Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Glebov, Oleg O.; Bright, Nicholas A.; Nichols, Benjamin J.

doi:10.1038/ncb1342

Cited by 493 publications

(550 citation statements)

References 35 publications

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“…This idea is supported by the fact that flotillin-1 expression in insect cells can induce the formation of large caveolae-like vesicles (Volonte et al, 1999). Moreover, flotillin-1 has recently been described as a determinant of a clathrin-independent endocytic pathway in mammalian cells, which implicates this protein in vesicular trafficking (Glebov et al, 2006). Despite a striking sequence similarity between flotillin-1 and flotillin-2, specific recruitment of flotillin-1 to lipid bodies upon fatty-acid loading could also underline a specific function in lipid regulation or trafficking towards these organelles.…”

Section: Discussionsupporting

confidence: 53%

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

Rajendran

Lay

Illges

2007

Biological Chemistry

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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.

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Section: Discussionsupporting

confidence: 53%

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

Rajendran

Lay

Illges

2007

Biological Chemistry

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“…These data do not rule out the possibility that MNV-1 infection may occur through a caveolae-dependent pathway in other cell types that express caveolin. The recently described flotillindependent pathway is also raft-associated (Glebov et al, 2006) and may be inhibited by cholesterol depletion; however, our results showed that flotillin-1 depletion had no effect on MNV-1 infection, suggesting that this pathway is also not used for MNV-1 infection.The role of dynamin in both CME and caveolae-dependent endocytosis is well established, whereas its role in other endocytic pathways is less clear (Marsh & Helenius, 2006;Miaczynska & Stenmark, 2008). Here, we show that inhibition of dynamin using dynasore decreased MNV-1 infection of RAW264.7 cells, but only when added at the entry stage of infection, indicating that MNV-1 entry is dynamin-dependent.…”

contrasting

confidence: 56%

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

Gerondopoulos

Jackson

Monaghan

et al. 2010

Journal of General Virology

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For many viruses, endocytosis and exposure to the low pH within acidic endosomes is essential for infection. It has previously been reported that feline calicivirus uses clathrin-mediated endocytosis for entry into mammalian cells. Here, we report that infection of RAW264.7 macrophages by the closely related murine norovirus-1 (MNV-1) does not require the clathrin pathway, as infection was not inhibited by expression of dominant-negative Eps15 or by knockdown of the adaptin-2 complex. Further, infection was not inhibited by reagents that raise endosomal pH. RAW264.7 macrophages were shown not to express caveolin, and flotillin depletion did not inhibit infection, suggesting that caveolae and the flotillin pathway are not required for cell entry. However, MNV-1 infection was inhibited by methyl-b-cyclodextrin and the dynamin inhibitor, dynasore. Addition of these drugs to the cells after a period of virus internalization did not inhibit infection, suggesting the involvement of cholesterol-sensitive lipid rafts and dynamin in the entry mechanism. Macropinocytosis (MPC) was shown to be active in RAW264.7 macrophages (as indicated by uptake of dextran) and could be blocked by 5-(N-ethyl-N-isopropyl) amiloride (EIPA), which is reported to inhibit this pathway. However, infection was enhanced in the presence of EIPA. Similarly, actin disruption, which also inhibits MPC, resulted in enhanced infection. These results suggest that MPC could contribute to virus degradation or that inhibition of MPC could lead to the upregulation of other endocytic pathways of virus uptake. INTRODUCTIONThe family Caliciviridae is divided into four genera: Vesivirus, Lagovirus, Sapovirus and Norovirus. The human noroviruses are the most common cause of acute viral gastroenteritis, especially in industrialized countries (Lopman et al., 2003); there is currently no treatment or vaccine for these viruses. In addition, there is still no routine tissue culture system for the propagation of human noroviruses, but the recent discovery of murine norovirus-1 (MNV-1) has provided an efficient model system for the study of norovirus replication, as this virus replicates efficiently in murine macrophages and dendritic cells (DCs) (Karst et al., 2003;Wobus et al., 2004). MNV-1 is highly prevalent in laboratory mice and has been shown to be lethal to mice with impaired innate immunity (Hsu et al., 2006;Karst et al., 2003;Muller et al., 2007).Noroviruses are non-enveloped and contain a singlestranded RNA genome of about 7.3 kb, which is linked to the viral VPg protein at the 59 end and is polyadenylated at the 39 end (Karst et al., 2003;Wobus et al., 2004). The genome is organized into four open reading frames (ORF-1-4). ORF-1 encodes the non-structural proteins, whereas ORF-2 and ORF-3 encode structural proteins (Sosnovtsev et al., 2006). ORF-4 was recently identified within the MNV-1 genome and encodes a single protein of unknown function (Thackray et al., 2007).For many viruses, infection requires virus uptake by endocytosis. A number of different endo...

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“…The observation of reggie-dependent cargo trafficking has been considered as reggie/flotillin-dependent and lipid raft bound endocytosis (Glebov et al, 2006). Our present assay with a ␤1-integrin antibody as well as earlier experiments with E-cadherin, transferrin receptor and the T-cell receptor did not detect a reggie-mediated endocytosis but discovered instead a reggie-assisted Rab11a-dependent recycling route (Solis et al, 2012Stuermer, 2010).…”

Section: Reggie In Vesicle Traffickingsupporting

confidence: 52%

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

Hülsbusch

Solis

Katanaev

et al. 2015

European Journal of Cell Biology

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a b s t r a c tReggies/flotillins are implicated in trafficking of membrane proteins to their target sites and in the regulation of the Rab11a-dependent targeted recycling of E-cadherin to adherens junctions (AJs). Here we demonstrate a function of reggies in focal adhesion (FA) formation and ␣5-and ␤1-integrin recycling to FAs. Downregulation of reggie-1 in HeLa and A431 cells by siRNA and shRNA increased the number of FAs, impaired their distribution and modified FA turnover. This was coupled to enhanced focal adhesion kinase (FAK) and Rac1 signaling and gain in plasma membrane motility. Wild type and constitutively-active (CA) Rab11a rescued the phenotype (normal number of FAs) whereas dominant-negative (DN) Rab11a mimicked the loss-of-reggie phenotype in control cells. That reggie-1 affects integrin trafficking emerged from the faster loss of internalized antibody-labeled ␤1-integrin in reggie-deficient cells. Moreover, live imaging using TIRF microscopy revealed vesicles containing reggie-1 and ␣5-or ␤1-integrin, trafficking close to the substrate-near membrane and making kiss-and-run contacts with FAs. Thus, reggie-1 in interaction with Rab11a controls Rac1 and FAK activation and coordinates the targeted recycling of ␣5-and ␤1-integrins to FAs to regulate FA formation and membrane dynamics.

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Flotillin-1 defines a clathrin-independent endocytic pathway in mammalian cells

Cited by 493 publications

References 35 publications

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

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

Murine norovirus-1 cell entry is mediated through a non-clathrin-, non-caveolae-, dynamin- and cholesterol-dependent pathway

Reggie-1/Flotillin-2 regulates integrin trafficking and focal adhesion turnover via Rab11a

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