiments revealed that B. subtilis YuaG can oligomerize into large clusters via the PHB domain. This illustrates an important difference in the setup of prokaryotic flotillins compared to the organization of eukaryotic flotillins.     

Spatio-temporal Remodeling of Functional Membrane Microdomains Organizes the Signaling Networks of a Bacterium

Lipid rafts are membrane microdomains specialized in the regulation of numerous cellular processes related to membrane organization, as diverse as signal transduction, protein sorting, membrane trafficking or pathogen invasion. It has been proposed that this functional diversity would require a heterogeneous population of raft domains with varying compositions. However, a mechanism for such diversification is not known. We recently discovered that bacterial membranes organize their signal transduction pathways in functional membrane microdomains (FMMs) that are structurally and functionally similar to the eukaryotic lipid rafts. In this report, we took advantage of the tractability of the prokaryotic model Bacillus subtilis to provide evidence for the coexistence of two distinct families of FMMs in bacterial membranes, displaying a distinctive distribution of proteins specialized in different biological processes. One family of microdomains harbors the scaffolding flotillin protein FloA that selectively tethers proteins specialized in regulating cell envelope turnover and primary metabolism. A second population of microdomains containing the two scaffolding flotillins, FloA and FloT, arises exclusively at later stages of cell growth and specializes in adaptation of cells to stationary phase. Importantly, the diversification of membrane microdomains does not occur arbitrarily. We discovered that bacterial cells control the spatio-temporal remodeling of microdomains by restricting the activation of FloT expression to stationary phase. This regulation ensures a sequential assembly of functionally specialized membrane microdomains to strategically organize signaling networks at the right time during the lifespan of a bacterium.     

The NfeD Protein Family and Its Conserved Gene Neighbours Throughout Prokaryotes: Functional Implications for Stomatin-Like Proteins

NfeD-like proteins are widely distributed throughout prokaryotes and are frequently associated with genes encoding stomatin-like proteins (slipins). Here, we reveal that the NfeD family is ancient and comprises three major groups: NfeD1a, NfeD1b and truncated NfeD1b. Members of each group are associated with one of four conserved gene partners, three of which have eukaryotic homologues that are membrane raft associated, namely stomatin, paraslipin (previously SLP-2) and flotillin. The first NfeD group (NfeD1b), comprises proteins of approximately 460-aa long that have three functional domains: an N-terminal protease, a middle membrane-spanning region and a soluble C-terminal region rich in β-strands. The nfeD1b gene is adjacent to eoslipin in prokaryotic genomes except in Firmicutes and Deinococci, where yqfA replaces eoslipin. Proteins in the second major group (NfeD1a) are homologous to the C-terminus of NfeD1b which forms a β-barrel-like domain, and their genes are associated with paraslipin. Using OrthoMCL clustering, we show that nfeD1b genes have become truncated on many independent occasions giving rise to the third major group. These short NfeD homologues frequently remain associated with their ancestral gene neighbour, resembling NfeD1a in structure, yet are much more related to full-length NfeD1b; we term these “truncated NfeD1b”. These conserved associations suggest that NfeD proteins are dependent on gene partners for their function and that the site of interaction may lie within the C-terminal portion that is common to all NfeD homologues. Although NfeD homologues are confined to prokaryotes, this conserved association could represent an excellent system to study slipin and flotillin proteins.     

Raft association and lipid droplet targeting of flotillins are independent of caveolin

