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Originally published In Press as doi:10.1074/jbc.M708776200 on December 20, 2007

J. Biol. Chem., Vol. 283, Issue 10, 6476-6488, March 7, 2008
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Caveolin Regulates Endocytosis of the Muscle Repair Protein, Dysferlin*Formula

Delia J. Hernández-Deviez{ddagger}, Mark T. Howes{ddagger}, Steven H. Laval§, Kate Bushby§, John F. Hancock{ddagger}, and Robert G. Parton{ddagger}1

From the {ddagger}Institute for Molecular Bioscience, Centre for Microscopy and Microanalysis, University of Queensland, Brisbane, Queensland 4072, Australia and the §Institute of Human Genetics, International, Centre for Life, Newcastle NE1 3BZ, United Kingdom

Received for publication, October 24, 2007 , and in revised form, November 26, 2007.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dysferlin and Caveolin-3 are plasma membrane proteins associated with muscular dystrophy. Patients with mutations in the CAV3 gene show dysferlin mislocalization in muscle cells. By utilizing caveolin-null cells, expression of caveolin mutants, and different mutants of dysferlin, we have dissected the site of action of caveolin with respect to dysferlin trafficking pathways. We now show that Caveolin-1 or -3 can facilitate exit of a dysferlin mutant that accumulates in the Golgi complex of Cav1-/- cells. In contrast, wild type dysferlin reaches the plasma membrane but is rapidly endocytosed in Cav1-/- cells. We demonstrate that the primary effect of caveolin is to cause surface retention of dysferlin. Caveolin-1 or Caveolin-3, but not specific caveolin mutants, inhibit endocytosis of dysferlin through a clathrin-independent pathway colocalizing with internalized glycosylphosphatidylinositol-anchored proteins. Our results provide new insights into the role of this endocytic pathway in surface remodeling of specific surface components. In addition, they highlight a novel mechanism of action of caveolins relevant to the pathogenic mechanisms underlying caveolin-associated disease.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dysferlin and Caveolin-3 (muscle-specific caveolin, Cav3) are sarcolemmal proteins whose role in muscle has gained clinical attention because mutations in their genes are associated with a number of muscle pathologies. Patients with mutations in the dysferlin (DYSF) gene develop disorders such as limb girdle muscular dystrophy type 2B, miyoshi myopathy, and distal myopathy (1-5). Whereas disruption in the Caveolin-3 (CAV3) gene has been linked to limb girdle muscular dystrophy 1C, Rippling muscle diseases, hyperCKemia, and distal myopathy among other myopathies (6-15). Dysferlin and Cav3 have been co-purified from muscle cells (16, 17) and shown to localize to adjacent membrane domains at the surface in mature muscle fibers (18). Moreover, dysferlin is depleted from the plasma membrane (PM)2 when Cav3 is mutated (8, 9, 14, 17, 19). We have recently demonstrated a role for caveolin in dysferlin localization at the PM (18). However, the interplay of dysferlin and caveolin membrane trafficking dynamics remains to be examined.

Dysferlin belongs to the ferlin family of proteins comprising otoferlin, myoferlin, and fer1L3 (20-22). The DYSF gene encodes a 230-kDa skeletal muscle membrane protein (2, 5, 23) with homology to the Caenorhabditis elegans sperm-vesicle fusion factor, fer-1 (2). Because of this dysferlin has been suggested to play a role in vesicle fusion in skeletal muscle (2, 24). Moreover, in the absence of dysferlin muscle cells show defective resealing of membrane disruptions (25). Dysferlin has a single transmembrane domain at the C terminus and a long N-terminal cytoplasmic region containing six C2 domains. C2 domains are a common feature of the synaptotagmin family of proteins implicated in vesicular traffic and membrane fusion events through calcium-dependent interactions with phospholipids and proteins (26-29). Interestingly, dysferlin and synaptotagmins share structural similarities (20, 24) further implicating dysferlin in membrane trafficking processes.

In mammalian cells, the CAV gene family consists of three isoforms: Caveolin-1, -2, and -3 (30-35), which are crucial structural components of caveolar membranes (~65 nm, uncoated flask-shaped PM pits). Caveolins are 21-24-kDa integral membrane proteins, Caveolin-1 (Cav1) and -2 (Cav2) are mainly co-expressed in non-muscle cells, whereas Cav3 is largely expressed in skeletal and cardiac muscle but is also found in some smooth muscle (36, 37). Caveolins are cholesterol- and fatty acid-binding proteins, and are thought to play a role in vesicular traffic and signal transduction events (38-42). The protein structure of caveolins is characterized by a hairpin loop topology with a hydrophobic region immersed in the lipid bilayer (the intramembrane domain), and both N and C terminus regions facing the cytoplasm (43-45). Additionally, the conserved juxtamembrane region, the caveolin scaffolding domain (CSD), has been shown to bind in vitro to a consensus sequence ({varphi}X{varphi}XXXX{varphi}XX{varphi}, {varphi} aromatic residues, X any amino acid) (46) present in various proteins. Dysferlin has several putative CSD binding motifs (17).

We have recently described the subcellular distribution of dysferlin with respect to Cav3 and showed that dysferlin association with the PM is impaired in the absence of caveolin, or in the presence of dystrophy-associated mutant forms of Cav3 (18). Although Cav3 and dysferlin copurify (16, 17), the precise interacting domains and roles in their trafficking dynamics are poorly understood. We show here that in the absence of caveolin, dysferlin reaches the PM but is rapidly endocytosed through a caveolin-, clathrin-, and dynamin-independent pathway. Wild type caveolin, but not mutant forms of caveolin associated with muscle disease, specifically inhibit dysferlin endocytosis causing its retention at the cell surface.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs, Reagents, and Antibodies—Cell culture reagents were obtained from Invitrogen. The antibodies used were: mouse anti-LBPA, mouse anti-LAMP-1 (Southern Biotech), mouse anti-GM130 (BD Biosciences), rabbit antibody made against the conserved region of Cav3 (47), rabbit anti-HA (Dr. T. Nilsson, Gothenburg University, Gothenburg, Sweden), rabbit anti-GFP (48), mouse anti-myc 9B11 (Cell Signaling Technology), and anti-protein-disulfide isomerase. Secondary antibodies conjugated to Alexa Fluor 488, 350, 546, 647, and CTB conjugated to Alexa Fluor 555 and Tf-Alexa Fluor 647 (Molecular Probes), CY3-conjugated anti-mouse antibody (Jackson Immunoresearch), and HRP-conjugated secondary antibodies (Zymed Laboratories) were used. SuperSignal substrate was obtained from Pierce Chemical Company. All other chemicals and reagents were obtained from Sigma.

GFP-dysferlin cDNA was used as a template to generate different truncation mutants by restriction digestion at unique enzymatic sites (49) (see Fig. 1), GFP{Delta}-C2 (GFP-TM), GFP{Delta}-1 (GFP3'GFP-2), GFP{Delta}-2 (GFP-Tth), GFP{Delta}-3 (GFP-Xho), and GFP-{Delta}-TM. Expression of the complete fusion proteins was confirmed in multiple cell lines. Cav3G55S-HA, Cav3C71W-HA, and Flotillin-HA were made as described (50, 51). The glycosylphosphatidylinositol (GPI)-GFP, Cav1{Delta}81-100-HA, and transferrin receptor constructs were gifts from C. Zurzolo (Institut Pasteur, France), D. Brown (State University of New York), S. L. Schmid (Scripps Research Institute), and V. Gerke (Center for Molecular Biology of Inflammation, ZMBE), respectively. Dynamin inhibitor, dynasore, was a kind gift from T. Kirchhausen (Harvard Medical School).

Cell Culture and Transfection—We utilized immortalized mouse embryonic fibroblasts (MEF) cell lines derived from Cav1 WT or knock-out mice as in previous studies (18, 52, 53). Cells were grown on glass coverslips and cDNAs were transiently expressed utilizing Lipofectamine 2000 (Invitrogen) according to the manufacturer's directions.

