Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-e... more Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform-incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.
UAS-GKVK Campus proteins are likely to be concentrated (Simons and Iko-GKVK PO nen, 1997; Simons ... more UAS-GKVK Campus proteins are likely to be concentrated (Simons and Iko-GKVK PO nen, 1997; Simons and Toomre, 2000), (2) rafts are dy-Bangalore 560 065 namic assemblies of small size, constituted by compo-India nents that are preferentially associated with lipids; 2 Raman Research Institute functional organization is dictated by the induction of CV Raman Avenue stable large-scale structures (Anderson and Jacob-Bangalore 560 080 son, 2002). India The prevailing operational description of rafts is based 3 Department of Chemical Sciences on the observation that detergent-resistant membranes Tata Institute of Fundamental Research (TIFR) (DRMs) obtained by the extraction of living cells with Homi Bhabha Road cold nonionic detergents (e.g. Triton X-100), retain a Mumbai 400005 specific set of proteins and lipids, including GPI-anchored India proteins (GPI-APs), signaling proteins such as lipidlinked nonreceptor tyrosine kinases, (glyco)sphingolipids, and cholesterol (Brown and London, 2000). DRM-Summary association has also been shown to correlate with the sorting and signaling properties of some proteins Cholesterol and sphingolipid-enriched "rafts" have (Brown and London, 2000; Simons and Ikonen, 1997; long been proposed as platforms for the sorting of Simons and Toomre, 2000). More recently however, the specific membrane components including glycosylcorrelation of DRMs with preexisting lipid domains in phosphatidylinositol-anchored proteins (GPI-APs), howmembranes is being seriously contested. For instance, ever, their existence and physical properties have in homogeneous fluid membrane systems, Triton X-100 been controversial. Here, we investigate the size of treatment was found to induce large-scale ordered dolipid-dependent organization of GPI-APs in live cells, mains (Heerklotz, 2002) and in some cases the addition using homo and hetero-FRET-based experiments, of the detergent severely perturbs preexisting l o domains combined with theoretical modeling. These studies (Heerklotz et al., 2003). Consequently, in the more comreveal an unexpected organization wherein cell surplex environment of the cell membrane, DRM-associaface GPI-APs are present as monomers and a smaller tion should not be relied upon to provide information fraction (20%-40%) as nanoscale (Ͻ5 nm) cholesterolregarding any kind of preexisting organization of comsensitive clusters. These clusters are composed of at ponents (Zurzolo et al., 2003). Thus, new methodologies most four molecules and accommodate diverse GPIare necessary for establishing the existence and proper-AP species; crosslinking GPI-APs segregates them ties of in vivo rafts involved in cellular function. from preexisting GPI-AP clusters and prevents endo-Here, we focus on the cell surface organization of a cytosis of the crosslinked species via a GPI-AP-seleccommon raft-marker, GPI-APs, which are a diverse set tive pinocytic pathway. In conjunction with an analysis of exoplasmic, eukaryotic proteins exhibiting specific of the statistical distribution of the clusters, these obintracellular sorting and signaling properties regulated servations suggest a mechanism for functional lipidby alterations in cholesterol and sphingolipid levels in dependent clustering of GPI-APs. cell membranes (Chatterjee and Mayor, 2001; Mayor and Riezman, 2004). The lipid-dependent organization Introduction of GPI-APs at the cell surface will directly reveal the structure of rafts and establish a direct functional signifi-Rafts have been hypothesized as lateral heterogeneities cance for this concept. in membranes of living cells that are enriched in (glyco) While experiments on artificial membrane containing sphingolipids, cholesterol, and specific membrane proa lipid composition resembling that of DRMs exhibit teins. They have been proposed to be responsible for ordered domains with sizes ranging from the nanometer the sorting of associated proteins and for providing sites to the micron scale (Silvius, 2003), both fluorescence for the assembly of cytoplasmic signaling complexes in microscopy and conventional electron microscopy have a variety of biological contexts (Anderson and Jacobconsistently failed to reveal large-scale laterally segregated structures enriched in GPI-APs in living cells (Hao *Correspondence: [email protected] (M.R.), [email protected] (S.M) et al., 2001; Mayor and Maxfield, 1995; Mayor et al., 4 These authors contributed equally to this work. 1994; Parton et al., 1994); viewed at optical resolution 5 Present address: Program in Molecular Pathogenesis, Skirball Insti-004ف( nm), GPI-APs appear uniformly distributed in cell In native cell membranes, methods designed to detect Cell 578 proximity between molecules have observed inhomoge-phore; Figure 1A) to show clustered distribution of diverse GPI-APs in multiple cell lines. Second, time re-neous distributions of