Biomedical subjects

Robert G Parton

Publications and source records attributed to Robert G Parton.

At least 55 records · Page 3Linked to original sources

PubMed2004

Lipid rafts and plasma membrane microorganization: insights from Ras.

The spatial organization of plasma membrane components in discrete microdomains is thought to be a key factor in the generation of distinct signal outputs. A detailed characterization of plasma membrane microdomains, including descriptions of their size, dynamics and abundance, has proved to be a taxing problem for cell biologists and biophysicists. The use of novel techniques is providing exciting new insights into the challenging problem of plasma membrane microstructure and has allowed the visualization of domains with the characteristics expected of lipid rafts - microdomains of the plasma membrane enriched in cholesterol and sphingolipids. This review focuses on some of these recent advances and uses Ras signaling as a paradigm for understanding inner plasma membrane organization and the role of lipid rafts in cellular function.

Animals↗

PubMed2004

Three separable domains regulate GTP-dependent association of H-ras with the plasma membrane.

The microlocalization of Ras proteins to different microdomains of the plasma membrane is critical for signaling specificity. Here we examine the complex membrane interactions of H-ras with a combination of FRAP on live cells to measure membrane affinity and electron microscopy of intact plasma membrane sheets to spatially map microdomains. We show that three separable forces operate on H-ras at the plasma membrane. The lipid anchor, comprising a processed CAAX motif and two palmitic acid residues, generates one attractive force that provides a high-affinity interaction with lipid rafts. The adjacent hypervariable linker domain provides a second attractive force but for nonraft plasma membrane microdomains. Operating against the attractive interaction of the lipid anchor for lipid rafts is a repulsive force generated by the N-terminal catalytic domain that increases when H-ras is GTP loaded. These observations lead directly to a novel mechanism that explains how H-ras lateral segregation is regulated by activation state: GTP loading decreases H-ras affinity for lipid rafts and allows the hypervariable linker domain to target to nonraft microdomains, the primary site of H-ras signaling.

Amino Acid Sequence↗

PubMed2003

The trans-membrane protein p25 forms highly specialized domains that regulate membrane composition and dynamics.

Trans-membrane proteins of the p24 family are abundant, oligomeric proteins predominantly found in cis-Golgi membranes. They are not easily studied in vivo and their functions are controversial. We found that p25 can be targeted to the plasma membrane after inactivation of its canonical KKXX motif (KK to SS, p25SS), and that p25SS causes the co-transport of other p24 proteins beyond the Golgi complex, indicating that wild-type p25 plays a crucial role in retaining p24 proteins in cis-Golgi membranes. We then made use of these observations to study the intrinsic properties of these proteins, when present in a different membrane context. At the cell surface, the p25SS mutant segregates away from both the transferrin receptor and markers of lipid rafts, which are enriched in cholesterol and glycosphingolipids. This suggests that p25SS localizes to, or contributes to form, specialized membrane domains, presumably corresponding to oligomers of p25SS and other p24 proteins. Once at the cell surface, p25SS is endocytosed, together with other p24 proteins, and eventually accumulates in late endosomes, where it remains confined to well-defined membrane regions visible by electron microscopy. We find that this p25SS accumulation causes a concomitant accumulation of cholesterol in late endosomes, and an inhibition of their motility - two processes that are functionally linked. Yet, the p25SS-rich regions themselves seem to exclude not only Lamp1 but also accumulated cholesterol. One may envision that p25SS accumulation, by excluding cholesterol from oligomers, eventually overloads neighboring late endosomal membranes with cholesterol beyond their capacity (see Discussion). In any case, our data show that p25 and presumably other p24 proteins are endowed with the intrinsic capacity to form highly specialized domains that control membrane composition and dynamics. We propose that p25 and other p24 proteins control the fidelity of membrane transport by maintaining cholesterol-poor membranes in the Golgi complex.

Amino Acid Sequence↗

PubMed2003

Dynamic and regulated association of caveolin with lipid bodies: modulation of lipid body motility and function by a dominant negative mutant.