Immunofluorescence and Microscopy—Immunolabeling of MEFs were carried out as described previously (18). Confocal images were acquired with an inverted Zeiss LSM 510 META microscope system (Axiovert 200M, Carl Zeiss MicroImaging) under a Plan apochromatic x63 1.4 NA oil immersion objective. Images were processed and merged using Adobe Photoshop 9.0 software. Identical imaging and processing parameters were used for all figures.

Surface Labeling and Single Cell Fluorescence Quantification—Surface labeling and quantification of the fluorescence intensity of dysferlin pool at the PM was performed as described previously (18). The average pixel intensity for PM and IC/Golgi pools were measured using Adobe Photoshop 9.0 software. Experiments were repeated three times.

Quantifications shown in Figs. 1D, 2C, 3B, and 7B are representative of 3 individual experiments and were performed once on 250-350 cells and twice on a total of 30 cells for each construct. Subcellular phenotypes were determined based on colabeling with relevant IC markers. Results are presented as percentage of cells showing a common phenotype.

Myc, Tfn, CTB, and GPI Uptake Assays—Uptake assays were performed as described previously (54). In brief, 20 mg/ml monoclonal anti-myc antibody, 1 mg/ml CTxB-Alexa Fluor 555, or 5 mg/ml Tfn Alexa Fluor 647 was bound to cells on ice for 30 min in CO2-independent medium. Cells were washed with ice-cold CO2-independent medium to remove unbound reagent prior to uptake in growth media (10% fetal bovine serum, 2 mM L-glutamine/Dulbecco's modified Eagle's medium) at 37 °C, for the times indicated. Cells were placed on ice-cold CO2-independent medium and washed 2 times for 30 s in 0.5 M glycine (pH 2.2). The cells were fixed in 2% paraformaldehyde and processed for immunofluorescence. For inhibition of dynamin-dependent uptake, cells were preincubated in either 80 mM dynasore/growth media or 0.4% Me2SO/growth media for 30 min at 37 °C, followed by Tfn and anti-myc uptake in the presence or absence of dynasore.

Ultrastructural Analysis of Dysferlin Endocytosis in WT Cav1 and Cav1-/- MEFs—WT Cav1 or Cav1-/- MEFs were transfected with GFPDysf. After overnight incubation, to allow expression of the constructs, the cells were incubated with mouse anti-myc antibodies at 4 °C for 20 min, washed, and further incubated with anti-mouse HRP at 4 °C for 20 min. The cells were warmed to 37 °C for 2 min to allow uptake and then incubated in DAB, with or without ascorbic acid (AA), fixed, and processed for resin embedding, exactly as described previously (54). Due to the low transfection efficiency, GFP-expressing cells were identified by light microscopy before processing. They were marked to allow subsequent location for sectioning. Quantitation of PM coverage of the HRP reaction product was by intersection counting. The number of intersections of a square lattice grid with unlabeled and DAB-covered areas of the PM in random areas of transfected WT Cav1 or Cav1-/- MEFs was measured on digital images to gain an estimate of PM coverage by the HRP reaction.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Subcellular Distribution of Dysferlin Truncation Mutants in WT Cav1 and Cav1-/- MEF Cells—To better understand the functional link between dysferlin and Cav3 we examined the subcellular distribution of truncation mutants of dysferlin and analyzed their trafficking dependence on caveolin. We used WT Cav1 and Cav1-/- MEFs as a model system. Cav1-/- cells have no detectable caveolae as they lack Cav1 (as well as the muscle-specific isoform Cav3). This represents a powerful model system to analyze dysferlin trafficking with respect to caveolin as caveolin re-expression rescues the dysferlin trafficking defects (18).


Figure 1
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  		FIGURE 1.
Subcellular distribution of dysferlin truncation mutants in Cav1-/- cells. A, schematic representations of full-length and truncation mutants of GFPDysf. B, baby hamster kidney cells were transfected with either GFP or different GFPDysf constructs. Total cell lysates were separated in a 5.5% SDS gel, electrotransferred, and immunoblotted using anti-GFP antibody. Truncated GFP-tagged mutants appear as single polypeptides of predicted sizes. C, full-length dysferlin, GFPDysf, is mainly targeted to the PM or to intracellular vesicles in WT Cav1 or Cav1-/- MEFs, respectively. GFP{Delta}-C2 is mainly targeted to the PM and to fine punctate structures. GFP{Delta}-1 predominantly accumulates in the Golgi as demonstrated by colocalization with the Golgi marker, GM130. GFP{Delta}-2 is mostly concentrated in endoplasmic reticulum shown by the extensive overlay with protein-disulfide isomerase, an endoplasmic reticulum marker, whereas GFP{Delta}-3 and GFP{Delta}-TM are predominantly cytosolic. D, predominant subcellular phenotypes were scored based on colabeling with relevant intracellular markers. Results are presented as percentage of cells showing a prevalent phenotype, and are representative of three individual experiments (n = 30-350/for each construct). E, summary table of dysferlin constructs and subcellular localizations. Bar, 10 µm.

 
Truncated versions of dysferlin with an N-terminal GFP tag were generated (see summary in Fig. 1). Expression of these mutants in baby hamster kidney cells showed single bands of the predicted molecular weight for each of the truncation mutants (Fig. 1B). We compared the subcellular distribution of the mutant proteins to the WT protein by heterologous expression of truncation mutant forms of dysferlin in WT Cav1 or Cav1-/- MEFs (refer to summary table in Fig. 1). Full-length dysferlin efficiently reaches the PM in WT Cav1 MEFs (Fig. 1C), consistent with previous results (18). In contrast, dysferlin localized to intracellular structures but not to the Golgi complex in Cav1-/- cells (Fig. 1C). GFP{Delta}-2, which lacks the three first C2 domains, was found to mainly localize to the endoplasmic reticulum as judged by a large overlap with the endoplasmic reticulum marker, protein-disulfide isomerase (Fig. 1, C-E). Truncated versions of dysferlin lacking the TM domain, GFP{Delta}-3 and GFP{Delta}-TM, were however, mainly cytosolic (Fig. 1, C-E) and were indistinguishable in WT Cav1 and Cav1-/- cells.

In contrast to WT dysferlin and to the above mutants, GFP{Delta}-1, which lacks the first four C2 domains, mostly accumulated in the Golgi complex of Cav1-/- MEFs, as demonstrated by colocalization with the Golgi marker, GM130 (Fig. 1, C-E), but associated with the PM in WT Cav1 MEFs (Figs. 1C and 2). To examine whether this reflected a direct role of caveolin in facilitating the transport of GFP{Delta}-1 to the PM, we co-expressed GFP{Delta}-1 and HA-tagged Cav1 (Cav1-HA) or Cav3 (Cav3-HA) in Cav1-/- MEFs. GFP{Delta}-1 efficiently exited the Golgi apparatus and reached the PM (Fig. 2). Quantitation showed that in ~66% of Cav1-/- MEFs expressing Cav1-HA or Cav3-HA, GFP{Delta}-1 was localized to the PM and to intracellular puncta (Fig. 2B). GFP{Delta}-C2, a mutant lacking all six C2 domains was predominantly targeted to the PM in both WT Cav1 (not shown) and Cav1-/- MEFs (Figs. 1, C-E, and 3, A and B). Quantitation of Cav1-/- MEFs expressing GFP{Delta}-C2 showed that in ~97% of the cells, GFP{Delta}-C2 localized to the PM.