GPI-APs. Chemical crosslinking with short (1.1 nm) crosslinkers indicate that cholesterol-solved anisotropy decay experiments provide evidence of high density in clusters. Third, steady-state anisot-sensitive complexes of GPI-APs exist at the cell surface (Friedrichson and Kurzchalia, 1998). Using a fluores-ropy as a function of photobleaching, in conjunction with a detailed theoretical analysis suggests that these cence resonance energy transfer (FRET) method called homo-FRET which detects proximity between like fluor-dense clusters are small and only a fraction of GPI-APs are clustered. Finally, theoretical modeling of hetero-ophores at 1-10 nm scale we had suggested that GPI-APs occur in cholesterol-sensitive, submicron-sized FRET measurements confirm the presence of a fraction of small, dense clusters. We have kept the analytical "domains" at the surface of living cells (Varma and Mayor, 1998). In contrast, studies based on detecting methods used for these experiments in a single location (Supplemental Data available on Cell website), thus, if FRET between different fluorophores (hetero-FRET) reported the absence of detectable clustering of GPI-APs, the reader wishes to obtain the details of these calculations they are urged to refer to this section. putting an upper bound on the fraction (if any) of clustered GPI-APs (Kenworthy and Edidin, 1998; Kenworthy et al.,
Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediato... more Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediators of sorting and signaling functions of associated molecules. Apart from a limited number of biophysical studies in living cell membranes, raft-association has been defined by a simple biochemical criterion, namely the ability to partition with detergent-resistant membranes (DRMs). Here we examine the evidence for the specification of internalization mechanisms and endocytic pathways by rafts as defined by this criterion. We have surveyed the endocytic trafficking of a variety of molecules such as lipids, toxins, glycosylphosphatidylinositol (GPI)-anchored proteins, and DRM-associated transmembrane proteins.
Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-e... more Hedgehog (Hh) plays crucial roles in tissue-patterning and activates signaling in Patched (Ptc)-expressing cells. Paracrine signaling requires release and transport over many cell diameters away by a process that requires interaction with heparan sulfate proteoglycans (HSPGs). Here, we examine the organization of functional, fluorescently tagged variants in living cells by using optical imaging, FRET microscopy, and mutational studies guided by bioinformatics prediction. We find that cell-surface Hh forms suboptical oligomers, further concentrated in visible clusters colocalized with HSPGs. Mutation of a conserved Lys in a predicted Hh-protomer interaction interface results in an autocrine signaling-competent Hh isoform-incapable of forming dense nanoscale oligomers, interacting with HSPGs, or paracrine signaling. Thus, Hh exhibits a hierarchical organization from the nanoscale to visible clusters with distinct functions.
UAS-GKVK Campus proteins are likely to be concentrated (Simons and Iko-GKVK PO nen, 1997; Simons ... more UAS-GKVK Campus proteins are likely to be concentrated (Simons and Iko-GKVK PO nen, 1997; Simons and Toomre, 2000), (2) rafts are dy-Bangalore 560 065 namic assemblies of small size, constituted by compo-India nents that are preferentially associated with lipids; 2 Raman Research Institute functional organization is dictated by the induction of CV Raman Avenue stable large-scale structures (Anderson and Jacob-Bangalore 560 080 son, 2002). India The prevailing operational description of rafts is based 3 Department of Chemical Sciences on the observation that detergent-resistant membranes Tata Institute of Fundamental Research (TIFR) (DRMs) obtained by the extraction of living cells with Homi Bhabha Road cold nonionic detergents (e.g. Triton X-100), retain a Mumbai 400005 specific set of proteins and lipids, including GPI-anchored India proteins (GPI-APs), signaling proteins such as lipidlinked nonreceptor tyrosine kinases, (glyco)sphingolipids, and cholesterol (Brown and London, 2000). DRM-Summary association has also been shown to correlate with the sorting and signaling properties of some proteins Cholesterol and sphingolipid-enriched "rafts" have (Brown and London, 2000; Simons and Ikonen, 1997; long been proposed as platforms for the sorting of Simons and Toomre, 2000). More recently however, the specific membrane components including glycosylcorrelation of DRMs with preexisting lipid domains in phosphatidylinositol-anchored proteins (GPI-APs), howmembranes is being seriously contested. For instance, ever, their existence and physical properties have in homogeneous fluid membrane systems, Triton X-100 been controversial. Here, we investigate the size of treatment was found to induce large-scale ordered dolipid-dependent organization of GPI-APs in live cells, mains (Heerklotz, 2002) and in some cases the addition using homo and hetero-FRET-based experiments, of the detergent severely