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Colocalization of the lipid dye Nile Red with anti-reggie1 and-2 and PrP c labeling. In Jurkat T cells, the lipid dye Nile Red (first column) colocalizes with reggie-1 (first row), reggie-2 (second row) and PrP c (third row). However, there is no colocalization with Thy-1 (fourth row), the endosomal marker EEA1 (fifth row) or the lysosomal marker protein Lamp-2 (last row). Scale bar, 5 µm.
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A new model of caveolin association with lipid body cores has recently been proposed which may be relevant to a number of cellular processes, e.g. lipid body generation. Here we show that PrPc and reggie-1 and reggie-2 also occur in the cores of Nile Red/Bodipy-stained (neutral lipid-containing) vesicular structures and, in immunoblots, in the lipi...
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... analysis of Jurkat T cells revealed the presence of vesicles which are labeled by Nile Red ( fig. 1), Bodipy 495/503, Oil Red O and Sudan Black (data not shown) at the light microscopic (LM) level and which appear elec- tron lucent at the EM level ( fig. 2). Such features are typ- ical of lipid-rich vesicles which are known to exist in many eukaryotic cells. Staining with filipin, a marker for cholesterol, did not show any ...
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... ± 0.36 SD mm (± 0.07 SE, n = 25), in good agree- ment with LM estimations. We examined the localization of reggie-1, reggie-2 and PrP c in conjunction with Nile Red by LSM analysis. Reg- gie-1 and reggie-2 were contained in a subpopulation of the Nile Red-labeled vesicle-like structures ( fig. 1, 1st and 2nd row). Moreover, a subpopulation of the Nile Red- stained vesicles was labeled by PrP c pAB ( fig. 1, 3rd row). Clearly, Nile Red-labeled vesicles are more abun- dant than reggie/Nile Red or PrP c /Nile Red doubly la- beled vesicles. Thy-1, another GPI-anchored protein of Jurkat T cells that is colocalized with reggie rafts, was ...
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... with LM estimations. We examined the localization of reggie-1, reggie-2 and PrP c in conjunction with Nile Red by LSM analysis. Reg- gie-1 and reggie-2 were contained in a subpopulation of the Nile Red-labeled vesicle-like structures ( fig. 1, 1st and 2nd row). Moreover, a subpopulation of the Nile Red- stained vesicles was labeled by PrP c pAB ( fig. 1, 3rd row). Clearly, Nile Red-labeled vesicles are more abun- dant than reggie/Nile Red or PrP c /Nile Red doubly la- beled vesicles. Thy-1, another GPI-anchored protein of Jurkat T cells that is colocalized with reggie rafts, was not detected in Nile Red-positive vesicles ( fig. 1, fourth row). More- over, fyn and lck, two src family ...
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... of the Nile Red- stained vesicles was labeled by PrP c pAB ( fig. 1, 3rd row). Clearly, Nile Red-labeled vesicles are more abun- dant than reggie/Nile Red or PrP c /Nile Red doubly la- beled vesicles. Thy-1, another GPI-anchored protein of Jurkat T cells that is colocalized with reggie rafts, was not detected in Nile Red-positive vesicles ( fig. 1, fourth row). More- over, fyn and lck, two src family kinases, associated with reggie-1, reggie-2 and PrP c in Jurkat T cell caps, also showed no colocalization with Nile Red-positive vesi- cles (data not shown). To exclude the possibility that the observed organelles are early endosomes or late endosomes/lysosomes which are known to contain reg- ...
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... over, fyn and lck, two src family kinases, associated with reggie-1, reggie-2 and PrP c in Jurkat T cell caps, also showed no colocalization with Nile Red-positive vesi- cles (data not shown). To exclude the possibility that the observed organelles are early endosomes or late endosomes/lysosomes which are known to contain reg- gie-1 and -2 as well as PrP c , we stained Jurkat T cells with an Ab against EEA1 ( fig. 1 fifth row) and Lamp-2 ( fig. 1, sixth row) in conjunction with Nile Red. In this case, no colocalization was detected, so that the lipid vesicles in Jurkat T cells are unlikely to be endosomes or lysosomes. ...
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... T cell caps, also showed no colocalization with Nile Red-positive vesi- cles (data not shown). To exclude the possibility that the observed organelles are early endosomes or late endosomes/lysosomes which are known to contain reg- gie-1 and -2 as well as PrP c , we stained Jurkat T cells with an Ab against EEA1 ( fig. 1 fifth row) and Lamp-2 ( fig. 1, sixth row) in conjunction with Nile Red. In this case, no colocalization was detected, so that the lipid vesicles in Jurkat T cells are unlikely to be endosomes or ...
