Orit Gutman

Tel Aviv University, Tel Aviv, Tel Aviv, Israel

Are you Orit Gutman?

Claim your profile

Publications (24)130.23 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Src interactions with the plasma membrane are an important determinant of its activity. In turn, Src activity modulates its association with the membrane through binding of activated Src to phosphotyrosylated proteins. Caveolin-1 (Cav-1), a major component of caveolae, is a known Src phosphorylation target, and both were reported to regulate cell transformation. However, the nature of Src-Cav-1 interactions, a potential mechanism of their coregulation, remained unclear.Here, we used FRAP beam-size analysis, coimmunoprecipitation, quantitative imaging and Far-Western studies with cells expressing wild-type as well as structural and activity mutants of Src-GFP and Cav-1-mRFP to measure their interactions with the membrane and with each other. We show dynamic Src-plasma membrane interactions, which are augmented and stabilized by Cav-1. The mechanism involves phosphorylation of Cav-1 at Tyr14 by Src, subsequent binding of the Src SH2 domain to phospho-Cav-1, leading to accumulation of activated Src in focal adhesions. This novel Cav-1 function potentially modulates focal adhesion dynamics.
    Molecular biology of the cell 10/2013; · 5.98 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Transforming growth factor-β (TGF-β) and bone morphogenetic protein (BMP) cytokines participate in a multiplicity of ways in the regulation of numerous physiological and pathological processes. Their wide-ranging biological functions are controlled by several mechanisms, including regulation of transcription, complex formation among the signaling receptors (oligomerization) and with co-receptors, binding of the receptors to scaffolding proteins or their targeting to specific membrane domains. Here, we address the generation of TGF-β and BMP receptor homo- and hetero-oligomers and its roles as a mechanism capable of fast regulation of signaling by these crucial cytokines. We examine the available biochemical, biophysical and structural evidence for the ternary structure of these complexes, and the possible roles of homomeric and heteromeric receptor oligomers in signaling.
    FEBS letters 01/2012; 586(14):1885-96. · 3.54 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Prenylation primarily by geranylgeranylation is required for membrane attachment and function of type I Rho of Plants (ROPs) and Gγ proteins, while type II ROPs are attached to the plasma membrane by S-acylation. Yet, it is not known how prenylation affects ROP membrane interaction dynamics and what are the functional redundancy and specificity of type I and type II ROPs. Here, we have used the expression of ROPs in mammalian cells together with geranylgeranylation and CaaX prenylation-deficient mutants to answer these questions. Our results show that the mechanism of type II ROP S-acylation and membrane attachment is unique to plants and likely responsible for the viability of plants in the absence of CaaX prenylation activity. The prenylation of ROPs determines their steady-state distribution between the plasma membrane and the cytosol but has little effect on membrane interaction dynamics. In addition, the prenyl group type has only minor effects on ROP function. Phenotypic analysis of the CaaX prenylation-deficient pluripetala mutant epidermal cells revealed that type I ROPs affect cell structure primarily on the adaxial side, while type II ROPs are functional and induce a novel cell division phenotype in this genetic background. Taken together, our studies show how prenyl and S-acyl lipid modifications affect ROP subcellular distribution, membrane interaction dynamics, and function.
    Plant physiology 02/2011; 155(2):706-20. · 6.56 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Rho GTPases are master regulators of cell polarity. For their function, Rhos must associate with discrete plasma membrane domains. Rho of Plants (ROPs) or RACs comprise a single family. Prenylation and S-acylation of hypervariable domain cysteines of Ras and Rho GTPases are required for their function; however, lipid modifications in the G domain have never been reported. Reversible S-acylation involves the attachment of palmitate (C16:0) or other saturated lipids to cysteines through a thioester linkage and was implicated in the regulation of signaling. Here we show that transient S-acylation of Arabidopsis AtROP6 takes place on two conserved G domain cysteine residues, C21 and C156. C21 is relatively exposed and is accessible for modification, but C156 is not, implying that its S-acylation involves a conformational change. Fluorescence recovery after photobleaching beam-size analysis shows that S-acylation of AtROP6 regulates its membrane-association dynamics, and detergent-solubilization studies indicate that it regulates AtROP6 association with lipid rafts. Site-specific acylation-deficient AtROP6 mutants can bind and hydrolyze GTP but display compromised effects on polar cell growth, endocytic uptake of the tracer dye FM4-64, and distribution of reactive oxygen species. These data reveal an S-acylation switch that regulates Rho signaling.