Lipid rafts are liquid ordered platforms that dynamically compartmentalize membranes. Caveolins and flotillins constitute a group of proteins that are enriched in these domains. Caveolin-1 has been shown to be an essential component of caveolae. Flotillins were also discovered as an integral component of caveolae and have since been suggested to interact with caveolins. However, flotillins are also expressed in non-caveolae-containing cells such as lymphocytes and neuronal cells. Hence, a discrepancy exists in the literature regarding the caveolin dependence of flotillin expression and their subcellular localization. To address this controversy, we used mouse embryonic fibroblasts (MEFs) from caveolin-1 knockout (Cav-1(-/-)) and wild-type mice to study flotillin expression and localization. Here we show that both membrane association and lipid raft partitioning of flotillins are not perturbed in Cav-1(-/-) MEFs, whereas membrane targeting and raft partitioning of caveolin-2, another caveolin family protein, is severely impaired. Moreover, we demonstrate that flotillin-1, but not flotillin-2, associates with lipid droplets upon oleic acid treatment and that this association is completely independent of caveolin. Taken together, our results show that flotillins are localized in lipid rafts independent of caveolin-1 and that translocation of flotillin-1 to lipid droplets is a caveolin-independent process.     

The Role of Flotillins in Regulating Aβ Production,Investigated Using Flotillin 1-/-, Flotillin 2-/- Double Knockout Mice

Flotillin 1 and flotillin 2 associate in the plasma membrane to form microdomains that have roles in cell signaling, regulation of cell-cell contacts, membrane-cytoskeletal interactions, and endocytosis. They are thought to be involved in the trafficking and hence processing of the Amyloid Precursor Protein, APP. In this study we set out to obtain in vivo confirmation of a link between flotillins and cleavage of APP to release amyloidogenic Aβ peptide, and to generate tools that would allow us to ask whether flotillins are functionally redundant. We used a mouse model for Aβ-dependent cerebral amyloidosis, APPPS1 mice, combined with deletion of either flotillin 1 singly, or flotillin 1 and flotillin 2 together. There was a small but significant reduction in Aβ levels, and the abundance of congo-red stained plaques, in brains of 12 week old mice lacking flotillin 1. A similar reduction in Aβ levels was observed in the flotillin 1-/-, flotillin 2-/- double knockouts. We did not observe large effects on the clustering or endocytosis of APP in flotillin 1-/- mouse embryonic fibroblasts. We conclude that flotillins are likely to play some role in APP trafficking or processing, but the relevant cellular mechanisms require more investigation. The availability of flotillin 1-/-, flotillin 2-/- mice, which have no overt phenotypes, will facilitate research into flotillin function in vivo.     

Flotillins Are Involved in the Polarization of Primitive and Mature Hematopoietic Cells

Background

Migration of mature and immature leukocytes in response to chemokines is not only essential during inflammation and host defense, but also during development of the hematopoietic system. Many molecules implicated in migratory polarity show uniform cellular distribution under non-activated conditions, but acquire a polarized localization upon exposure to migratory cues.

Methodology/Principal Findings

Here, we present evidence that raft-associated endocytic proteins (flotillins) are pre-assembled in lymphoid, myeloid and primitive hematopoietic cells and accumulate in the uropod during migration. Furthermore, flotillins display a polarized distribution during immunological synapse formation. Employing the membrane lipid-order sensitive probe Laurdan, we show that flotillin accumulation in the immunological synapse is concomittant with membrane ordering in these regions.

Conclusions

Together with the observation that flotillin polarization does not occur in other polarized cell types such as polarized epithelial cells, our results suggest a specific role for flotillins in hematopoietic cell polarization. Based on our results, we propose that in hematopoietic cells, flotillins provide intrinsic cues that govern segregation of certain microdomain-associated molecules during immune cell polarization.     

Flotillins Interact with PSGL-1 in Neutrophils and,upon Stimulation,Rapidly Organize into Membrane Domains Subsequently Accumulating in the Uropod

Background

Neutrophils polarize and migrate in response to chemokines. Different types of membrane microdomains (rafts) have been postulated to be present in rear and front of polarized leukocytes and disruption of rafts by cholesterol sequestration prevents leukocyte polarization. Reggie/flotillin-1 and -2 are two highly homologous proteins that are ubiquitously enriched in detergent resistant membranes and are thought to shape membrane microdomains by forming homo- and hetero-oligomers. It was the goal of this study to investigate dynamic membrane microdomain reorganization during neutrophil activation.