Citations
... On the other hand many studies have provided conclusive evidence for the strong association of flotillins with GPI-anchored proteins. In Jurkat T cells, close associations of flotillins with GPI-anchored proteins such as PrP and Thy-1 have been shown to participate in signaling leading to the recruitment of TCR components [11,[46][47][48] whereas in neurons, flotillins-GPI-anchored proteins interactions regulate the targeted delivery of cargo proteins to specific sites of the plasma membrane by triggering the activation of src tyrosine and MAP kinases, as well as small GTP hydrolizing enzymes, controlling therefore, extension and axon growth [49]. Detailed mechanism of how such interactions might be formed is missing, however, as flotillins contain putative cholesterol recognition amino acid consensus (CRAC) motifs [50] and hydrophobic stretches [51], it is conceivable that direct interactions of flotillins with cholesterol or/and other inner leaflet lipids might mediate their associations with GPI-anchored proteins. ...
Flotillins are prominent, oligomeric protein components of erythrocyte (RBC) membrane raft domains and are considered to play an important structural role in lateral organization of the plasma membrane. In our previous work on erythroid membranes and giant plasma membrane vesicles (GPMVs) derived from them we have shown that formation of functional domains (resting state rafts) depends on the presence of membrane palmitoylated protein 1 (MPP1/p55), pointing to its new physiological role. Exploration of the molecular mechanism of MPP1 function in organizing membrane domains described here, through searching for its molecular partners in RBC membrane by using different methods, led to the identification of the raft-marker proteins, flotillin 1 and flotillin 2, as hitherto unreported direct MPP1 binding-partners in the RBC membrane. These proteins are found in high molecular-weight complexes in native RBC membrane and, significantly, their presence was shown to be separate from the well-known protein 4.1-dependent interactions of MPP1 with membrane proteins. Furthermore, FLIM analysis revealed that loss of the endogenous MPP1-flotillins interactions resulted in significant changes in RBC membrane-fluidity, emphasizing the physiological importance of such interactions in vivo. Therefore, our data establish a new perspective on the role of MPP1 in erythroid cells and suggests that direct MPP1-flotillins interactions could be the major driving-force behind the formation of raft domains in RBC.
... Feeding-fasting dependent association of flotillin-1 with LDs in the liver Flotillin-1, a lipid raft associated marker protein, has been found on LDs in several cell culture studies [10,16,20]. We asked if association of flotillin-1 with hepatic LDs is correlated with the feeding/fasting state. ...
Lipid droplets (LDs) are cellular stores of neutral fat that facilitate lipid and protein trafficking in response to metabolic cues. Unlike other vesicles, the phospholipid membrane on the LD is a monolayer. Interestingly, this monolayer membrane has free cholesterol, and may therefore contain lipid microdomains that serve as a platform for assembling proteins involved in signal transduction, cell polarity, pathogen entry etc. In support of this, cell culture studies have detected microdomain-associated “raftophilic” proteins on LDs. However, the physiological significance of this observation has been unclear. Here we show that two proteins (Flotillin-1 and SNAP23) that bind to membrane microdomains associate differently with LDs purified from rat liver depending on the feeding/fasting state of the animal. Flotillin-1 increases on LDs in the fed state, possibly because LDs interact with the endoplasmic reticulum (ER), facilitating supply of flotillin-1 from ER to LDs. Interestingly, this increase in flotillin-1 is correlated with an increase in free cholesterol on the LDs in fed state. In opposite behaviour to flotillin-1, SNAP23 increases on LDs in the fasted state and this appears to mediate LD-mitochondria interactions. Such LD-mitochondria interactions may provide fatty acids to mitochondria for promoting beta-oxidation in hepatocytes in response to fasting. Our work brings out physiologically relevant aspects of lipid droplet biology that are different from, and may not be entirely possible to replicate and study in cell culture. © 2017 Sadh et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... The two types of flotillins that can be found in planar rafts, flotillin-1 and flotillin-2, are products of different genes; both have a molecular weight of 48 kDa and they share 44% identity in their primary sequence [5]. In addition to their prominent localization at the plasma membrane, both flotillins reside in intracellular compartments [6], where they localize to lipid droplets [7] and to compartments of the endocytic pathway, such as recycling endosomes [8]. Flotillins are involved in many cellular functions, including endocytosis, exocytosis, membrane cycling, formation and maintenance of lipid rafts in the membrane, cell signaling, cell migration and cell adhesion. ...