    Current biology: CB 05/2010; 20(10):914-20. · 10.99 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-β2 (PLCβ2) by G protein αq and βγ dimers. We show that activation by αq and βγ differ from activation by Rac2 and from each other. Stimulation by αq enhanced the plasma membrane association of PLCβ2, but not of PLCβ2Δ, which lacks the αq-interacting region. Although αq resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCβ2 and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCβ2 by αq occurred by enhancing PLCβ2 association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by βγ shifted the FRAP of PLCβ2 and PLCβ2Δ to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCβ2 membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP2) in localized versus dispersed populations. PLCβ2 activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCβ2 to act locally on PtdInsP2 at specific domains. Activation by αq leads to lipid-like diffusion of PLCβ2 accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by βγ recruits PLCβ2 to the membrane by transient interactions, leading to fast “surfing” diffusion along the membrane, sampling large regions for dispersed PtdInsP2 populations.
    Journal of Biological Chemistry 02/2010; 285(6):3905-3915. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: We combined fluorescence recovery after photobleaching (FRAP) beam-size analysis with biochemical assays to investigate the mechanisms of membrane recruitment and activation of phospholipase C-beta(2) (PLCbeta(2)) by G protein alpha(q) and betagamma dimers. We show that activation by alpha(q) and betagamma differ from activation by Rac2 and from each other. Stimulation by alpha(q) enhanced the plasma membrane association of PLCbeta(2), but not of PLCbeta(2)Delta, which lacks the alpha(q)-interacting region. Although alpha(q) resembled Rac2 in increasing the contribution of exchange to the FRAP of PLCbeta(2) and in enhancing its membrane association, the latter effect was weaker than with Rac2. Moreover, the membrane recruitment of PLCbeta(2) by alpha(q) occurred by enhancing PLCbeta(2) association with fast-diffusing (lipid-like) membrane components, whereas stimulation by Rac2 led to interactions with slow diffusing membrane sites. On the other hand, activation by betagamma shifted the FRAP of PLCbeta(2) and PLCbeta(2)Delta to pure lateral diffusion 3- to 5-fold faster than lipids, suggesting surfing-like diffusion along the membrane. We propose that these different modes of PLCbeta(2) membrane recruitment may accommodate contrasting functional needs to hydrolyze phosphatidylinositol 4,5-bisphosphate (PtdInsP(2)) in localized versus dispersed populations. PLCbeta(2) activation by Rac2, which leads to slow lateral diffusion and much faster exchange, recruits PLCbeta(2) to act locally on PtdInsP(2) at specific domains. Activation by alpha(q) leads to lipid-like diffusion of PLCbeta(2) accompanied by exchange, enabling the sampling of larger, yet limited, areas prior to dissociation. Finally, activation by betagamma recruits PLCbeta(2) to the membrane by transient interactions, leading to fast "surfing" diffusion along the membrane, sampling large regions for dispersed PtdInsP(2) populations.
    Journal of Biological Chemistry 12/2009; 285(6):3905-15. · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Calcium-dependent exocytosis is regulated by a vast number of proteins. DOC2B is a synaptic protein that translocates to the plasma membrane (PM) after small elevations in intracellular calcium concentration. The aim of this study was to investigate the role of DOC2B in calcium-triggered exocytosis. Using biochemical and biophysical measurements, we demonstrate that the C2A domain of DOC2B interacts directly with the PM in a calcium-dependent manner. Using a combination of electrophysiological, morphological, and total internal reflection fluorescent measurements, we found that DOC2B acts as a priming factor and increases the number of fusion-competent vesicles. Comparing secretion during repeated stimulation between wild-type DOC2B and a mutated DOC2B that is constantly at the PM showed that DOC2B enhances catecholamine secretion also during repeated stimulation and that DOC2B has to translocate to the PM to exert its facilitating effect, suggesting that its activity is dependent on calcium. The hypothesis that DOC2B exerts its effect at the PM was supported by the finding that DOC2B affects the fusion kinetics of single vesicles and interacts with the PM SNAREs (soluble NSF attachment receptors). We conclude that DOC2B is a calcium-dependent priming factor and its activity at the PM enables efficient expansion of the fusion pore, leading to increased catecholamine release.