Figure 2
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  		FIGURE 2.
Cav1 and Cav3 redistribute GFP{Delta}-1 from the Golgi to the plasma membrane in Cav1-/- cells. MEF cells were transfected with GFPDysf or GFP{Delta}-1 or co-transfected with GFP{Delta}-1 and epitope-tagged Cav3-HA, and colabeled with anti-HA or anti-Cav and anti-GM130 antibodies. A, in Cav1-/- cells dysferlin is mainly localized to punctate structures throughout the cytoplasm, whereas GFP{Delta}-1 mutant accumulates in the Golgi complex as demonstrated by colocalization with the Golgi marker, GM130. Interestingly, expression of epitope-tagged Cav3 redistributes GFP{Delta}-1 to the PM. Similarly in WT Cav1 MEFs endogenous caveolin is sufficient for GFP{Delta}-1 Golgi exit and PM targeting. B, phenotype quantification. Cav1-/- or WT Cav1 cells expressing GFP{Delta}-1 or co-expressing GFP{Delta}-1 and Cav1-HA or Cav3-HA were subject to phenotype scoring. Predominant subcellular phenotypes were scored based on colabeling with relevant intracellular markers. Results are presented as percentage of cells showing a prevalent phenotype, and are representative of three individual experiments (n = 30-350/for each construct). Bar, 10 µm.

 
Taken together these results show that GFP{Delta}-1, which lacks the first four C2 domains, predominantly accumulates in the Golgi complex in the absence of caveolin. This suggests that caveolin is required for GFP{Delta}-1 transport from the Golgi complex to the PM. In contrast, a mutant lacking all six C2 domains is not retained in the Golgi complex in the presence or absence of caveolin. The loss of all six C2 domains renders mutated dysferlin independent of caveolin for surface delivery.

The fact that GFP{Delta}-C2 efficiently reached the PM in Cav1-/- MEFs (Fig. 3) showed that this truncated protein was not dependent on caveolin for surface targeting. We investigated whether a Golgi-localized dystrophy mutant of Cav3 (Cav3P104L-HA), which causes retention of full-length dysferlin in the Golgi complex (18) would affect the traffic of GFP{Delta}-C2 to the PM. Cells co-expressing epitope-tagged Cav3P104L (Cav3P104L-HA) and GFP{Delta}-C2 showed a dramatic accumulation in the Golgi complex (66% of the cells) compared with cells expressing mutated dysferlin alone (4% of the cells) (Fig. 3). Thus GFP{Delta}-C2, which does not require caveolin for Golgi exit and PM targeting, is blocked in trafficking from the Golgi by the P104L caveolin mutant.

Characterization of Dysferlin Trafficking in Cells Lacking Caveolin—In the absence of caveolin dysferlin accumulates in an intracellular compartment of unknown nature. The identification of these structures should provide insights into the role of caveolins in dysferlin trafficking. We first examined whether dysferlin was targeted for degradation in Cav1-/- cells. However, dysferlin failed to co-localize significantly with markers of the late endocytic pathway such as LBPA and LAMP1 (supplemental Fig. S1A). Despite the low surface labeling in the Cav1-/- MEFs, we speculated that dysferlin is able to reach the PM but then is efficiently endocytosed in the absence of caveolin. If this was the case, antibodies to the lumenal myc epitope should be readily internalized by Cav1-/- cells expressing GFPDysf but not by WT Cav1 cells. WT Cav1 and Cav1-/- MEFs were transfected with a dysferlin cDNA containing an N-terminal GFP tag and a C-terminal (lumenal/extracellular) myc tag (GFPDysf). After 4 h post-transfection, antibodies against the dysferlin ectoplasmic myc tag were added to the culture medium and antibodies were allowed to internalize overnight. The cells were then fixed, permeabilized, and labeled with secondary antibodies. A striking accumulation of myc antibodies was observed in the Cav1-/- MEFs (Fig. 4A), very little was internalized in WT Cav1 and no uptake was observed in neighboring non-transfected cells (see Fig. 4A) indicating that the antibodies were taken up specifically after binding to the exposed lumenal myc epitope and not by fluid phase uptake. Consistent with this, the internalized antibodies colocalized with GFPDysf. Identical results were obtained when experiments were performed using Fab fragments against the myc epitope (supplemental Fig. S1B). Cav1-/- MEFs expressing GFP-dysf showed higher uptake of Fab fragments compared with WT Cav1 cells (supplemental Fig. S1B). There was no colocalization of Fab fragments and transferrin in WT Cav1 or Cav1-/- cells at any of the time points examined (data not shown).


Figure 3
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  		FIGURE 3.
Dystrophy-associated mutant of Cav3 retains GFP{Delta}-C2 in the Golgi complex. Cav1-/- cells were transfected with GFP{Delta}-C2 or co-transfected with GFP{Delta}-C2 or GFPDysf and HA-tagged Cav3P104L, and colabeled with anti-HA and anti-GM130 antibodies. A, GFP{Delta}-C2 is targeted to the PM and punctate structures throughout the cytoplasm. As seen with full-length dysferlin (18), expression of epitope-tagged Cav3P104L-HA causes a dramatic redistribution of GFP{Delta}-C2 to the Golgi complex as judged by triple labeling with anti-GM130 antibody. B, phenotype quantification. Cav1-/- cells expressing GFP{Delta}-C2 or co-expressing GFP{Delta}-C2 and Cav3P104L-HA or GFPDysf and Cav3P104L-HA were subject to phenotype scoring. Predominant subcellular phenotypes were scored based on colabeling with relevant intracellular markers. Results are presented as percentage of cells showing a prevalent phenotype and are representative of three individual experiments (n = 30-350/for each construct). Bar, 10 µm.

 
To investigate this in more detail, myc antibodies were bound to the surface of GFPDysf expressing Cav1-/- or WT Cav1 cells at 4 °C and warmed for 30 min at 37 °C to allow internalization of the antibodies. Surface antibodies were removed by an acid wash. Interestingly, in WT Cav1 or Cav1-/- MEFs, significant GFPDysf internalization was observed after 30 min. Despite the low level of PM dysferlin, Cav1-/- MEFs contained many more vesicles positive for both GFP and myc (Fig. 4B) suggesting a much higher endocytic rate. This was confirmed by quantitation of the intracellular/PM (IC/PM) ratio after uptake at 10 min (WT Cav1, IC/PM ratio = 0.05 ± 0.001; Cav1-/-, IC/PM ratio = 0.52 ± 0.04) and 40 min (WT Cav1, IC/PM ratio = 0.10 ± 0.009; Cav1-/-, IC/PM ratio = 1.07 ± 0.12) (Fig. 4C). Furthermore, and consistent with our previous work (18), we predicted that re-expression of Cav1-HA in Cav1-/- MEFs would inhibit dysferlin internalization (Fig. 4D). Cav1-/- MEFs co-expressing GFPDysf and Cav1-HA showed a distribution of dysferlin similar to that seen in WT Cav1 MEFs (refer to supplemental Fig. S2A); the ability of dysferlin to reside at the PM membrane has been rescued by expression of Cav-1 (Fig. 4D and supplemental Fig. S2A). This was further confirmed by quantifying the amount of internalized myc antibodies in WT Cav1 and Cav1-/- MEFs expressing GFPDysf (WT Cav1, 9.06 ± 0.67; Cav1-/-, 24.04 ± 1.89) or Cav1-/- cells co-expressing GFP-dysf and Cav1-HA (9.33 ± 0.56) (refer to "Experimental Procedures") (Fig. 4D). Thus these results demonstrate that the major effect of caveolin is to retain dysferlin at the PM and inhibit its internalization. These results demonstrate that dysferlin is not absolutely dependent on caveolin to reach the PM but is efficiently retained at the PM in the presence of caveolin. Taken together, these results show a hitherto unexpected dynamic cycling of dysferlin in Cav1-/- cells.

Dysferlin Is Internalized Through a Clathrin-independent Endocytic Pathway—We next examined the pathway by which dysferlin is internalized in Cav1-/- MEFs using transferrin to label the clathrin pathway and cholera toxin binding subunit (CTB) or GPI-anchored proteins (AP) as markers of other pathways. Myc antibodies taken up by expressed GFP-dysferlin for various times did not colocalize significantly with transferrin (Fig. 5A). However, significant colocalization of internalized myc antibodies and CTB was evident after 2, 10, and 40 min of internalization (Fig. 5A). No significant difference in the internalization rate of transferrin or CTB was seen between WT Cav1 or Cav1-/- MEFs (supplemental Fig. S2B).