... Our data are consistent with the work published by Langhorst and colleagues [6] where they show that flotillin-2 is present in vesicles in subconfluent HeLa cell cultures. Flotillin-2 has been identified in different cellular compartments, including lipid rafts at the plasma membrane, lipid droplets [7], and vesicles of the endocytic pathway [8]. Flotillin-2 distribution varies among different cell types. ...
Myoblasts undergo a series of changes in the composition and dynamics of their plasma membranes during the initial steps of skeletal muscle differentiation. These changes are crucial requirements for myoblast fusion and allow the formation of striated muscle fibers. Membrane microdomains, or lipid rafts, have been implicated in myoblast fusion. Flotillins are scaffold proteins that are essential for the formation and dynamics of lipid rafts. Flotillins have been widely studied over the last few years, but still little is known about their role during skeletal muscle differentiation. In the present study, we analyzed the expression and distribution of flotillin-2 in chick, mice and human muscle cells grown in vitro. Primary cultures of chick myogenic cells showed a decrease in the expression of flotillin-2 during the first 72 hours of muscle differentiation. Interestingly, flotillin-2 was found to be highly expressed in chick myogenic fibroblasts and weakly expressed in chick myoblasts and multinucleated myotubes. Flotillin-2 was distributed in vesicle-like structures within the cytoplasm of chick myogenic fibroblasts, in the mouse C2C12 myogenic cell line, and in neonatal human muscle cells. Cryo-immunogold labeling revealed the presence of flotillin-2 in vesicles and in Golgi stacks in chick myogenic fibroblasts. Further, brefeldin A induced a major reduction in the number of flotillin-2 containing vesicles which correlates to a decrease in myoblast fusion. These results suggest the involvement of flotillin-2 during the initial steps of skeletal myogenesis.
... Lipid microdomains, also called lipid rafts, are known as the platform for the spatial concentration of some glycosylphosphatidylinositol (GPI)-anchored proteins, such as PrP C and Thy-1, as well as for signal transduction [17]. PrP C and flotillins have been identified in the membrane microdomains during the induction of T cell activation, in the intracellular lipid-rich vesicles in Jurkat T cells [18,19], and in SN56 cell after induced by stress-inducible protein-1 [20]. Moreover, some studies even speculated that the conversion of PrP C into PrP Sc may occur in the lipid rafts or during its intracellular process [21,22]. ...
Flotillins are membrane association proteins consisting of two homologous members, flotillin-1 (Flot-1) and flotillin-2 (Flot-2). They define a clathrin-independent endocytic pathway in mammal cells, which are also distinct from some other endocytosis mechanisms. The implicated cargoes of the flotillin-dependent pathway are mainly some GPI-anchored proteins, such as CD59 and Thy-1, which positionally colocalize with flotillins at the plasma membrane microdomains. To see whether flotillins are involved in the endocytosis of PrP(C), the potential molecular interaction between PrP(C) and flotillins in a neuroblastoma cell line SK-N-SH was analyzed. Co-immunoprecipitation assays did not reveal a detectable complex in the cell lysates of a normal feeding situation. After stimulation of Cu(2+), PrP(C) formed a clear complex with Flot-1, but not with Flot-2. Immunofluorescent assays illustrated that PrP(C) colocalized well with Flot-1, and the complexes of PrP(C)-Flot-1 shifted from the cell membrane to the cytoplasm along with the treatment of Cu(2+). Down-regulating the expression of Flot-1 in SK-N-SH cells by Flot-1-specific RNAi obviously abolished the Cu(2+)-stimulated endocytosis process of PrP(C). Moreover, we also found that in the cell line human embryonic kidney 293 (HEK293) without detectable PrP(C) expression, the distribution of cellular Flot-1 maintained almost unchanged during Cu(2+) treatment. Cu(2+)-induced PrP(C)-Flot-1 molecular interaction and endocytosis in HEK293 cells were obtained when expressing wild-type human PrP (PrP(PG5)), but not in the preparation expressing octarepeat-deleted PrP (PrP(PG0)). Our data here provide direct evidences for the molecular interaction and endocytosis of PrP(C) with Flot-1 in the presence of copper ions, and the octarepeat region of PrP(C) is critical for this process, which strongly indicates that the Flot-1-dependent endocytic pathway seems to mediate the endocytosis process of PrP(C) in the special situation.
