Flotillin proteins recruit sphingosine to membranes and maintain cellular sphingosine-1-phosphate levels.

Sphingosine-1-phosphate (S1P) is an important lipid signalling molecule. S1P is produced via intracellular phosphorylation of sphingosine (Sph). As a lipid with a single fatty alkyl chain, Sph may diffuse rapidly between cellular membranes and through the aqueous phase.

Rnd3-induced cell rounding requires interaction with Plexin-B2.

Rnd proteins are atypical members of the Rho GTPase family that induce actin cytoskeletal reorganization and cell rounding. Rnd proteins have been reported to bind to the intracellular domain of several plexin receptors, but whether plexins contribute to the Rnd-induced rounding response is not known. Here we show that Rnd3 interacts preferentially with plexin-B2 of the three plexin-B proteins, whereas Rnd2 interacts with all three B-type plexins, and Rnd1 shows only very weak interaction with plexin-B proteins in immunoprecipitations.

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.

Functional genomic screen identifies novel mediators of collagen uptake.

Tissue fibrosis occurs when matrix production outpaces matrix degradation. Degradation of collagen, the main component of fibrotic tissue, is mediated through an extracellular proteolytic pathway and intracellular pathway of cellular uptake and lysosomal digestion. Recent studies demonstrate that disruption of the intracellular pathways can exacerbate fibrosis.

Deletion of cavin genes reveals tissue-specific mechanisms for morphogenesis of endothelial caveolae.

Caveolae are abundant in endothelial cells and are thought to have important roles in endothelial cell biology. The cavin proteins are key components of caveolae, and are expressed at varied amounts in different tissues. Here we use knockout mice to determine the roles of cavins 2 and 3 in caveolar morphogenesis in vivo.

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.

Endocytosis of flotillin-1 and flotillin-2 is regulated by Fyn kinase.

Flotillin-1 and flotillin-2 co-assemble into plasma membrane microdomains that are involved in the endocytosis of molecules such as glycosyl phosphatidylinositol (GPI)-linked proteins. Previous studies suggest that budding of flotillin microdomains from the plasma membrane is a tightly regulated process. Here, we demonstrate that endocytosis of flotillins is regulated by the Src family kinase Fyn.

N-terminus-mediated dimerization of ROCK-I is required for RhoE binding and actin reorganization.

ROCK-I (Rho-associated kinase 1) is a serine/threonine kinase that can be activated by RhoA and inhibited by RhoE. ROCK-I has an N-terminal kinase domain, a central coiled-coil region and a RhoA-binding domain near the C-terminus. We have previously shown that RhoE binds to the N-terminal 420 amino acids of ROCK-I, which includes the kinase domain as well as N-terminal and C-terminal extensions.

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.

Casein kinase I epsilon associates with and phosphorylates the tight junction protein occludin.

Occludin is an integral-membrane protein that contributes to tight junction function. We have identified casein kinase I epsilon (CKI epsilon) as a binding partner for the C-terminal cytoplasmic domain of occludin by yeast two-hybrid screening. CKI epsilon phosphorylated occludin and co-localised and co-immunoprecipitated with occludin from human endothelial cells.

Inhibition of ROCK by RhoE.

RhoE belongs to the Rnd subfamily of small Rho-related GTP-binding proteins. Similar to other Rho proteins, RhoE regulates actin cytoskeleton dynamics. Expression of RhoE induces loss of actin stress fibers, and it also increases cell migration speed.

RhoE function is regulated by ROCK I-mediated phosphorylation.

The Rho GTPase family member RhoE regulates actin filaments partly by binding to and inhibiting ROCK I, a serine/threonine kinase that induces actomyosin contractility. Here, we show that ROCK I can phosphorylate multiple residues on RhoE in vitro. In cells, ROCK I-phosphorylated RhoE localizes in the cytosol, whereas unphosphorylated RhoE is primarily associated with membranes.

RhoE inhibits cell cycle progression and Ras-induced transformation.

Rho GTPases are major regulators of cytoskeletal dynamics, but they also affect cell proliferation, transformation, and oncogenesis. RhoE, a member of the Rnd subfamily that does not detectably hydrolyze GTP, inhibits RhoA/ROCK signaling to promote actin stress fiber and focal adhesion disassembly. We have generated fibroblasts with inducible RhoE expression to investigate the role of RhoE in cell proliferation.

Rocks: multifunctional kinases in cell behaviour.

ROCKs, or Rho kinases, are serine/threonine kinases that are involved in many aspects of cell motility, from smooth-muscle contraction to cell migration and neurite outgrowth. Recent experiments have defined new functions of ROCKs in cells, including centrosome positioning and cell-size regulation, which might contribute to various physiological and pathological states.

RhoE binds to ROCK I and inhibits downstream signaling.

RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets.

Crystal structure of the core domain of RhoE/Rnd3: a constitutively activated small G protein.

We report the 2.1 A crystal structure of the core G protein domain of the unusual Rho family member RhoE/Rnd3 in complex with endogenous GTP and magnesium. Unlike other small G proteins, RhoE, along with two other proteins Rnd1/Rho6 and Rnd2/RhoN, does not hydrolyze GTP.