Caveolins are a crucial component of caveolae but have also been localized to the Golgi complex, and, under some experimental conditions, to lipid bodies (LBs). The physiological relevance and dynamics of LB association remain unclear. We now show that endogenous caveolin-1 and caveolin-2 redistribute to LBs in lipid loaded A431 and FRT cells. Association with LBs is regulated and reversible; removal of fatty acids causes caveolin to rapidly leave the lipid body. We also show by subcellular fractionation, light and electron microscopy that during the first hours of liver regeneration, caveolins show a dramatic redistribution from the cell surface to the newly formed LBs. At later stages of the regeneration process (when LBs are still abundant), the levels of caveolins in LBs decrease dramatically. As a model system to study association of caveolins with LBs we have used brefeldin A (BFA). BFA causes rapid redistribution of endogenous caveolins to LBs and this association was reversed upon BFA washout. Finally, we have used a dominant negative LB-associated caveolin mutant (cavDGV) to study LB formation and to examine its effect on LB function. We now show that the cavDGV mutant inhibits microtubule-dependent LB motility and blocks the reversal of lipid accumulation in LBs.

Animals↗

PubMed2003

Annexin II regulates multivesicular endosome biogenesis in the degradation pathway of animal cells.

Proteins of the annexin family are believed to be involved in membrane-related processes, but their precise functions remain unclear. Here, we have made use of several experimental approaches, including pathological conditions, RNA interference and in vitro transport assays, to study the function of annexin II in the endocytic pathway. We find that annexin II is required for the biogenesis of multivesicular transport intermediates destined for late endosomes, by regulating budding from early endosomes-but not the membrane invagination process. Hence, the protein appears to be a necessary component of the machinery controlling endosomal membrane dynamics and multivesicular endosome biogenesis. We also find that annexin II interacts with cholesterol and that its subcellular distribution is modulated by the subcellular distribution of cholesterol, including in cells from patients with the cholesterol-storage disorder Niemann-Pick C. We conclude that annexin II forms cholesterol-containing platforms on early endosomal membranes, and that these platforms regulate the onset of the degradation pathway in animal cells.

Animals↗

PubMed2003

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane.

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.

Animals↗

PubMed2003

Observing cell surface signaling domains using electron microscopy.

The plasma membrane is made up of a complex mosaic of different functional microdomains, but the tools required to accurately visualize them have always been limited. We present a protocol that allows both inner and outer leaflet domains to be visualized on a nanometer scale. With a combination of electron microscopic and statistical analysis approaches, it is possible to screen proteins associating with, and co-localizing within, lipid rafts and other morphologically featureless microdomains. The approach has enormous potential to determine plasma membrane organization and the spatial dynamics of regulated signaling and membrane trafficking events associated with the cell surface.

3T3 Cells↗

PubMed2003

Characterization of E-cadherin endocytosis in isolated MCF-7 and chinese hamster ovary cells: the initial fate of unbound E-cadherin.

The endocytosis of E-cadherin has recently emerged as an important determinant of cadherin function with the potential to participate in remodeling adhesive contacts. In this study we focused on the initial fate of E-cadherin when it predominantly exists free on the cell surface prior to adhesive binding or incorporation into junctions. Surface-labeling techniques were used to define the endocytic itinerary of E-cadherin in MCF-7 cells and in Chinese hamster ovary cells stably expressing human E-cadherin. We found that in this experimental system E-cadherin entered a transferrin-negative compartment before transport to the early endosomal compartment, where it merged with classical clathrin-mediated uptake pathways. E-cadherin endocytosis was inhibited by mutant dynamin, but not by an Eps15 mutant that effectively blocked transferrin internalization. Furthermore, sustained signaling by the ARF6 GTPase appeared to trap endocytosed E-cadherin in large peripheral structures. We conclude that in isolated cells unbound E-cadherin on the cell surface is predominantly endocytosed by a clathrin-independent pathway resembling macropinocytotic internalization, which then fuses with the early endosomal system. Taken with earlier reports, this suggests the possibility that multiple pathways exist for E-cadherin entry into cells that are likely to reflect cell context and regulation.

ADP-Ribosylation Factor 6↗

PubMed2003

Involvement of caveolin-2 in caveolar biogenesis in MDCK cells.

Caveolins have been identified as key components of caveolae, specialized cholesterol-enriched raft domains visible as small flask-shaped invaginations of the plasma membrane. In polarized MDCK cells caveolin-1 and -2 are found together on basolateral caveolae whereas the apical membrane, where only caveolin-1 is present, lacks caveolae. Expression of a caveolin mutant prevented the formation of the large caveolin-1/-2 hetero-oligomeric complexes, and led to intracellular retention of caveolin-2 and disappearance of caveolae from the basolateral membrane. Correspondingly, in MDCK cells over-expressing caveolin-2 the basolateral membrane exhibited an increased number of caveolae. These results indicate the involvement of caveolin-2 in caveolar biogenesis.

Animals↗

PubMed2003

Direct visualization of Ras proteins in spatially distinct cell surface microdomains.