    Journal of Neuroscience 08/2008; 28(27):6794-806. · 6.91 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src-GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src.
    The Journal of Cell Biology 09/2007; 178(4):675-86. · 10.82 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: www.jcb.org/cgi/doi/10.1083/jcb.200701133 JCB 675
    The Journal of Cell Biology 02/2007; 178:675-686. · 10.82 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Cholesterol and glycosphingolipid-enriched membrane domains, termed lipid rafts, were proposed to play important roles in trafficking and signaling events. These functions are inhibited following putative disruption of rafts by cholesterol depletion, commonly induced by treatment with methyl-beta-cyclodextrin (MbetaCD). However, several studies showed that the lateral diffusion of membrane proteins is inhibited by MbetaCD, suggesting that it may have additional effects on membrane organization unrelated to cholesterol removal. Here, we investigated this possibility by comparison of the effects of cholesterol depletion by MbetaCD and by metabolic inhibition (compactin), and of treatment with alpha-CD, which does not bind cholesterol. The studies employed two series of proteins (Ras and influenza hemagglutinin), each containing as internal controls related mutants that differ in raft association. Mild MbetaCD treatment retarded the lateral diffusion of both raft and non-raft mutants, whereas similar cholesterol reduction (30-33%) by metabolic inhibition enhanced selectively the diffusion of the raft-associated mutants. Moreover, alpha-CD also inhibited the diffusion of raft and non-raft mutants, despite its lack of effect on cholesterol content. These findings suggest that the widely used treatment with CD to reduce cholesterol has additional, cholesterol-independent effects on membrane protein mobility, which do not necessarily distinguish between raft and non-raft proteins.
    Traffic 08/2006; 7(7):917-26. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase C-beta (PLCbeta) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCbeta(2) isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCbeta(2) with the plasma membrane. For either GFP-PLCbeta(2) or GFP-PLCbeta(2)Delta, a C-terminal deletion mutant lacking the region required for stimulation by Galpha(q), these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCbeta(2) and GFP-PLCbeta(2)Delta in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCbeta(2). Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCbeta(2), but had the opposite effect on GFP-PLCbeta(2)Delta, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCbeta isozyme by a GTP-binding protein and assign a novel function to the PLCbeta(2) C-terminal region, regulating its exchange between membrane-bound and cytosolic states.
    Journal of Biological Chemistry 04/2003; 278(10):8645-52. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Phospholipase C-β (PLCβ) isozymes play important roles in transmembrane signaling. Their activity is regulated by heterotrimeric G proteins. The PLCβ2 isozyme is unique in being stimulated also by Rho GTPases (Rac and Cdc42). However, the mechanism(s) of this stimulation are still unclear. Here, we employed fluorescence recovery after photobleaching to investigate the interaction of green fluorescent protein (GFP)-PLCβ2with the plasma membrane. For either GFP-PLCβ2 or GFP-PLCβ2Δ, a C-terminal deletion mutant lacking the region required for stimulation by Gαq, these interactions were characterized by a mixture of exchange with a cytoplasmic pool and lateral diffusion. Constitutively active Rac2(12V) stimulated the activity of both GFP-PLCβ2 and GFP-PLCβ2Δ in live cells, and enhanced their membrane association as evidenced by the marked reduction in their fluorescence recovery rates. Both effects required the putative N-terminal pleckstrin homology (PH) domain of PLCβ2. Importantly, Rac2(12V) dramatically increased the contribution of exchange to the fluorescence recovery of GFP-PLCβ2, but had the opposite effect on GFP-PLCβ2Δ, where lateral diffusion became dominant. Our results demonstrate for the first time the regulation of membrane association of a PLCβ isozyme by a GTP-binding protein and assign a novel function to the PLCβ2C-terminal region, regulating its exchange between membrane-bound and cytosolic states.