Methodology/Principal Findings

We show now, using immunofluorescence staining and co-immunoprecipitation, that endogenous flotillin-1 and -2 colocalize and associate in resting spherical and polarized primary neutrophils. Flotillins redistribute very early after chemoattractant stimulation, and form distinct caps in more than 90% of the neutrophils. At later time points flotillins accumulate in the uropod of polarized cells. Chemotactic peptide-induced redistribution and capping of flotillins requires integrity and dynamics of the actin cytoskeleton, but does not involve Rho-kinase dependent signaling related to formation of the uropod. Both flotillin isoforms are involved in the formation of this membrane domain, as uropod location of exogenously expressed flotillins is dramatically enhanced by co-overexpression of tagged flotillin-1 and -2 in differentiated HL-60 cells as compared to cells expressing only one tagged isoform.Flotillin-1 and -2 associate with P-selectin glycoprotein ligand 1 (PSGL-1) in resting and in stimulated neutrophils as shown by colocalization and co-immunoprecipitation. Neutrophils isolated from PSGL-1-deficient mice exhibit flotillin caps to the same extent as cells isolated from wild type animals, implying that PSGL-1 is not required for the formation of the flotillin caps. Finally we show that stimulus-dependent redistribution of other uropod-located proteins, CD43 and ezrin/radixin/moesin, occurs much slower than that of flotillins and PSGL-1.

Conclusions/Significance

These results suggest that flotillin-rich actin-dependent membrane microdomains are importantly involved in neutrophil uropod formation and/or stabilization and organize uropod localization of PSGL-1.     

The biofilm formation defect of a Bacillus subtilis flotillin‐defective mutant involves the protease FtsH

Biofilm formation in Bacillus subtilis requires the differentiation of a subpopulation of cells responsible for the production of the extracellular matrix that structures the biofilm. Differentiation of matrix‐producing cells depends, among other factors, on the FloT and YqfA proteins. These proteins are present exclusively in functional membrane microdomains of B. subtilis and are homologous to the eukaryotic lipid raft‐specific flotillin proteins. In the absence of FloT and YqfA, diverse proteins normally localized to the membrane microdomains of B. subtilis are not functional. Here we show that the absence of FloT and YqfA reduces the level of the septal‐localized protease FtsH. The flotillin homologues FloT and YqfA are occasionally present at the midcell in exponentially growing cells and the absence of FloT and YqfA negatively affects FtsH concentration. Biochemical experiments indicate a direct interaction between FloT/YqfA and FtsH. Moreover, FtsH is essential for the differentiation of matrix producers and hence, biofilm formation. This molecular trigger of biofilm formation may therefore be used as a target for the design of new biofilm inhibitors. Accordingly, we show that the small protein SpoVM, known to bind to and inhibit FtsH activity, inhibits biofilm formation in B. subtilis and other distantly related bacteria.     

Flotillins Regulate Membrane Mobility of the Dopamine Transporter but Are Not Required for Its Protein Kinase C Dependent Endocytosis

Flotillins were proposed to mediate clathrin‐independent endocytosis, and recently, flotillin‐1 was implicated in the protein kinase C (PKC)‐triggered endocytosis of the dopamine transporter (DAT). Since endocytosis of DAT was previously shown to be clathrin‐mediated, we re‐examined the role of clathrin coat proteins and flotillin in DAT endocytosis using DAT tagged with the hemagglutinin epitope (HA) in the extracellular loop and a quantitative HA antibody uptake assay. Depletion of flotillin‐1, flotillin‐2 or both flotillins together by small interfering RNAs (siRNAs) did not inhibit PKC‐dependent internalization and degradation of HA‐DAT. In contrast, siRNAs to clathrin heavy chain and μ2 subunit of clathrin adaptor complex AP‐2 as well as a dynamin inhibitor Dyngo‐4A significantly decreased PKC‐dependent endocytosis of HA‐DAT. Similarly, endocytosis and degradation of DAT that is not epitope‐tagged were highly sensitive to the clathrin siRNAs and dynamin inhibition but were not affected by flotillin knockdown. Very little co‐localization of DAT with flotillins was observed in cells ectopically expressing DAT and in cultured mouse dopaminergic neurons. Depletion of flotillins increased diffusion rates of HA‐DAT in the plasma membrane, suggesting that flotillin‐organized microdomains may regulate the lateral mobility of DAT. We propose that clathrin‐mediated endocytosis is the major pathway of PKC‐dependent internalization of DAT, and that flotillins may modulate functional association of DAT with plasma membrane rafts rather than mediate DAT endocytosis .     