... Moreover, cross-linking of PrP c resulted in its clustering in the region of the preformed flotillin cap [42]. Flotillins were also found in lipid-rich vesicles from Jurkat T cells together with PrP c [52]. Furthermore, scrapie prion protein (PrP Sc ) is localized in flotillin-1 positive late endosomes in the central nervous system cells [53]. ...
The proteins of flotillin-1 and flotillin-2 were originally discovered in axon regeneration of goldfish retinal ganglion cells. They are generally used as marker proteins of lipid rafts and considered to be scaffolding proteins of lipid microdomains. Although they are ubiquitously expressed and well-conserved from fly to man, their exact functions remain controversial. In this review, we summarize the structure of flotillins and some functions of them, such as regulating axon regeneration, endocytosis, T cell activation, insulin signaling, membrane protein recruitment, roles in the progression of some diseases and so on.
... Indeed, the majority of T-cell lines, previously characterized as Sudan Black B negative [31], do not accumulate fat. But some studies presented rare cases of SBB positive acute lymphoblastic leukemia (ALL) [44] as the Jurkat T cell line [45]. This rare occurrence might explain why small lipids droplets are observed in contact with PUFA. ...
It is suspected that bone marrow (BM) microenvironmental factors may influence the evolution of chronic myeloid leukaemia (CML). In this study, we postulated that adipocytes and lipids could be involved in the progression of CML. To test this hypothesis, adipocytes were co-cultured with two BCR-ABL positive cell lines (PCMDS and K562). T cell (Jurkat) and stroma cell (HS-5) lines were used as controls. In the second set of experiments, leukemic cell lines were treated with stearic, oleic, linoleic or α-linolenic acids in presence or absence of leptin. Survival, proliferation, leptin production, OB-R isoforms (OB-Ra and OB-Rb), phosphoinositide 3-kinase (PI3k) and BCL-2 expression have been tested after 24h, 48h and 72h of treatment. Our results showed that adipocytes induced a decrease of CML proliferation and an increase in lipid accumulation in leukemic cells. In addition, CML cell lines induced adipocytes cell death. Chromatography analysis showed that BM microenvironment cells were full of saturated (SFA) and monounsaturated (MUFA) fatty acids, fatty acids that protect tumor cells against external agents. Stearic acid increased Bcl-2 expression in PCMDS, whereas oleic and linoleic acids had no effects. In contrast, α-linolenic acid decreased the proliferation and the survival of CML cell lines as well as BCL-2 and OB-R expression. The effect of α-linolenic acids seemed to be due to PI3K pathway and Bcl-2 inhibition. Leptin production was detected in the co-culture medium. In the presence of leptin, the effect of α-linolenic acid on proliferation, survival, OB-R and BCl-2 expression was reduced.
... The hypothesis that reggies might participate in cargo trafficking was supported by observations that reggies reside at many intracellular compartments including lysosomes, recycling endosomes (but not early endosome antigen 1positive early endosomes) (Langhorst et al. 2008a), post-Golgi vesicles (Morrow et al. 2002;Langhorst et al. 2008a), lipid bodies (Reuter et al. 2004) and phagosomes in macrophages (Dermine et al. 2001). Moreover, it has been demonstrated that reggies interact with actin (Langhorst et al. 2007) in addition to communicating with the Rho-type GTPases and their effectors for the regulation of actin dynamics (Langhorst et al. 2008a;Munderloh et al. 2009). ...
J. Neurochem. (2011) 116, 708–713.
The two proteins reggie-1/flotillin-2 and reggie-2/flotillin-1 form microdomains at the plasma membrane and at intracellular compartments where src tyrosine kinases associate with them. Specific GPI-anchored proteins, in particular prion protein and Thy-1, co-cluster with reggie microdomains at the plasma membrane and elicit signal transduction in association with reggies which regulates the activation of several GTPases involved in the recruitment of specific membrane proteins from intracellular carriers to target sites of the cell membrane in a cell type-specific manner. For example, prion protein and reggie regulate the recruitment and targeted delivery of the T cell receptor complex to the T cell cap, of E-cadherin to cell–cell contact sites in epithelial cells, and of bulk membrane and growth receptors to the growth cone in developing neurons. Evidence is accumulating that reggies are involved in guiding the cell-type-specific membrane proteins from the intracellular compartments to their target sites at the cell membrane, a function required in all cells which explains why reggies are expressed in many or all cells in invertebrates and vertebrates.