    Journal of Biological Chemistry 03/2003; 278(10):8645-8652. · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Ras-membrane interactions play important roles in signaling and oncogenesis. H-Ras and K-Ras have nonidentical membrane anchoring moieties that can direct them to different membrane compartments. Ras-lipid raft interactions were reported, but recent studies suggest that activated K-Ras and H-Ras are not raft resident. However, specific interactions of activated Ras proteins with nonraft sites, which may underlie functional differences and phenotypic variation between different Ras isoforms, are unexplored. Here we used lateral mobility studies by FRAP to investigate the membrane interactions of green fluorescent protein-tagged H- and K-Ras in live cells. All Ras isoforms displayed stable membrane association, moving by lateral diffusion and not by exchange with a cytoplasmic pool. The lateral diffusion rates of constitutively active K- and H-Ras increased with their expression levels in a saturable manner, suggesting dynamic association with saturable sites or domains. These sites are distinct from lipid rafts, as the activated Ras mutants are not raft resident. Moreover, they appear to be different for H- and K-Ras. However, wild-type H-Ras, the only isoform preferentially localized in rafts, displayed cholesterol-sensitive interactions with rafts that were independent of its expression level. Our findings provide a mechanism for selective signaling by different Ras isoforms.
    The Journal of Cell Biology 06/2002; 157(5):865-72. · 10.82 Impact Factor
  • H Niv, O Gutman, Y I Henis, Y Kloog
    [Show abstract] [Hide abstract]
    ABSTRACT: Membrane anchorage of Ras proteins in the inner leaflet of the plasma membrane is an important factor in their signaling and oncogenic potential. Despite these important roles, the precise mode of Ras-membrane interactions is not yet understood. It is especially important to characterize these interactions at the surface of intact cells. To investigate Ras-membrane interactions in live cells, we employed studies on the lateral mobility of a constitutively active Ras isoform to characterize its membrane dynamics, and examined the effects of the Ras-displacing antagonist S-trans, trans-farnesylthiosalicylic acid (FTS) (Haklai, R., Gana-Weisz, M., Elad, G., Paz, A., Marciano, D., Egozi, Y., Ben-Baruch, G., and Kloog, Y. (1998) Biochemistry 37, 1306-1314) on these parameters. A green fluorescent protein (GFP) was fused to the N terminus of constitutively active Ki-Ras 4B(12V) to generate GFP-Ki-Ras(12V). When stably expressed in Rat-1 cells, this protein was preferentially localized to the plasma membrane and displayed transforming activity. The lateral mobility studies demonstrated that GFP-Ki-Ras(12V) undergoes fast lateral diffusion at the plasma membrane, rather than exchange between membrane-bound and unbound states. Treatment of the cells with FTS had a biphasic effect on GFP-Ki-Ras(12V) lateral mobility. At the initial phase, the lateral diffusion rate of GFP-Ki-Ras(12V) was elevated, suggesting that it is released from some constraints on its lateral mobility. This was followed by dislodgment of the protein into the cytoplasm, and a reduction in the diffusion rate of the fraction of GFP-Ki-Ras(12V) that remained associated with the plasma membrane. Control experiments with other S-prenyl analogs showed that these effects are specific for FTS. These results have implications for the interactions of Ki-Ras with specific membrane anchorage domains or sites.