perturbs preexisting l o domains combined with theoretical modeling. These studies (Heerklotz et al., 2003). Consequently, in the more comreveal an unexpected organization wherein cell surplex environment of the cell membrane, DRM-associaface GPI-APs are present as monomers and a smaller tion should not be relied upon to provide information fraction (20%-40%) as nanoscale (Ͻ5 nm) cholesterolregarding any kind of preexisting organization of comsensitive clusters. These clusters are composed of at ponents (Zurzolo et al., 2003). Thus, new methodologies most four molecules and accommodate diverse GPIare necessary for establishing the existence and proper-AP species; crosslinking GPI-APs segregates them ties of in vivo rafts involved in cellular function. from preexisting GPI-AP clusters and prevents endo-Here, we focus on the cell surface organization of a cytosis of the crosslinked species via a GPI-AP-seleccommon raft-marker, GPI-APs, which are a diverse set tive pinocytic pathway. In conjunction with an analysis of exoplasmic, eukaryotic proteins exhibiting specific of the statistical distribution of the clusters, these obintracellular sorting and signaling properties regulated servations suggest a mechanism for functional lipidby alterations in cholesterol and sphingolipid levels in dependent clustering of GPI-APs. cell membranes (Chatterjee and Mayor, 2001; Mayor and Riezman, 2004). The lipid-dependent organization Introduction of GPI-APs at the cell surface will directly reveal the structure of rafts and establish a direct functional signifi-Rafts have been hypothesized as lateral heterogeneities cance for this concept. in membranes of living cells that are enriched in (glyco) While experiments on artificial membrane containing sphingolipids, cholesterol, and specific membrane proa lipid composition resembling that of DRMs exhibit teins. They have been proposed to be responsible for ordered domains with sizes ranging from the nanometer the sorting of associated proteins and for providing sites to the micron scale (Silvius, 2003), both fluorescence for the assembly of cytoplasmic signaling complexes in microscopy and conventional electron microscopy have a variety of biological contexts (Anderson and Jacobconsistently failed to reveal large-scale laterally segregated structures enriched in GPI-APs in living cells (Hao *Correspondence: [email protected] (M.R.), [email protected] (S.M) et al., 2001; Mayor and Maxfield, 1995; Mayor et al., 4 These authors contributed equally to this work. 1994; Parton et al., 1994); viewed at optical resolution 5 Present address: Program in Molecular Pathogenesis, Skirball Insti-004ف( nm), GPI-APs appear uniformly distributed in cell In native cell membranes, methods designed to detect Cell 578 proximity between molecules have observed inhomoge-phore; Figure 1A) to show clustered distribution of diverse GPI-APs in multiple cell lines. Second, time re-neous distributions of GPI-APs. Chemical crosslinking with short (1.1 nm) crosslinkers indicate that cholesterol-solved anisotropy decay experiments provide evidence of high density in clusters. Third, steady-state anisot-sensitive complexes of GPI-APs exist at the cell surface (Friedrichson and Kurzchalia, 1998). Using a fluores-ropy as a function of photobleaching, in conjunction with a detailed theoretical analysis suggests that these cence resonance energy transfer (FRET) method called homo-FRET which detects proximity between like fluor-dense clusters are small and only a fraction of GPI-APs are clustered. Finally, theoretical modeling of hetero-ophores at 1-10 nm scale we had suggested that GPI-APs occur in cholesterol-sensitive, submicron-sized FRET measurements confirm the presence of a fraction of small, dense clusters. We have kept the analytical "domains" at the surface of living cells (Varma and Mayor, 1998). In contrast, studies based on detecting methods used for these experiments in a single location (Supplemental Data available on Cell website), thus, if FRET between different fluorophores (hetero-FRET) reported the absence of detectable clustering of GPI-APs, the reader wishes to obtain the details of these calculations they are urged to refer to this section. putting an upper bound on the fraction (if any) of clustered GPI-APs (Kenworthy and Edidin, 1998; Kenworthy et al.,
Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediato... more Membrane rafts enriched in cholesterol and sphingolipids have been hypothesized to be key mediators of sorting and signaling functions of associated molecules. Apart from a limited number of biophysical studies in living cell membranes, raft-association has been defined by a simple biochemical criterion, namely the ability to partition with detergent-resistant membranes (DRMs). Here we examine the evidence for the specification of internalization mechanisms and endocytic pathways by rafts as defined by this criterion. We have surveyed the endocytic trafficking of a variety of molecules such as lipids, toxins, glycosylphosphatidylinositol (GPI)-anchored proteins, and DRM-associated transmembrane proteins.
Uploads
Papers by Pranav Sharma