Flotillin microdomains interact with the cortical cytoskeleton to control uropod formation and neutrophil recruitment

We studied the function of plasma membrane microdomains defined by the proteins flotillin 1 and flotillin 2 in uropod formation and neutrophil chemotaxis. Flotillins become concentrated in the uropod of neutrophils after exposure to chemoattractants such as N-formyl-Met-Leu-Phe (fMLP). Here, we show that mice lacking flotillin 1 do not have flotillin microdomains, and that recruitment of neutrophils toward fMLP in vivo is reduced in these mice. Ex vivo, migration of neutrophils through a resistive matrix is reduced in the absence of flotillin microdomains, but the machinery required for sensing chemoattractant functions normally. Flotillin microdomains specifically associate with myosin IIa, and spectrins. Both uropod formation and myosin IIa activity are compromised in flotillin 1 knockout neutrophils. We conclude that the association between flotillin microdomains and cortical cytoskeleton has important functions during neutrophil migration, in uropod formation, and in the regulation of myosin IIa.     

Identification, sequence and developmental expression of invertebrate flotillins from Drosophila melanogaster

Caveolae are vesicular organelles that represent a sub-compartment of the plasma membrane. Caveolins (Cav-1, -2 and -3) and flotillins {FLO-1 and FLO-2 [also known as epidermal surface antigens (ESAs)]} are two families of mammalian caveolae-associated integral membrane proteins. Although a caveolin gene family has recently been described in the invertebrate Caenorhabditis elegans, it remains unknown as to whether flotillin homologues exist in invertebrates.

Here, we report the identification, cDNA sequence and embryonic expression pattern of the first invertebrate flotillin, i.e. flotillin from Drosophila melanogaster (FLO^Dm). FLO^Dm is most closely related to mammalian flotillin-1. Remarkably, the invertebrate FLO^Dm protein behaves like mammalian flotillins and is targeted to the caveolae-enriched membrane fraction after transient expression in mammalian cells. Localization of the FLO^Dm message in D. melanogaster embryos reveals that expression of FLO^Dm is confined primarily to the developing nervous system. This is consistent with our previous observation that mammalian flotillin-1 mRNA and protein is expressed abundantly in brain tissue. Interestingly, the FLO^Dm gene is localized to chromosomal region 52 B1–B2. In addition, we find that at least two flotillin-related genes are expressed in D. melanogaster. Our current results provide a starting point and systematic basis for dissecting the role of flotillin in caveolae and neuronal development using Drosophila as a genetic system.     

Chromosomal localization, genomic organization and evolution of the genes encoding human phosphatidylinositol transfer protein membrane-associated (PITPNM) 1, 2 and 3

Eukaryotic proteins containing a phosphatidylinositol transfer (PITP) domain can be divided into two groups, one consisting of small soluble 35-kDa proteins and the other those that are membrane-associated and show sequence similarities to the Drosophila retinal degeneration B (rdgB) protein. The rdgB protein consists of four domains, an amino terminal PITP domain, a Ca2+-binding domain, a transmembrane domain and a carboxyl terminal domain that interacts with the protein tyrosine kinase PYK2. Three mammalian phosphatidylinositol transfer protein membrane-associated genes (PITPNM1, 2 and 3) with homology to Drosophila rdgB have previously been described and shown to be expressed in the mammalian retina. These findings and the demonstration that the rdgB gene plays a critical role in the invertebrate phototransduction pathway have led to the mammalian genes being considered as candidate genes for human eye diseases. In order to facilitate the analysis of these genes we have used radiation hybrid mapping and fluorescence in situ hybridization to localize the PITPNM2 and 3 genes to human chromosomes 12p24 and 17p13 respectively and hybrid mapping to confirm the localization of PITPNM1 to chromosome 11q13. We have also determined the genomic organization of both the soluble and membrane-associated Drosophila and human PITP domain-containing genes. Phylogenetic analysis indicates that the two groups arose by gene duplication that occurred very early in animal evolution.     