Localization of signaling complexes to specific microdomains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains, including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent microdomain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Animals↗

PubMed2003

Lipid rafts and caveolae as portals for endocytosis: new insights and common mechanisms.

Clathrin-coated pits and caveolae are two of the most recognizable features of the plasma membrane of mammalian cells. While our understanding of the machinery regulating and driving clathrin-coated pit-mediated endocytosis has progressed dramatically, including the elucidation of the structure of individual components and partial in vitro reconstitution, the role of caveolae as alternative endocytic carriers still remains elusive 50 years after their discovery. However, recent work has started to provide new insights into endocytosis by caveolae and into apparently related pathways involving lipid raft domains. These pathways, distinguished by their exquisite sensitivity to cholesterol-sequestering agents, can involve caveolae but also exist in cells devoid of caveolins and caveolae. This review examines the current evidence for the involvement of rafts and caveolae in endocytosis and the molecular players involved in their regulation.

Adipocytes↗

PubMed2003

Caveolae--from ultrastructure to molecular mechanisms.

Almost 50 years after the first sighting of small pits that covered the surface of mammalian cells, investigators are now getting to grips with the detailed workings of these enigmatic structures that we now know as caveolae.

Animals↗

PubMed2003

Rab23, a negative regulator of hedgehog signaling, localizes to the plasma membrane and the endocytic pathway.

The regulation of hedgehog signaling by vesicular trafficking was exemplified by the finding that Rab23, a Rab-GTPase vesicular transport protein, is mutated in open brain mice. In this study, the localization of Rab23 was analyzed by light and immunoelectron microscopy after expression of wild-type (Rab23-GFP), constitutively active Rab23 (Rab23Q68L-GFP), and inactive Rab23 (Rab23S23N-GFP) in a range of mammalian cell types. Rab23-GFP and Rab23Q68L-GFP were predominantly localized to the plasma membrane but were also associated with intracellular vesicular structures, whereas Rab23S23N-GFP was predominantly cytosolic. Vesicular Rab23-GFP colocalized with Rab5Q79L and internalized transferrin-biotin, but not with a marker of the late endosome or the Golgi complex. To investigate Rab23 with respect to members of the hedgehog signaling pathway, Rab23-GFP was coexpressed with either patched or smoothened. Patched colocalized with intracellular Rab23-GFP but smoothened did not. Analysis of patched distribution by light and immunoelectron microscopy revealed it is primarily localized to endosomal elements, including transferrin receptor-positive early endosomes and putative endosome carrier vesicles and, to a lesser extent, with LBPA-positive late endosomes, but was excluded from the plasma membrane. Neither patched or smoothened distribution was altered in the presence of wild-type nor mutant Rab23-GFP, suggesting that despite the endosomal colocalization of Rab23 and patched, it is likely that Rab23 acts more distally in regulating hedgehog signaling.

Animals↗

PubMed2002

Flotillin-1/reggie-2 traffics to surface raft domains via a novel golgi-independent pathway. Identification of a novel membrane targeting domain and a role for palmitoylation.

Flotillins are lipid raft-associated proteins, which have been implicated in neuronal regeneration and insulin signaling. We now show that newly synthesized flotillin-1 reaches the plasma membrane via a Sar1-independent and brefeldin A-resistant targeting pathway. Consistent with post-translational membrane association of flotillin, protease sensitivity experiments suggest that flotillin-1 is not a transmembrane protein but is associated with the cytoplasmic face of the plasma membrane. The N terminus of flotillin contains a prohibitin-like domain (PHB), which shows homology to a number of proteins associated with raft domains including stomatin, podocin, and prohibitin. We show that the PHB domain of flotillin can efficiently target a heterologous protein, green fluorescent protein, to the plasma membrane. Another PHB-containing protein, stomatin, traffics to the plasma membrane via the conventional secretory pathway. Plasma membrane association of both full-length flotillin and the green fluorescent protein-tagged PHB domain of flotillin is dependent on palmitoylation and requires a conserved cysteine residue, Cys-34, in the PHB domain. The results identify a novel targeting mechanism for plasma membrane association of flotillin-1 involving a Golgi-independent trafficking pathway, the PHB domain, and palmitoylation.

Amino Acid Sequence↗

PubMed2002

Characterization of a distinct plasma membrane macrodomain in differentiated adipocytes.