    Journal of Biological Chemistry 02/1999; 274(3):1606-13. · 4.65 Impact Factor
  • E Fire, O Gutman, M G Roth, Y I Henis
    [Show abstract] [Hide abstract]
    ABSTRACT: Measurements of the lateral mobility of native and mutated membrane proteins, combined with treatments that alter clathrin lattice structure, are capable of characterizing their interactions with coated pits in live cells (Fire, E., Zwart, D. E., Roth, M. G., and Henis, Y. I. (1991) J. Cell Biol. 115, 1585-1594). To explore the dependence of these interactions on the internalization signal and the aggregation state of the protein, we have extended this approach to investigate the interactions between coated pits and several influenza hemagglutinin (HA) mutants, which differ in the internalization signals in their short cytoplasmic tails. The lack of internalization signals in the trimeric wild-type HA enables a direct comparison between specific internalization signals introduced singly in each mutant. We have selected for these studies HA mutants that showed different internalization rates and varied in their tendency to aggregate into complexes larger than trimers. Our results indicate that the mode of interaction with coated pits (transient association-dissociation versus stable entrapment) depends on the internalization signal and affects the internalization efficiency. Mutants that contain a strong internalization signal and undergo fast endocytosis were entrapped in coated pits for the entire duration of the lateral mobility measurement, suggesting stable association with (slow dissociation from) coated pits. A mutant with a suboptimal internalization signal, which was internalized 10-fold slower, exhibited transient interactions with coated pits. Both types of interactions disappeared or were significantly reduced upon disruption of the clathrin lattices under hypertonic conditions, and were modulated following the "freezing" of coated pits by cytosol acidification. Unlike the dependence on the cytoplasmic internalization signal, the interactions with coated pits did not depend on the aggregation state (measured by sucrose gradient centrifugation after solubilization in n-octylglucoside) of the mutants.
    Journal of Biological Chemistry 10/1995; 270(36):21075-81. · 4.65 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: To investigate the possible role of viral glycoprotein mobility in membrane fusion, fluorescence photobleaching recovery was employed to study the effects of exposure to mildly acidic pH (required to convert many viral fusion proteins to the fusion-active form) on the lateral mobility of influenza hemagglutinin (HA) proteins expressed at the surface of transfected cells. HA proteins from two different strains were compared: X:31 HA, which is activated by a brief exposure to pH 4.9 but is irreversibly inactivated at longer exposure times, and HA from A/Japan/305/57, which is relatively stable to inactivation at this pH [Puri, A., Booy, F.P., Doms, R.W., White, J.M., & Blumenthal, R. (1990) J. Virol. 64, 3824-3832]. The HA proteins from both strains, expressed in CV-1 cells using VS-40 vectors, exhibited relatively unrestricted lateral diffusion at the cell surface. The high mobility persisted following a brief exposure (1 min) to pH 4.9 to mediate conversion to the fusogenic state. Longer times (up to 15 min) of preincubation at pH 4.9 inhibited the lateral mobility of X:31 HA (the lateral diffusion rate was markedly reduced, followed by immobilization) but not of A/Japan HA, whose fusion activity is resistant to such treatment. Inhibition of the lateral mobility of X:31 HA due to preincubation at low pH was not specific to the CV-1 cells and was found also in a CHO cell line stably expressing this protein. The results presented demonstrate a close correlation between loss of mobility and inactivation of fusogenic activity, in accord with the notion that lateral motion of the HA proteins is required for fusion.(ABSTRACT TRUNCATED AT 250 WORDS)
    Biochemistry 01/1993; 32(1):101-6. · 3.38 Impact Factor
  • Source
    B Aroeti, O Gutman, Y I Henis
    [Show abstract] [Hide abstract]
    ABSTRACT: Destabilization of the target membrane structure by fusion-promoting viral glycoproteins is assumed to be an essential part of the fusion mechanism. To explore this possibility, we employed fluorescence photobleaching recovery to investigate changes in the lateral mobility of native membrane constituents in human red blood cells (RBCs) during the course of Sendai virus-mediated fusion. The mobile fraction of RBC membrane proteins labeled with 5-(4,6-dichloro-5-triazin-2-yl)aminofluorescein increased significantly in the course of fusion, relaxing back to the original values upon completion of the fusion process. A different effect was observed on the lateral mobility of a fluorescent lipid probe, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine, incorporated initially into the external monolayer. In this case, the lateral diffusion coefficient (rather than the mobile fraction) increased during fusion; this increase was permanent in the absence of Mg-ATP and transient in its presence. An active viral fusion protein was required to mediate the effects on both protein and lipid mobility. These effects, which take place on the same time scale as that of the fusion process, suggest that the organization of the RBC membrane is perturbed during fusion and that the observed changes may be related to the fusion mechanism.