Coassembly of flotillins induces formation of membrane microdomains, membrane curvature, and vesicle budding

Endocytosis has a crucial role in many cellular processes. The best-characterized mechanism for endocytosis involves clathrin-coated pits [1], but evidence has accumulated for additional endocytic pathways in mammalian cells [2]. One such pathway involves caveolae, plasma-membrane invaginations defined by caveolin proteins. Plasma-membrane microdomains referred to as lipid rafts have also been associated with clathrin-independent endocytosis by biochemical and pharmacological criteria [3]. The mechanisms, however, of nonclathrin, noncaveolin endocytosis are not clear [4, 5]. Here we show that coassembly of two similar membrane proteins, flotillin1 and flotillin2 [6-8], is sufficient to generate de novo membrane microdomains with some of the predicted properties of lipid rafts [9]. These microdomains are distinct from caveolin1-positive caveolae, are dynamic, and bud into the cell. Coassembly of flotillin1 and flotillin2 into microdomains induces membrane curvature, the formation of plasma-membrane invaginations morphologically similar to caveolae, and the accumulation of intracellular vesicles. We propose that flotillin proteins are defining structural components of the machinery that mediates a clathrin-independent endocytic pathway. Key attributes of this machinery are the dependence on coassembly of both flotillins and the inference that flotillin microdomains can exist in either flat or invaginated states.     

Interplay between Toxin Transport and Flotillin Localization

The flotillin proteins are localized in lipid domains at the plasma membrane as well as in intracellular compartments. In the present study, we examined the importance of flotillin-1 and flotillin-2 for the uptake and transport of the bacterial Shiga toxin (Stx) and the plant toxin ricin and we investigated whether toxin binding and uptake were associated with flotillin relocalization. We observed a toxin-induced redistribution of the flotillins, which seemed to be regulated in a p38-dependent manner. Our experiments provide no evidence for a changed endocytic uptake of Stx or ricin in cells silenced for flotillin-1 or -2. However, the Golgi-dependent sulfation of both toxins was significantly reduced in flotillin knockdown cells. Interestingly, when the transport of ricin to the ER was investigated, we obtained an increased mannosylation of ricin in flotillin-1 and flotillin-2 knockdown cells. The toxicity of both toxins was twofold increased in flotillin-depleted cells. Since BFA (Brefeldin A) inhibits the toxicity even in flotillin knockdown cells, the retrograde toxin transport is apparently still Golgi-dependent. Thus, flotillin proteins regulate and facilitate the retrograde transport of Stx and ricin.     

Linking membrane microdomains to the cytoskeleton: regulation of the lateral mobility of reggie-1/flotillin-2 by interaction with actin

The reggies/flotillins are oligomeric scaffolding proteins for membrane microdomains. We show here that reggie-1/flotillin-2 microdomains are organized along cortical F-actin in several cell types. Interaction with F-actin is mediated by the SPFH domain as shown by in vivo co-localization and in vitro binding experiments. Reggie-1/flotillin-2 microdomains form independent of actin, but disruption or stabilization of the actin cytoskeleton modulate the lateral mobility of reggie-1/flotillin-2 as shown by FRAP. Furthermore, reggie/flotillin microdomains can efficiently be immobilized by actin polymerisation, while exchange of reggie-1/flotillin-2 molecules between microdomains is enhanced by actin disruption as shown by tracking of individual microdomains using TIRF microscopy.     