... Several Journal of Lipids independent methods have been used to detect them there. Proteins now acknowledged to be inside the lipid droplet core are PAT family proteins [32,33], caveolins [34], flotillins [35] and cyclooxygenase [36], among others [37,38]. ...
Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.
... Interestingly, PrP C was also found to interact with flotillin-2 and flotillin-1 in T-cells [41], but not in testis [20]. As flotillins have been proposed to have a role both in the endocytosis and function of PrP C424344, we then analysed whether flotillin-2 interacted with Dpl and/or PrP C in singly and/or doubly transfected FRT cells. To this end, we performed co-immunoprecipitation experiments using antibodies against GFP–Dpl or PrP C in the precipitation step, and used an antibody against flotillin-2 to reveal this protein in the immunoprecipitate by Western blot analysis. ...
... Moreover, on one hand flotillin-1, which forms a heterocomplex with flotillin-2 in N2a cells, has been reported to interact with neuroglobin, a protein involved in neuroprotective pathways [47], and on the other hand flotillin levels are increased in Alzheimers-disease-affected brains [48] . Interestingly PrP Sc was shown to accumulate in flotillin-1-positive vesicles in infected GT1-7 cells [42] , suggesting a possible involvement of these raft proteins in neurotoxic processes. Thus considering the antagonistic behaviour of Dpl and PrP C regarding the onset of neurodegeneration, it will be interesting to investigate further whether and how this mutual or exclusive interaction with flotillin is involved in the signalling function of both proteins and/or in the pathways leading to neurodegeneration. ...
Dpl (doppel) is a paralogue of the PrPC (cellular prion protein), whose misfolded conformer (the scrapie prion protein, PrPSc) is responsible for the onset of TSEs (transmissible spongiform encephalopathies) or prion diseases. It has been shown that the ectopic expression of Dpl in the brains of some lines of PrP-knockout mice provokes cerebellar ataxia, which can be rescued by the reintroduction of the PrP gene, suggesting a functional interaction between the two proteins. It is, however, still unclear where, and under which conditions, this event may occur. In the present study we addressed this issue by analysing the intracellular localization and the interaction between Dpl and PrPC in FRT (Fischer rat thyroid) cells stably expressing the two proteins separately or together. We show that both proteins localize prevalently on the basolateral surface of FRT cells, in both singly and doubly transfected clones. Interestingly we found that they associate with DRMs (detergent-resistant membranes) or lipid rafts, from where they can be co-immunoprecipitated in a cholesterol-dependent fashion. Although the interaction between Dpl and PrPC has been suggested before, our results provide the first clear evidence that this interaction occurs in rafts and is dependent on the integrity of these membrane microdomains. Furthermore, both Dpl and PrPC could be immunoprecipitated with flotillin-2, a raft protein involved in endocytosis and cell signalling events, suggesting that they share the same lipid environment.
... Examples are the preformed reggie cap in T cells (Figure 3), the condensation of reggies along cell-cell contact sites [46] and their selective accumulation in growth cones ( Figure 3) and growing processes in general. In addition to the plasma membrane, reggie/flotillin proteins are found in association with vesicles in intracellular compartments, namely dense core vesicles in PC12 cells, Golgi-associated vesicles, the Rab11 recycling compartment, multivesicular bodies [17], late endosomes/ lysosomes [32,35], phagosomes of macrophages [31], lipid bodies [54] and in the non-vesicular aggresomes [17] but not in early endosomes. Interestingly, reggie/flotillin vesicles have been observed to traffic rapidly bidirectionally between intracellular sites and the plasma membrane [17], a process that is accelerated by epidermal growth factor (EGF) that is known to promote lamellipodia formation, cell migration and elongation of processes. ...
The proteins reggie-1 and reggie-2 were originally discovered in neurons during axon regeneration. Subsequently, they were independently identified as markers of lipid rafts in flotation assays and were hence named flotillins. Since then, reggie/flotillin proteins have been found to be evolutionarily conserved and are present in all vertebrate cells - yet their function has remained elusive and controversial. Recent results now show that reggie/flotillin proteins are indeed necessary for axon regeneration and growth: no axons form when reggies/flotillins are downregulated and signaling pathways controlling actin dynamics are perturbed. Their widespread expression and conservation, however, suggest that these proteins regulate basic cellular functions beyond regeneration. It is argued here that the reggie/flotillin proteins regulate processes vital to all cells - the targeted delivery of bulk membrane and specific membrane proteins from internal vesicle pools to strategically important sites including cell contact sites, the T cell cap, regenerating axons and growth cones and other protrusions.
