Luna Lab

The intermediate monoclinic phase of phosphatidylcholines

Elizabeth J. Luna et al. — Fri, 15 Mar 2013 07:05:14 PDT

Two pure phospholipids, dimyristoyl phosphatidylcholine and dipalmitoyl phosphatidylcholine, have been studied using freeze-fracture electron microscopy and the partitioning of the spin label, TEMPO. It is found that the characteristic band pattern, corresponding to monoclinic symmetry in multilamellar liposomes, is observed only in freeze-fracture electron microphotographs when samples are quenched from temperatures intermediate between the chain melting transition temperature and the pretransition temperature of the membrane. Markings are also observed on fracture faces of samples quenched from below the pretransition, but these "bands" are few in number and are widely and irregularly spaced. The lipid membranes used for freeze-fracture were prepared using detergent dialysis and are thought to consist of one, two, or some small number of concentric bilayer shells. These observations are in excellent accord with the recent, prior studies of Janiak, M.J., Small, D.M. and Shirley, G.G., ((1976) Biochemistry 15, 4575--4580), who found monoclinic symmetry (Pbeta' structure) in multilamellar liposomes of these phospholipids only when the sample temperature was intermediate between the main, chain melting transition temperature, and the pretransition temperature. The significance of these results for relating freeze-fracture electron microphotographis to phase diagrams derived from spin label or calorimetric data is discussed briefly. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) partitioning data show distinct differences between liposomal preparations of these lipids, and other preparations having fewer bilayers per vesicular structure, with respect to the position, width, and hysteresis of the pretransition.

Lateral phase separations in binary mixtures of phospholipids having different charges and different crystalline structures

Elizabeth J. Luna et al. — Fri, 15 Mar 2013 07:05:13 PDT

Synthetic dipalmitoyl phosphatidylserine exhibits a sharp chain-melting transition temperature at 51 degrees C as judged by partitioning of the spin label 2,2,6,6-tetramethylpiperidine-1-oxyl. Phase diagrams representing lateral phase separations in binary mixtures of dipalmitoyl phosphatidylserine with dipalmitoyl phosphatidylcholine as well as with dimyristoyl phosphatidylcholine are derived from paramagnetic resonance determinations of 2,2,6,6,-tetramethylpiperidine-1-oxyl partitioning, freeze-fracture electron microscopic studies and theoretical arguments that limit the general form of acceptable phase diagrams. The reported phase diagrams are the first to describe binary mixtures in which one lipid is charged and the second lipid uncharged. These phase diagrams also are the first to include the problem of solid phases with different crystalline conformations as it relates to the occurrence of a pretransition in phosphatidylcholines and its absence in phosphatidylserines. In addition to the phase diagrams reported here for these two binary mixtures, a brief theoretical discussion is given of other possible phase diagrams that may be appropriate to other lipid mixtures with particular consideration given to the problem of crystalline phases of different structures and the possible occurrence of second-order phase transitions in these mixtures.

Multiple phase equilibria in binary mixtures of phospholipids

Elizabeth J. Luna et al. — Fri, 15 Mar 2013 07:05:12 PDT

Approximate phse diagrams describing lateral phase separations are given for binary mixtures of dimyristoyl phosphatidylcholine with dipalmitoyl phosphatidylcholine, distearoyl phosphatidycholine, and dipalmitoyl phosphatidylethanolamine. These diagrams are based in part on freeze-fracture electron microscopic data. These phase diagrams represent an improvement over previous studies in that both solid phses (Pbeta' and Lbeta') of the phosphatidylcholines are included. Further consideration is given to the problem of binary mixtures in which there are two Pbeta' phases that do not form a continuous range of solid solutions.