GPI-AP are internalized via a clathrin- and dynamin-independent endocytic pathway (54-56). To test if dysferlin was trafficking from the PM via this pathway we co-internalized antibodies against the extracellular tags (mouse anti-myc for dysferlin and rabbit anti-GFP for GPI-GFP). Cav1-/- MEFs co-expressing GFPDysf and GPI-GFP were labeled on ice with anti-myc and anti-GFP antibodies, warmed to 37 °C for 2, 10, and 40 min, and then acid-washed. Internalized antibodies were detected with anti-mouse Alexa Fluor 546 and anti-rabbit Alexa Fluor 647 antibodies after permeabilization. At all time points endocytic vesicles containing myc and GFP antibodies were readily detectable (Fig. 5A), demonstrating that internalized dysferlin was targeted to a GPI-AP enriched compartment. We further investigated if dysferlin vesicular traffic followed a dynamin-dependent route using the dynamin inhibitor, Dynasore (57, 58). Whereas transferrin uptake was blocked, dysferlin endocytosis was unaffected by incubation with Dynasore (Fig. 5B). We conclude that the major endocytic pathway involved in dysferlin endocytosis in Cav1-/- cells is dynamin-independent.


Figure 4
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  		FIGURE 4.
Dysferlin cycles between an intracellular compartment and the plasma membrane. WT Cav1 and Cav1-/- MEFs were transfected with GFPDysf and allowed to internalized anti-myc antibodies overnight or for 30 min at 37 °C. A, in both WT and Cav1-/- cells dysferlin is cycling between the PM and an intracellular endocytic compartment. Inset shows extensive colocalization between GFPDysf and internalized myc antibodies in Cav1-/- cells (*, untransfected cell; **, cell expressing low levels of dysferlin) but in WT Cav1 cells there is very little internalization. B, in WT Cav1 MEFs dysferlin is mainly localized at the PM although some internalized myc can be seen after 30 min (see inset). In contrast after 30 min of myc antibody internalization, dysferlin shows a highly dynamic endocytic traffic in Cav1-/- cells; inset, extensive overlay between GFPDysf and internalized myc. C, time course of myc antibodies uptake in WT or Cav1-/- MEFs expressing GFPDysf. The mean fluorescence intensity of dysferlin associated with the PM (myc surface labeling) and internalized myc (2, 10, and 40 min chase at 37 °C) was measured and expressed as IC/PM ratio. D, dysferlin internalization is rescued to WT levels by expression of epitope-tagged Cav1 in Cav1-/- MEFs. WT Cav1 and Cav1-/- MEFs were transfected with GFPDysf or co-transfected with GFPDysf and Cav1-HA and anti-myc antibodies were internalized for 20 min at 37 °C. The mean fluorescence intensity of internalized dysferlin (myc labeling) was quantified. Error bars are S.E. of three experiments (n = 30). Bars, 10 µm.

 
Ultrastructural Analysis of Dysferlin Trafficking—To gain further insights into dysferlin endocytosis, Cav1-/- or WT Cav1 MEFs were transfected with GFP-dysf, then incubated with anti-myc antibodies followed by an anti-mouse HRP-labeled antibody at 4 °C. The cells were then warmed for 2 min at 37 °C, and the DAB reaction visualized in the presence or absence of AA to identify internal structures, as in previous studies (54). The cells were then fixed and processed for correlative light and electron microscopy, identifying GFP-dysf-expressing cells by light microscopy and then sectioning the plastic embedded cells for EM. Consistent with the light microscopy, WT cells showed a uniform, almost continuous, layer of HRP reaction product over the entire cell surface (WT Cav1 - AA; Fig. 6, A and B) and little internal staining. No preferential staining of caveolae was observed, consistent with our previous immunoelectromicrograph studies (18). In contrast, Cav1-/- MEFs showed very patchy sparse labeling over the cell surface (KO - AA; Fig. 6, C and D) but with some tubular profiles apparently enriched in reaction product (Fig. 6, E and G). Quantitation of the surface coverage of the HRP reaction product in WT Cav1 versus Cav1-/- cells showed a far higher surface coverage in the WT Cav1 cells (see Fig. 6, A and C) consistent with the low level of surface labeling in Cav1-/- cells as observed by light microscopy. Endocytic structures were clearly observed in the Cav1-/- cells treated with AA (KO + AA, Fig. 6, H and I). The ring-shaped morphology and size of the labeled elements are consistent with structures labeled by CTB HRP in WT and Cav1-/- MEFs in the previous studies (54). These studies show that dysferlin is retained over the entire cell surface in the presence of caveolin but is rapidly internalized in its absence.


Figure 5
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  		FIGURE 5.
Dysferlin co-internalizes with the early endocytic markers CTB and GPI-AP in a dynamin-independent manner. A, Cav1-/- cells were transfected with GFP-dysf or co-transfected with GFP-dysf and TFR or GPI-GFP. Anti-myc antibodies were co-internalized with fluorophore-conjugated Tfn or CTB or anti-GFP antibodies for various times at 37 °C. Dysferlin is internalized mainly via a non-clathrin pathway. No significant colocalization is seen between internalized myc antibodies and Tfn. Dysferlin follows CTB and GPI in their endocytic traffic. Extensive co-internalization is visualized with anti-myc (GFP-dysf) and CTB and GFP (GPI-GFP) after 2, 10, and 40 min uptake. B, dynamin inhibitor, Dynasore, does not block dysferlin endocytic traffic. Cav1-/- MEFs were transfected with GFPDysf and incubated in 80 mM Dynasore, 0.4% Me2SO, Dulbecco's modified Eagle's medium or 0.4% Me2SO, Dulbecco's modified Eagle's medium alone. Anti-myc antibody or Tfn were internalized for 20 min at 37 °C. Inhibition of dynamin does not block the internalization of anti-myc antibodies by dysferlin, whereas Tfn internalization was blocked. Bars, 10 µm (A); 20 µm (B).

 
Dysferlin Trafficking Is Not Rescued by Caveolin Scaffolding Domain Mutants in Cav1-/- MEF Cells—To gain further insights into the functional interaction between dysferlin and caveolin and the relevance of these observations to muscle disease, we examined the effect of caveolin mutants on dysferlin endocytosis as compared with wild type caveolin. We made use of HA-tagged CSD point mutants, Cav3G55S (Cav3G55S-HA) and Cav3C71W (Cav3C71W-HA) (50, 59), which have been linked to muscular dystrophy (11, 13) and the CSD deletion mutant, Cav1{Delta}81-100 (Cav1{Delta}-81-100-HA) (60). Quantitation of surface versus intracellular dysferlin was performed using antibodies to the lumenal myc tag as in previous studies (18). Cav3G55S-HA or Cav3C71W-HA expressed in Cav1-/- MEFs localized predominantly at the surface in a similar fashion to the wild type protein (Fig. 7), whereas Cav1{Delta}81-100-HA mainly targeted to the Golgi complex as judged by colocalization with the Golgi marker, GM130 (Fig. 7). GFP-dysf was largely localized to intracellular puncta in Cav1-/- cells (PM/IC ratio 0.67 ± 0.07) but PM association was restored by re-expression of either Cav1-HA (PM/IC ratio 1.99 ± 0.96) or Cav3-HA (PM/IC ratio 2.3 ± 0.55) (18) (Fig. 8, A and B). In contrast, expression of Cav1{Delta}81-100-HA (PM/IC ratio 0.61 ± 0.10), Cav3G55S-HA (PM/IC ratio 0.65 ± 0.15), or Cav3C71W-HA (PM/IC ratio 1.16 ± 0.57) did not affect dysferlin traffic to the PM and dysferlin remained enriched in intracellular vesicles of Cav1-/- MEFs (Fig. 8B). The lack of an inhibitory effect of these mutants on dysferlin endocytosis is also shown by the uptake of myc antibodies when these mutants are expressed together with GFP-dysf, as compared with the WT Cav3 protein (Fig. 8C). The above results suggest that this conserved domain of caveolin is required for inhibition of dysferlin endocytosis and its retention at the PM.