Stomatin is highly expressed in exosomes of different origin and is a promising candidate as an exosomal marker

Proteins involved in the organizing of lipid rafts can be found in exosomes, as shown for caveolin‐1, and they could contribute to exosomal cargo sorting, as shown for flotillins. Stomatin belongs to the same stomatin/prohibitin/flotillin/HflK/C family of lipid rafts proteins, but it has never been studied in exosomes except for extracellular vesicles (EVs) originating from blood cells. Here we first show the presence of stomatin in exosomes produced by epithelial cancer cells (non–small cell lung cancer, breast, and ovarian cancer cells) as well as in EVs from biological fluids, including blood plasma, ascitic fluids, and uterine flushings. A high abundance of stomatin in EVs of various origins and its enrichment in exosomes make stomatin a promising exosomal marker. Comparison with other lipid raft proteins and exosomal markers showed that the level of stomatin protein in exosomes from different sources corresponds well to that of CD9, while it differs essentially from flotillin‐1 and flotillin‐2 homologs, which in turn are present in exosomes in nearly equal proportions. In contrast, the level of vesicular caveolin‐1 as well as its EV‐to‐cellular ratio vary drastically depending on cell type.     

Flotillins/cavatellins are differentially expressed in cells and tissues and form a hetero-oligomeric complex with caveolins in vivo. Characterization and epitope-mapping of a novel flotillin-1 monoclonal antibody probe

Caveolae are vesicular organelles that represent a subcompartment of the plasma membrane. Caveolins and flotillins are two families of mammalian caveolae-associated integral membrane proteins. However, it remains unknown whether flotillins interact with caveolin proteins to form a stable caveolar complex or if expression of flotillins can drive vesicle formation. Here, we examine the cell type and tissue-specific expression of the flotillin gene family. For this purpose, we generated a novel monoclonal antibody probe that recognizes only flotillin-1. A survey of cell and tissue types demonstrates that flotillins 1 and 2 have a complementary tissue distribution. At the cellular level, flotillin-2 was ubiquitously expressed, whereas flotillin-1 was most abundant in A498 kidney cells, muscle cell lines, and fibroblasts. Using three different models of cellular differentiation, we next examined the expression of flotillins 1 and 2. Taken together, our data suggest that the expression levels of flotillins 1 and 2 are independently regulated and does not strictly correlate with known expression patterns of caveolin family members. However, when caveolins and flotillins are co-expressed within the same cell, as in A498 cells, they form a stable hetero-oligomeric "caveolar complex." In support of these observations, we show that heterologous expression of murine flotillin-1 in Sf21 insect cells using baculovirus-based vectors is sufficient to drive the formation of caveolae-like vesicles. These results suggest that flotillins may participate functionally in the formation of caveolae or caveolae-like vesicles in vivo. Thus, flotillin-1 represents a new integral membrane protein marker for the slightly larger caveolae-related domains (50-200 nm) that are observed in cell types that fail to express caveolin-1. As a consequence of these findings, we propose the term "cavatellins" be used (instead of flotillins) to describe this gene family.     

Flotillins functionally organize the bacterial membrane

Proteins and lipids are heterogeneously distributed in biological membranes. The correct function of membrane proteins depends on spatiotemporal organization into defined membrane areas, called lipid domains or rafts. Lipid microdomains are therefore thought to assist compartmentalization of membranes. However, how lipid and protein assemblies are organized and whether proteins are actively involved in these processes remains poorly understood. We now have identified flotillins to be responsible for lateral segregation of defined membrane domains in the model organism Bacillus subtilis. We show that flotillins form large, dynamic assemblies that are able to influence membrane fluidity and prevent condensation of Laurdan stained membrane regions. Absence of flotillins in vivo leads to coalescence of distinct domains of high membrane order and, hence, loss of flotillins in the bacterial plasma‐membrane reduces membrane heterogeneity. We show that flotillins interact with various proteins involved in protein secretion, cell wall metabolism, transport and membrane‐related signalling processes. Importantly, maintenance of membrane heterogeneity is critical for vital cellular processes such as protein secretion.