An N-terminal, 830 residues intrinsically disordered region of the cytoskeleton-regulatory protein supervillin contains Myosin II- and F-actin-binding sites

Stanislav O. Fedechkin et al. — Mon, 28 Jan 2013 11:16:08 PST

Supervillin, the largest member of the villin/gelsolin family, is a cytoskeleton regulating, peripheral membrane protein. Supervillin increases cell motility and promotes invasive activity in tumors. Major cytoskeletal interactors, including filamentous actin and myosin II, bind within the unique supervillin amino terminus, amino acids 1-830. The structural features of this key region of the supervillin polypeptide are unknown. Here, we utilize circular dichroism and bioinformatics sequence analysis to demonstrate that the N-terminal part of supervillin forms an extended intrinsically disordered region (IDR). Our combined data indicate that the N-terminus of human and bovine supervillin sequences (positions 1-830) represents an IDR, which is the largest IDR known to date in the villin/gelsolin family. Moreover, this result suggests a potentially novel mechanism of regulation of myosin II and F-actin via the intrinsically disordered N-terminal region of hub protein supervillin.

Human Genome-Wide Association and Mouse Knockout Approaches Identify Platelet Supervillin as an Inhibitor of Thrombus Formation under Shear Stress

Leonard C. Edelstein et al. — Fri, 04 May 2012 08:19:18 PDT

BACKGROUND: High shear force critically regulates platelet adhesion and thrombus formation during ischemic vascular events. To identify genetic factors that influence platelet thrombus formation under high shear stress, we performed a genome-wide association study (GWAS) and confirmatory experiments in human and animal platelets.

METHODS AND RESULTS: Closure times in the shear-dependent Platelet Function Analyzer (PFA)-100® were measured on healthy, non-diabetic European Americans (n=125) and African Americans (n=116). A GWAS significant association (p<5X10(-8)) was identified with 2 SNPs within the SVIL gene (chr 10p11.23) in African-Americans but not European Americans. Microarray analyses of human platelet RNA demonstrated the presence of SVIL isoform 1 (supervillin) but not muscle-specific isoforms 2 and 3 (archvillin, SmAV). SVIL mRNA levels were associated with SVIL genotypes (p≤0.02) and were inversely correlated with PFA-100 closure times (p<0.04) and platelet volume (p<0.02). Leukocyte-depleted platelets contained abundant levels of the ~205 kD supervillin polypeptide. To assess functionality, mice lacking platelet supervillin were generated and back-crossed onto a C57BL/6 background. Compared to controls, murine platelets lacking supervillin were larger by flow cytometry and confocal microscopy, and exhibited enhanced platelet thrombus formation under high shear, but not low shear, conditions.

CONCLUSIONS: We show for the first time that 1) platelets contain supervillin, 2) platelet thrombus formation in the PFA-100® is associated with human SVIL variants and low SVIL expression, and 3) murine platelets lacking supervillin exhibit enhanced platelet thrombus formation at high shear stress. These data are consistent with an inhibitory role for supervillin in platelet adhesion and arterial thrombosis.

Supervillin couples myosin-dependent contractility to podosomes and enables their turnover

Ridhirama Bhuwania et al. — Fri, 30 Mar 2012 07:40:51 PDT

Podosomes are actin-rich adhesion and invasion structures. Especially in macrophages, podosomes exist in two subpopulations, large precursors at the cell periphery and smaller podosomes (successors) in the cell interior. To date, the mechanisms that differentially regulate these subpopulations are largely unknown. Here, we show that the membrane-associated protein supervillin localizes preferentially to successor podosomes and becomes enriched at precursors immediately prior to their dissolution. Consistently, podosome numbers are inversely correlated with supervillin protein levels. Using deletion constructs, we find that the myosin II-regulatory N-terminus of supervillin (SV 1-174) is crucial for these effects. Phosphorylated myosin light chain (pMLC) localizes at supervillin-positive podosomes, and time-lapse analyses show that enrichment of GFP-supervillin at podosomes coincides with their coupling to contractile myosin IIA-positive cables. We also show that supervillin binds only to activated myosin IIA, and a dysregulated N-terminal construct (SV 1-830) enhances pMLC levels at podosomes. Thus, preferential recruitment of supervillin to podosome subpopulations may both require and induce actomyosin contractility. Using siRNA and pharmacological inhibition, we demonstrate that supervillin and myosin IIA cooperate to regulate podosome lifetime, podosomal matrix degradation and cell polarization. In sum, we show here that podosome subpopulations differ in their molecular composition and identify supervillin, in cooperation with myosin IIA, as a critical factor in the regulation of podosome turnover and function.