Dominant acting mutants of Cav3 cause a reduction in surface Cav3, retention of Cav3 in the Golgi complex, and increased degradation (61, 62). We have previously shown that these mutants cause an accumulation of dysferlin in the Golgi complex. We investigated whether this was a result of a block of dysferlin exit from the Golgi rather than a consequence of redistribution due to dysferlin instability at the PM in the absence of caveolin. No myc antibody uptake was evident in the perinuclear region or intracellular vesicles (Fig. 9A) suggesting that dysferlin exit from the Golgi complex was blocked by the dystrophy-associated mutant caveolin and that these mutants have a dominant inhibitory role on Golgi exit. This effect of caveolin-dystrophy mutants on dysferlin exit from the Golgi complex is specific and not a result of their Golgi localization. Expression of a CSD deletion mutant, Cav1{Delta}81-100, which is also Golgi localized does not restrain dysferlin from exiting the Golgi (Fig. 9B). Taken together, these results show two distinct effects of caveolin mutants on trafficking of dysferlin.


Figure 6
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  		FIGURE 6.
Ultrastructural characterization of dysferlin endocytosis in Cav1-/- and WT Cav1 MEFs. WT Cav1 or Cav1-/- MEFs were transfected with GFPDysf and then after 14 h were incubated sequentially with antibodies to the lumenal myc tag and then HRP-labeled secondary (anti-mouse) antibodies at 4 °C. The cells were then warmed to 37 °C for 2 min to allow endocytosis to occur. The DAB reaction was performed on the living cells at 4 °C in the presence (+AA) or absence (-AA) of ascorbic acid as indicated. After fixation the transfected cells were identified under the light microscope by their GFP fluorescence and were marked to allow subsequent identification after embedding in resin. The marked areas were sectioned and viewed unstained. In the absence of AA, allowing visualization of both surface and intracellular pools of HRP, WT Cav1 cells showed HRP reaction product over the entire cell surface (A and B). In striking contrast, all transfected Cav1-/- cells showed sparse patchy surface labeling (C and D; arrowheads) consistent with greatly reduced retention of dysferlin at the plasma membrane. Quantitation of surface coverage by intersection counting (see "Experimental Procedures") showed that 10.3% of the PM of Cav1-/- cells was covered in HRP reaction product and 89.0% of the PM of WT cells. Neighboring untransfected cells showed no trace of HRP labeling (results not shown) demonstrating the specificity of the antibody labeling. Potential clathrin-independent early endocytic carriers (arrowheads) were frequently observed in the Cav1-/- cells (E-G). In the presence of ascorbic acid to quench extracellular HRP, internal ring-shaped endocytic elements were clearly demonstrated in the Cav1-/- cells (H and I). Bars, A-D, 1 µm; E-I, 200 nm.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In this work we have provided novel insights into dysferlin trafficking dynamics with respect to caveolin. Through the use of cells lacking caveolin, by expression of caveolin mutants, and by using different mutants of dysferlin, we have now identified the precise steps in dysferlin trafficking that are regulated by caveolin. We show directly that caveolins can facilitate exit of dysferlin mutants from the Golgi complex. In addition, dominant-acting caveolin mutants inhibit Golgi exit of mutant or wild type dysferlin. However, most unexpectedly, we now show that the primary effect of caveolin is to inhibit dysferlin endocytosis, implicating an endocytic mechanism in caveolin-associated muscle pathology.

Caveolin Modulation of Dysferlin Exit from the Golgi Complex—In cells devoid of caveolin dysferlin accumulates in intracellular vesicular structures, which we now show are endocytic in nature. No accumulation was observed in the Golgi complex and we demonstrated that despite the lack of surface labeling, dysferlin is rapidly transiting the cell surface in these cells, as shown by uptake of extracellular antibodies to a lumenal tag. These results suggest that dysferlin does not absolutely depend on caveolin for Golgi exit. Recent studies have suggested that novel exocytic carriers, containing defined quanta of caveolin, leave the Golgi complex and fuse directly with the PM (63). These carriers, termed exocytic caveolar carriers (64), form a novel exocytic pathway distinct from classical exocytic carriers (see scheme in Fig. 10); these carriers would presumably be absent in cells lacking caveolin. Our data provide new insights into these pathways. Full-length dysferlin can clearly utilize a non-caveolar carrier pathway to reach the PM, as shown in Cav1-/- cells. Similarly, a dysferlin mutant lacking all six C2 domains ({Delta}-C2) can also efficiently reach the PM, both in the absence or presence of caveolin again showing use of a non-caveolar carrier. However, in stark contrast to these two constructs, a protein of intermediate length, which lacks four C2 domains, shows an absolute dependence on caveolin for exit from the Golgi complex (Fig. 2 and scheme in Fig. 10). Whereas this is an artificially generated construct, these results clearly demonstrate a role for caveolin in Golgi exit, as suggested previously for a number of proteins including the angiotensin receptor (59), insulin receptor (65), and the stretch-activated channel, TRPC1 (66). The structural features that make these proteins dependent on caveolin for Golgi exit are as yet, unclear. In the case of dysferlin, it appears that the shorter construct is absolutely dependent on the caveolar pathway, whereas the additional cytoplasmic region of the full-length protein allows the protein to use multiple pathways. This might involve interaction of the terminal C2 domains with cellular machinery involved in trafficking via these caveolin-independent pathways. One candidate protein is Ahnak, which interacts with the C2A domain of dysferlin (67). Ahnak is a marker of a distinct exocytic vesicle, the enlargeosome (68, 69). Consistent with a role for dysferlin in membrane repair (24), enlargeosomes are proposed to be the source of membrane during PM resealing (68). Although enlargeosomes do not associate with caveolin-enriched detergent-resistant membranes (69) it remains to be examined whether dysferlin exits the Golgi via enlargeosomes. As C2 domains have also been implicated in phospholipid binding it is also possible that interaction with the distinct domains of the Golgi membrane allow segregation of dysferlin away from the caveolar domain.


Figure 7
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  		FIGURE 7.
Subcellular distribution of caveolin scaffolding domain mutants. Cav1-/- cells were transfected with HA-tagged Cav3, Cav3G55S, Cav3C71W, Cav1{Delta}81-100, and Cav3P104L, and colabeled with anti-HA and anti-GM130 antibodies. A, similarly to the wild type Cav3, the CSD mutants Cav3G55S and Cav3C71W CSD are targeted to the PM. In contrast, deletion of the CSD, Cav1{Delta}81-100, results in accumulation in the Golgi complex similarly to the dystrophy mutant Cav3P104L, demonstrated by the extensive overlap with the Golgi marker, GM130. B, CSD mutant predominant phenotypes were scored based on colabeling with relevant intracellular markers. Results are presented as percentage of cells showing a prevalent subcellular localization, and are representative of three individual experiments (n = 30-250/for each construct). Bar, 10 µm.