Caveolae are small invaginations of the cell surface that are abundant in mature adipocytes. A recent study (Kanzaki, M., and Pessin, J. E. (2002) J. Biol. Chem. 277, 25867-25869) described novel caveolin- and actin-containing structures associated with the adipocyte cell surface that contain specific signaling proteins. We have characterized these structures, here termed "caves," using light and electron microscopy and observe that they represent surface-connected wide invaginations of the basal plasma membrane that are sometimes many micrometers in diameter. Rather than simply a caveolar domain, these structures contain all elements of the plasma membrane including clathrin-coated pits, lipid raft markers, and non-raft markers. GLUT4 is recruited to caves in response to insulin stimulation. Caves can occupy a significant proportion of the plasma membrane area and are surrounded by cortical actin. Caveolae density in caves is similar to that on the bulk plasma membrane, but because these structures protrude much deeper into the plane of focus of the light microscope molecules such as caveolin and other plasma membrane proteins appear more concentrated in caves. We conclude that the adipocyte surface membrane contains numerous wide invaginations that do not represent novel caveolar structures but rather large surface caves.

3T3 Cells↗

PubMed2002

Differential sorting and fate of endocytosed GPI-anchored proteins.

In this paper, we studied the fate of endocytosed glycosylphosphatidyl inositol anchored proteins (GPI- APs) in mammalian cells, using aerolysin, a bacterial toxin that binds to the GPI anchor, as a probe. We find that GPI-APs are transported down the endocytic pathway to reducing late endosomes in BHK cells, using biochemical, morphological and functional approaches. We also find that this transport correlates with the association to raft-like membranes and thus that lipid rafts are present in late endosomes (in addition to the Golgi and the plasma membrane). In marked contrast, endocytosed GPI-APs reach the recycling endosome in CHO cells and this transport correlates with a decreased raft association. GPI-APs are, however, diverted from the recycling endosome and routed to late endosomes in CHO cells, when their raft association is increased by clustering seven or less GPI-APs with an aerolysin mutant. We conclude that the different endocytic routes followed by GPI-APs in different cell types depend on the residence time of GPI-APs in lipid rafts, and hence that raft partitioning regulates GPI-APs sorting in the endocytic pathway.

Animals↗

PubMed2002

Inhibition of lipid raft-dependent signaling by a dystrophy-associated mutant of caveolin-3.

Specific point mutations in caveolin-3, a predominantly muscle-specific member of the caveolin family, have been implicated in limb-girdle muscular dystrophy and in rippling muscle disease. We examined the effect of these mutations on caveolin-3 localization and function. Using two independent assay systems, Raf activation in fibroblasts and neurite extension in PC12 cells, we show that one of the caveolin-3 point mutants, caveolin-3-C71W, specifically inhibits signaling by activated H-Ras but not by K-Ras. To gain insights into the effect of the mutant protein on H-Ras signaling, we examined the localization of the mutant proteins in fibroblastic cells and in differentiating myotubes. Unlike the previously characterized caveolin-3-DGV mutant, the inhibitory caveolin-3-C71W mutant reached the plasma membrane and colocalized with wild type caveolins. In BHK cells, caveolin-3-C71W associated with caveolae and in differentiating muscle cells with the developing T-tubule system. In contrast, the caveolin-3-P104L mutant accumulated in the Golgi complex and had no effect on H-Ras-mediated Raf activation. Inhibition by caveolin-3-C71W was rescued by cholesterol addition, suggesting that the mutant protein perturbs cholesterol-rich raft domains. Thus, we have demonstrated that a naturally occurring caveolin-3 mutation can inhibit signaling involving cholesterol-sensitive raft domains.

Animals↗

PubMed2002

GPI-anchored proteins are delivered to recycling endosomes via a distinct cdc42-regulated, clathrin-independent pinocytic pathway.

Endocytosis of cell-surface proteins via specific pathways is critical for their function. We show that multiple glycosylphosphatidylinositol-anchored proteins (GPI-APs) are endocytosed to the recycling endosomal compartment but not to the Golgi via a nonclathrin, noncaveolae mediated pathway. GPI anchoring is a positive signal for internalization into rab5-independent tubular-vesicular endosomes also responsible for a major fraction of fluid-phase uptake; molecules merely lacking cytoplasmic extensions are not included. Unlike the internalization of detergent-resistant membrane (DRM)-associated interleukin 2 receptor, endocytosis of DRM-associated GPI-APs is unaffected by inhibition of RhoA or dynamin 2 activity. Inhibition of Rho family GTPase cdc42, but not Rac1, reduces fluid-phase uptake and redistributes GPI-APs to the clathrin-mediated pathway. These results describe a distinct constitutive pinocytic pathway, specifically regulated by cdc42.

Animals↗