Novel interactors and a role for supervillin in early cytokinesis

Tara C. Smith et al. — Mon, 14 Mar 2011 08:17:16 PDT

Supervillin, the largest member of the villin/gelsolin/flightless family, is a peripheral membrane protein that regulates each step of cell motility, including cell spreading. Most known interactors bind within its amino (N)-terminus. We show here that the supervillin carboxy (C)-terminus can be modeled as supervillin-specific loops extending from gelsolin-like repeats plus a villin-like headpiece. We have identified 27 new candidate interactors from yeast two-hybrid screens. The interacting sequences from 12 of these proteins (BUB1, EPLIN/LIMA1, FLNA, HAX1, KIF14, KIFC3, MIF4GD/SLIP1, ODF2/Cenexin, RHAMM, STARD9/KIF16A, Tks5/SH3PXD2A, TNFAIP1) co-localize with and mis-localize EGFP-supervillin in mammalian cells, suggesting associations in vivo. Supervillin-interacting sequences within BUB1, FLNA, HAX1, and MIF4GD also mimic supervillin over-expression by inhibiting cell spreading. Most new interactors have known roles in supervillin-associated processes, e.g. cell motility, membrane trafficking, ERK signaling, and matrix invasion; three (KIF14, KIFC3, STARD9/KIF16A) have kinesin motor domains; and five (EPLIN, KIF14, BUB1, ODF2/cenexin, RHAMM) are important for cell division. GST fusions of the supervillin G2-G3 or G4-G6 repeats co-sediment KIF14 and EPLIN, respectively, consistent with a direct association. Supervillin depletion leads to increased numbers of bi- and multi-nucleated cells. Cytokinesis failure occurs predominately during early cytokinesis. Supervillin localizes with endogenous myosin II and EPLIN in the cleavage furrow, and overlaps with the oncogenic kinesin, KIF14, at the midbody. We conclude that supervillin, like its interactors, is important for efficient cytokinesis. Our results also suggest that supervillin and its interaction partners coordinate actin and microtubule motor functions throughout the cell cycle.

The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility

Zhiyou Fang et al. — Mon, 14 Mar 2011 08:17:14 PDT

In migrating cells, the cytoskeleton coordinates signal transduction and redistribution of transmembrane proteins, including integrins and growth factor receptors. Supervillin is an F-actin- and myosin II-binding protein that tightly associates with signaling proteins in cholesterol-rich, 'lipid raft' membrane microdomains. We show here that supervillin also can localize with markers for early and sorting endosomes (EE/SE) and with overexpressed components of the Arf6 recycling pathway in the cell periphery. Supervillin tagged with the photoswitchable fluorescent protein, tdEos, moves both into and away from dynamic structures resembling podosomes at the basal cell surface. Rapid integrin recycling from EE/SE is inhibited in supervillin-knockdown cells, but the rates of integrin endocytosis and recycling from the perinuclear recycling center (PNRC) are unchanged. A lack of synergy between supervillin knockdown and the actin filament barbed-end inhibitor, cytochalasin D, suggests that both treatments affect actin-dependent rapid recycling. Supervillin also enhances signaling from the epidermal growth factor receptor (EGFR) to extracellular signal-regulated kinases (ERKs) 1 and 2 and increases the velocity of cell translocation. These results suggest that supervillin, F-actin and associated proteins coordinate a rapid, basolateral membrane recycling pathway that contributes to ERK signaling and actin-based cell motility.