 
Dystrophy Mutants of Caveolin Disrupt Golgi Exit of Both Full-length Dysferlin and Dysferlin Truncation Mutants—We have previously demonstrated that dystrophy-associated mutants of caveolin (Cav3P104L and Cav3R26Q) cause dysferlin accumulation within the Golgi complex (18). We find no evidence for dysferlin transport to the cell surface under these conditions suggesting that the caveolin mutants cause a complete block in Golgi exit. Interestingly, this was true for all the tested membrane-associated dysferlin constructs, including {Delta}-C2, which lacks most of the cytoplasmic domain and traffics to the PM in a caveolin-independent manner. This raises the possibility that mutant caveolin might perturb dysferlin trafficking at an earlier stage in the Golgi complex, before divergence of the two pathways. Consistent with this, Golgi caveolin mutants accumulate throughout the Golgi complex including the cis Golgi (18, 47). The specificity of this effect is shown by the fact that a Golgi-localized form of caveolin lacking amino acids 81-100 does not prevent dysferlin from exiting the Golgi. This suggests that a direct interaction between dysferlin and caveolin, at least, at the Golgi level may be taking place. If so, this narrows down the interacting domain of dysferlin to the TM domain and nearby cytoplasmic region, which contains four potential CSD binding motifs. However, these findings do not rule out perturbation of lipid domains of the Golgi complex, which may be influenced by expression of a form of caveolin with mutations in this potential lipid-binding domain (45). If so, these effects are restricted to specific cargo proteins as the transit of other proteins, such as GPI-anchored proteins, through the Golgi complex is unaffected by the expression of the mutant caveolin proteins (18).

Caveolins Inhibit Dysferlin Endocytosis—We show here for the first time that caveolins inhibit endocytosis of dysferlin. In cells lacking Cav1 and Cav3, dysferlin is rapidly cleared from the PM resulting in a low level of PM dysferlin in contrast to a large intracellular pool at steady state. Our results show a much higher endocytic rate for dysferlin in Cav1-/- cells as antibodies to a lumenal tag accumulate far more rapidly in Cav1-/- cells than in cells expressing Cav1 or Cav3 despite the higher level of surface dysferlin in these cells (see Figs. 4 and 8) (18). Expression of Cav1 or Cav3, but not specific caveolin mutants, inhibits dysferlin endocytosis resulting in its retention at the cell surface and a high level of PM dysferlin. Dysferlin endocytosis in Cav1-/- cells is via a dynamin (and caveolin-)-independent pathway. Colocalization with GPI-anchored proteins and CTB, but not transferrin, at early stages of endocytosis strongly implicates the CLIC/GEEC clathrin-independent pathway (54, 56) in dysferlin endocytosis. This is supported by ultrastructural analysis of the endocytic pathway showing dysferlin in tubular/ring-shaped early endosomal elements. These results suggest a novel role of this pathway in regulating dysferlin surface expression and, a role for caveolin in inhibiting the clathrin-independent endocytosis of specific markers.


Figure 8
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  		FIGURE 8.
Dysferlin retention at the PM is dependent on an intact CSD. Expression of CSD point mutants does not rescue dysferlin traffic to the PM in Cav1-/- MEF cells. Cav1-/- cells were transfected with GFPDysf or co-transfected with GFPDysf and HA-tagged Cav3, Cav3G55S, Cav3C71W, Cav1{Delta}81-100, and Cav3P104L and labeled with rabbit anti-HA and mouse anti-myc antibodies. Surface labeling of dysferlin and quantification of PM and intracellular pools of dysferlin were performed as described under "Experimental Procedures." A, in cells lacking caveolin dysferlin is mainly targeted to intracellular puncta throughout the cytoplasm. Whereas expression of epitope-tagged Cav3 rescues dysferlin traffic to the PM, expression of CSD mutants (i.e. Cav3G55S) does not rescue dysferlin traffic to the PM. B, the mean fluorescence intensity of dysferlin associated with the PM (myc labeling) and intracellular structures (GFP labeling) was measured and expressed as the PM/IC ratio. Error bars are S.E. of three experiments (n = 30); **, p < 0.001. C, Cav1-/- cells co-expressing GFPDysf and HA-tagged, Cav3C71W, Cav3G55S, or Cav3 were allowed to uptake anti-myc antibodies for 10 min at 37 °C. Expression of Cav3C71W or Cav3G55S, but not WT Cav3 protein, have no effect on dysferlin endocytosis as demonstrated by the internalization of anti-myc antibodies. Bars, 10 µm.

 
The inhibitory effect of caveolins on dysferlin endocytosis presents an interesting conundrum. We believe that a direct inhibitory effect of caveolin by binding dysferlin to immobile caveolar domains is unlikely; both electron microscopy and light microscopy show that dysferlin does not colocalize significantly with caveolin, even at the level of light microscopy, and both immuno-EM on frozen sections (18) and EM surface labeling experiments (this study) confirmed that dysferlin was not concentrated within caveolae. Thus, the inhibition by caveolin appears to be indirect. Yet our results suggest that the effect of caveolin on dysferlin endocytosis is specific; whereas acute Cav1 expression has been shown to inhibit clathrin-independent endocytosis of CTB (54, 70) and SV40 is efficiently internalized by cells devoid of caveolae (71) CTB and SV40 internalization was quantitatively identical in WT Cav1 and Cav1-/- MEFs as used here. Furthermore, no effect on GPI-AP internalization could be detected in Cav1-/- cells. This argues against a general negative inhibitory role of caveolins on clathrin-independent endocytosis, but suggests that caveolins specifically inhibit dysferlin entry into this pathway. We could also show that the increased uptake of dysferlin in Cav1-/- cells was not due to increased endocytosis caused by dysferlin expression (results not shown). Caveolae have been suggested to be negative regulators of clathrin-independent endocytosis (70, 72) but other work identifies caveolae as endocytic vehicles (73-75). A new concept described here is that caveolin is regulating the non-caveolar endocytosis of dysferlin as we have clearly demonstrated that dysferlin is not concentrated in caveolae.


Figure 9
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  		FIGURE 9.
Dystrophy mutant of Cav3 has a dominant inhibitory effect on dysferlin exit from the Golgi. Cav1-/- cells were co-transfected with GFP-Dysf and HA-tagged Cav3P104L or Cav1{Delta}81-100 and labeled with rabbit anti-HA (A and B) and mouse anti-GM130 (B) antibodies. Anti-myc antibody was internalized overnight (B). A, expression of Cav3P04L-HA blocks dysferlin exit from the Golgi apparatus. No significant Golgi pool of internalized myc antibodies is seen after overnight incubation at 37 °C. B, expression of Golgi-localized epitope-tagged Cav1{Delta}81-100 mutant does not affect GFP-dysf exit from the Golgi apparatus. Cav1{Delta}81-100, but not GFPDysf, is retained in the Golgi complex as demonstrated by colocalization with the Golgi marker, GM130. Bars, 10 µm.

 


Figure 10
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  		FIGURE 10.
Model for regulation of caveolin trafficking by caveolin. In WT Cav1 cells full-length dysferlin exit from the Golgi complex may take place via caveolar and noncaveolar exocytic carriers. In Cav1-/- cells dysferlin must use an alternative pathway(s). However, a truncation mutant lacking part of the cytoplasmic domain ({Delta}-1) is completely dependent on caveolin for Golgi exit as it cannot enter the non-caveolar pathway. A more severe mutant ({Delta}-C2) traffics to the PM in a caveolin-independent manner as it lacks information for incorporation into caveolar carriers. Retention of dysferlin at the PM is dependent on caveolin/caveolae due to inhibition of dysferlin endocytosis by caveolin. A truncation of dysferlin lacking all six C2 domains, {Delta}-C2, is not internalized suggesting a role for the cytoplasmic domain in endocytosis.

 
A model for the inhibitory effect of caveolin on endocytosis must take into account the intriguing finding that two Cav3 single point mutants, which occur naturally in the human population, did not inhibit dysferlin endocytosis in complete contrast to the wild type protein. This implicates the scaffolding domain of caveolin in its inhibitory activity. The two inhibitory mutants were initially reported as dystrophy mutants (11) but subsequently have been shown to occur as polymorphisms in the population (76, 77). Yet two previous studies have shown specific effects of these mutant proteins in cultured cells (50, 59). Further studies should elucidate the underlying mechanisms involved in the inhibitory activity of the wild type protein in comparison to these single point mutants and whether these mutations can contribute to disease under certain conditions.