    Journal of Biological Chemistry 08/1992; 267(19):13272-7. · 4.65 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Fluorescence photobleaching recovery was employed to study the effects of specific immobilization of Sendai virus envelope glycoproteins (F, the fusion protein, and HN, the hemagglutinin-neuraminidase) on the virally mediated fusion of human erythrocytes. Lateral immobilization of varying fractions of F and/or HN (after virus adsorption and hemagglutination, but before fusion) was achieved by cross-linking them with succinyl concanavalin A (inhibiting both F and HN) or with specific rabbit IgG directed against either F or HN. Alternatively, agglutinated cells were treated with low concentrations of the above proteins (inducing only minor inhibition of either mobility or fusion), and immobilization of F and/or HN was induced by cross-linking with a secondary antibody; this protocol ensured a minimal contribution of direct binding to the viral proteins to the inhibition of fusion. Our results demonstrate that lateral immobilization of either F or HN results in a strong inhibition of cell-cell fusion and a much weaker inhibition of virus-cell fusion. The level of cell-cell fusion was directly correlated with the level of laterally mobile viral glycoproteins in the cell membrane (either F or HN). We conclude that lateral mobility of both F and HN in the red cell membrane is essential for cell-cell fusion and that not only F but also HN has a role in this fusion event. The possible reasons for the different dependence of cell-cell and virus-cell fusion on viral glycoprotein mobility are discussed.
    Journal of Biological Chemistry 11/1989; 264(29):17119-25. · 4.65 Impact Factor
  • Z Katzir, O Gutman, Y I Henis
    [Show abstract] [Hide abstract]
    ABSTRACT: Fusion of human erythrocytes by Sendai virions is accompanied by lateral mobilization of the viral envelope proteins (F, the fusion protein, and HN, the hemagglutinin/neuraminidase protein) in the target cell membrane; the dynamic parameters characterizing the lateral diffusion of F and HN in the fused cell membrane are identical [Henis, Y. I., & Gutman, O. (1987) Biochemistry 26, 812-819; Aroeti, B., & Henis, Y. I. (1988) Biochemistry 27, 5654-5661]. This identity raised the possibility that F and HN diffuse together in the cell membrane in mutual heterocomplexes. In order to investigate the possible formation of F-HN complexes in the target cell membrane, which could be important for the fusion process mediated by the viral envelope proteins, we combined fluorescence photobleaching recovery (FPR) measurements of the lateral mobility of the viral glycoproteins with antibody-mediated cross-linking of F or HN. After fusion, one viral glycoprotein type was immobilized by cross-linking with highly specific bivalent polyclonal IgG. The other glycoprotein type was labeled with fluorescence monovalent Fab' fragments that do not induce cross-linking, and its mobility was measured by FPR. Neither the mobile fraction nor the lateral diffusion coefficient of the Fab'-labeled viral glycoproteins was affected by immobilization of the second viral envelope protein, demonstrating that F and HN diffuse independently in the target cell membrane and are not associated in mutual complexes.
    Biochemistry 08/1989; 28(15):6400-5. · 3.38 Impact Factor
  • Source
    Y I Henis, O Gutman
    [Show abstract] [Hide abstract]
    ABSTRACT: Two independent methods demonstrated that resealed human erythrocyte ghosts undergo Sendai virus-mediated cell-cell fusion to a much lower degree (about 4%) than intact erythrocytes, in spite of similar levels of viral envelope-cell fusion in the two preparations. Fluorescence photobleaching recovery (FPR) showed similar lateral mobilities of the viral glycoproteins following fusion with either ghosts or whole erythrocytes. It is suggested that although viral glycoprotein mobilization in the cell membrane is essential for cell-cell fusion, the target cell properties are also important; in the absence of the required cellular parameters, the mobilization may not be a sufficient condition.
    FEBS Letters 03/1988; 228(2):281-4. · 3.58 Impact Factor