In conclusion, these studies have elucidated distinct roles of caveolin in regulating dysferlin trafficking pathways, both in positively regulating exocytosis and negatively regulating endocytosis via a clathrin-independent pathway for which dysferlin acts as a new marker. An interesting possibility is that this inhibitory activity of caveolin on endocytosis is regulated in vivo, allowing modulation of the surface levels of dysferlin and membrane remodeling. Whether caveolin acts in a similar fashion on other surface proteins will require further investigation. The involvement of endocytosis in muscle disease may be a more general phenomenon. Another sarcolemmal protein, {alpha}-sarcoglycan, which is linked to a subset of muscle disease limb girdle muscular dystrophy 2D (78, 79), is translocated from the cell surface to endosomes upon perturbation of its PM stability (80). {alpha}-Sarcoglycan stability at the PM relies on a proper assembly of the sarcoglycan complex (80). Thus dysferlin and {alpha}-sarcoglycan represent examples of sarcolemmal proteins where endocytic mechanisms play a central role in maintaining the integrity of the PM.

These results provide new insights into the functions of caveolins and the mechanisms underlying caveolin-related diseases. In addition, they provide fundamental insights into the regulation of exocytic and endocytic trafficking pathways of membrane proteins in mammalian cells and the importance of this poorly characterized clathrin-independent endocytic pathway in surface remodeling of specific PM components.


   FOOTNOTES
 
* This work was supported by grants from the National Health and Medical Research Council of Australia (to R. G. P. and J. F. H.), the Muscular Dystrophy Campaign, Association Francais contre les Myopathies, and the Jain Foundation (to K. B. and S. H. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1 and S2. Back

1 To whom correspondence should be addressed: Institute for Molecular Bioscience, University of Queensland, Brisbane QLD 4072, Australia. Tel.: 61-7-3346-2032; Fax: 61-7-3346-2339; E-mail: R.Parton{at}imb.uq.edu.au.

2 The abbreviations used are: PM, plasma membrane; Cav, Caveolin; Cav1-/-, Cav1-/- immortalized cell lines; CSD, caveolin scaffolding domain; CTB, cholera toxin binding subunit; GPI-AP, glycosylphosphatidylinositol-anchored proteins; GEECs, GPI-AP-enriched early endosomal compartments; Tfn, transferrin; WT, wild type; AA, ascorbic acid; HA, hemagglutinin; GFP, green fluorescent protein; HRP, horseradish peroxidase; MEF, mouse embryonic fibroblasts; DAB, dimethylaminoazobenzene; IC, intracellular; TM, transmembrane. Back


   ACKNOWLEDGMENTS
 
We thank Michelle Hill for preparation of immortalized mouse embryonic fibroblast cell lines and Rachel Hancock for technical assistance in EM processing. We also thank members of the Parton group for critical reading of the manuscript. Confocal microscopy was performed at the ACRF/IMB Dynamic Imaging Facility for Cancer Biology, established with funding from the Australian Cancer Research Foundation. The Institute for Molecular Bioscience is a Special Research Centre of the Australian Research Council.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Aoki, M., Liu, J., Richard, I., Bashir, R., Britton, S., Keers, S. M., Oeltjen, J., Brown, H. E., Marchand, S., Bourg, N., Beley, C., McKenna-Yasek, D., Arahata, K., Bohlega, S., Cupler, E., Illa, I., Majneh, I., Barohn, R. J., Urtizberea, J. A., Fardeau, M., Amato, A., Angelini, C., Bushby, K., Beckmann, J. S., and Brown, R. H., Jr. (2001) Neurology 57, 271-278[Abstract/Free Full Text]
  2. Bashir, R., Britton, S., Strachan, T., Keers, S., Vafiadaki, E., Lako, M., Richard, I., Marchand, S., Bourg, N., Argov, Z., Sadeh, M., Mahjneh, I., Marconi, G., Passos-Bueno, M. R., Moreira Ede, S., Zatz, M., Beckmann, J. S., and Bushby, K. (1998) Nat. Genet. 20, 37-42[CrossRef][Medline] [Order article via Infotrieve]
  3. Illa, I., Serrano-Munuera, C., Gallardo, E., Lasa, A., Rojas-Garcia, R., Palmer, J., Gallano, P., Baiget, M., Matsuda, C., and Brown, R. H. (2001) Ann. Neurol. 49, 130-134[CrossRef][Medline] [Order article via Infotrieve]
  4. Liu, J., Aoki, M., Illa, I., Wu, C., Fardeau, M., Angelini, C., Serrano, C., Urtizberea, J. A., Hentati, F., Hamida, M. B., Bohlega, S., Culper, E. J., Amato, A. A., Bossie, K., Oeltjen, J., Bejaoui, K., McKenna-Yasek, D., Hosler, B. A., Schurr, E., Arahata, K., de Jong, P. J., and Brown, R. H., Jr. (1998) Nat. Genet. 20, 31-36[CrossRef][Medline] [Order article via Infotrieve]
  5. Matsuda, C., Aoki, M., Hayashi, Y. K., Ho, M. F., Arahata, K., and Brown, R. H., Jr. (1999) Neurology 53, 1119-1122[Abstract/Free Full Text]
  6. Betz, R. C., Schoser, B. G., Kasper, D., Ricker, K., Ramirez, A., Stein, V., Torbergsen, T., Lee, Y. A., Nothen, M. M., Wienker, T. F., Malin, J. P., Propping, P., Reis, A., Mortier, W., Jentsch, T. J., Vorgerd, M., and Kubisch, C. (2001) Nat. Genet. 28, 218-219[CrossRef][Medline] [Order article via Infotrieve]
  7. Carbone, I., Bruno, C., Sotgia, F., Bado, M., Broda, P., Masetti, E., Panella, A., Zara, F., Bricarelli, F. D., Cordone, G., Lisanti, M. P., and Minetti, C. (2000) Neurology 54, 1373-1376[Abstract/Free Full Text]
  8. Figarella-Branger, D., Pouget, J., Bernard, R., Krahn, M., Fernandez, C., Levy, N., and Pellissier, J. F. (2003) Neurology 61, 562-564[Abstract/Free Full Text]
  9. Fischer, D., Schroers, A., Blumcke, I., Urbach, H., Zerres, K., Mortier, W., Vorgerd, M., and Schroder, R. (2003) Ann. Neurol. 53, 233-241[CrossRef][Medline] [Order article via Infotrieve]
  10. Kubisch, C., Schoser, B. G., von During, M., Betz, R. C., Goebel, H. H., Zahn, S., Ehrbrecht, A., Aasly, J., Schroers, A., Popovic, N., Lochmuller, H., Schroder, J. M., Bruning, T., Malin, J. P., Fricke, B., Meinck, H. M., Torbergsen, T., Engels, H., Voss, B., and Vorgerd, M. (2003) Ann. Neurol. 53, 512-520[CrossRef][Medline] [Order article via Infotrieve]
  11. McNally, E. M., de Sa Moreira, E., Duggan, D. J., Bonnemann, C. G., Lisanti, M. P., Lidov, H. G., Vainzof, M., Passos-Bueno, M. R., Hoffman, E. P., Zatz, M., and Kunkel, L. M. (1998) Hum. Mol. Genet. 7, 871-877[Abstract/Free Full Text]
  12. Merlini, L., Carbone, I., Capanni, C., Sabatelli, P., Tortorelli, S., Sotgia, F., Lisanti, M. P., Bruno, C., and Minetti, C. (2002) J. Neurol. Neurosurg. Psychiatry 73, 65-67[Abstract/Free Full Text]
  13. Minetti, C., Sotgia, F., Bruno, C., Scartezzini, P., Broda, P., Bado, M., Masetti, E., Mazzocco, M., Egeo, A., Donati, M. A., Volonte, D., Galbiati, F., Cordone, G., Bricarelli, F. D., Lisanti, M. P., and Zara, F. (1998) Nat. Genet. 18, 365-368[CrossRef][Medline] [Order article via Infotrieve]
  14. Tateyama, M., Aoki, M., Nishino, I., Hayashi, Y. K., Sekiguchi, S., Shiga, Y., Takahashi, T., Onodera, Y., Haginoya, K., Kobayashi, K., Iinuma, K., Nonaka, I., Arahata, K., Itoyama, Y., and Itoyoma, Y. (2002) Neurology 58, 323-325[Abstract/Free Full Text]
  15. Vorgerd, M., Ricker, K., Ziemssen, F., Kress, W., Goebel, H. H., Nix, W. A., Kubisch, C., Schoser, B. G., and Mortier, W. (2001) Neurology 57, 2273-2277[Abstract/Free Full Text]
  16. Capanni, C., Sabatelli, P., Mattioli, E., Ognibene, A., Columbaro, M., Lattanzi, G., Merlini, L., Minetti, C., Maraldi, N. M., and Squarzoni, S. (2003) Exp. Mol. Med. 35, 538-544[Medline] [Order article via Infotrieve]
  17. Matsuda, C., Hayashi, Y. K., Ogawa, M., Aoki, M., Murayama, K., Nishino, I., Nonaka, I., Arahata, K., and Brown, R. H., Jr. (2001) Hum. Mol. Genet. 10, 1761-1766[Abstract/Free Full Text]
  18. Hernandez-Deviez, D. J., Martin, S., Laval, S. H., Lo, H. P., Cooper, S. T., North, K. N., Bushby, K., and Parton, R. G. (2006) Hum. Mol. Genet. 15, 129-142[Abstract/Free Full Text]
  19. Yabe, I., Kawashima, A., Kikuchi, S., Higashi, T., Fukazawa, T., Hamada, T., Sasaki, H., and Tashiro, K. (2003) Acta Neurol. Scand. 108, 47-51[CrossRef][Medline] [Order article via Infotrieve]
  20. Britton, S., Freeman, T., Vafiadaki, E., Keers, S., Harrison, R., Bushby, K., and Bashir, R. (2000) Genomics 68, 313-321[CrossRef][Medline] [Order article via Infotrieve]
  21. Davis, D. B., Delmonte, A. J., Ly, C. T., and McNally, E. M. (2000) Hum. Mol. Genet. 9, 217-226[Abstract/Free Full Text]
  22. Yasunaga, S., Grati, M., Cohen-Salmon, M., El-Amraoui, A., Mustapha, M., Salem, N., El-Zir, E., Loiselet, J., and Petit, C. (1999) Nat. Genet. 21, 363-369[CrossRef][Medline] [Order article via Infotrieve]
  23. Weiler, T., Bashir, R., Anderson, L. V., Davison, K., Moss, J. A., Britton, S., Nylen, E., Keers, S., Vafiadaki, E., Greenberg, C. R., Bushby, C. R., and Wrogemann, K. (1999) Hum. Mol. Genet. 8, 871-877[Abstract/Free Full Text]
  24. Bansal, D., and Campbell, K. P. (2004) Trends Cell Biol. 14, 206-213[CrossRef][Medline] [Order article via Infotrieve]
  25. Bansal, D., Miyake, K., Vogel, S. S., Groh, S., Chen, C. C., Williamson, R., McNeil, P. L., and Campbell, K. P. (2003) Nature 423, 168-172[CrossRef][Medline] [Order article via Infotrieve]
  26. Mikoshiba, K., Fukuda, M., Ibata, K., Kabayama, H., and Mizutani, A. (1999) Chem. Phys. Lipids 98, 59-67[CrossRef][Medline] [Order article via Infotrieve]
  27. Nalefski, E. A., and Falke, J. J. (1996) Protein Sci. 5, 2375-2390[Medline] [Order article via Infotrieve]
  28. Pallanck, L. (2003) Trends Neurosci. 26, 2-4[CrossRef][Medline] [Order article via Infotrieve]
  29. Rizo, J., and Sudhof, T. C. (1998) J. Biol. Chem. 273, 15879-15882[Free Full Text]
  30. Rothberg, K. G., Heuser, J. E., Donzell, W. C., Ying, Y. S., Glenney, J. R., and Anderson, R. G. (1992) Cell 68, 673-682[CrossRef][Medline] [Order article via Infotrieve]
  31. Scherer, P. E., Lewis, R. Y., Volonte, D., Engelman, J. A., Galbiati, F., Couet, J., Kohtz, D. S., van Donselaar, E., Peters, P., and Lisanti, M. P. (1997) J. Biol. Chem. 272, 29337-29346[Abstract/Free Full Text]
  32. Tang, Z., Scherer, P. E., Okamoto, T., Song, K., Chu, C., Kohtz, D. S., Nishimoto, I., Lodish, H. F., and Lisanti, M. P. (1996) J. Biol. Chem. 271, 2255-2261[Abstract/Free Full Text]
  33. Way, M., and Parton, R. G. (1995) FEBS Lett. 376, 108-112[CrossRef][Medline] [Order article via Infotrieve]
  34. Kurzchalia, T. V., Dupree, P., Parton, R. G., Kellner, R., Virta, H., Lehnert, M., and Simons, K. (1992) J. Cell Biol. 118, 1003-1014[Abstract/Free Full Text]
  35. Williams, T. M., and Lisanti, M. P. (2004) Genome Biol. 5, 214[CrossRef][Medline] [Order article via Infotrieve]
  36. Doyle, D. D., Upshaw-Earley, J., Bell, E., and Palfrey, H. C. (2003) Biochem. Biophys. Res. Commun. 304, 22-25[CrossRef][Medline] [Order article via Infotrieve]
  37. Song, K. S., Scherer, P. E., Tang, Z., Okamoto, T., Li, S., Chafel, M., Chu, C., Kohtz, D. S., and Lisanti, M. P. (1996) J. Biol. Chem. 271, 15160-15165[Abstract/Free Full Text]
  38. Ikonen, E., Heino, S., and Lusa, S. (2004) Biochem. Soc. Trans. 32, 121-123[CrossRef][Medline] [Order article via Infotrieve]
  39. Razani, B., Schlegel, A., and Lisanti, M. P. (2000) J. Cell Sci. 113, 2103-2109[Abstract]
  40. Cheng, Z. J., Singh, R. D., Marks, D. L., and Pagano, R. E. (2006) Mol. Membr. Biol. 23, 101-110[CrossRef][Medline] [Order article via Infotrieve]
  41. Parton, R. G., and Richards, A. A. (2003) Traffic 4, 724-738[CrossRef][Medline] [Order article via Infotrieve]
  42. Nichols, B. (2003) J. Cell Sci. 116, 4707-4714[Abstract/Free Full Text]
  43. Dupree, P., Parton, R. G., Raposo, G., Kurzchalia, T. V., and Simons, K. (1993) EMBO J. 12, 1597-1605[Medline] [Order article via Infotrieve]
  44. Monier, S., Parton, R. G., Vogel, F., Behlke, J., Henske, A., and Kurzchalia, T. V. (1995) Mol. Biol. Cell 6, 911-927[Abstract]
  45. Parton, R. G., Hanzal-Bayer, M., and Hancock, J. F. (2006) J. Cell Sci. 119, 787-796[Abstract/Free Full Text]
  46. Couet, J., Li, S., Okamoto, T., Ikezu, T., and Lisanti, M. P. (1997) J. Biol. Chem. 272, 6525-6533[Abstract/Free Full Text]
  47. Luetterforst, R., Stang, E., Zorzi, N., Carozzi, A., Way, M., and Parton, R. G. (1999) J. Cell Biol. 145, 1443-1459[Abstract/Free Full Text]
  48. Prior, I. A., Harding, A., Yan, J., Sluimer, J., Parton, R. G., and Hancock, J. F. (2001) Nat. Cell Biol. 3, 368-375[CrossRef][Medline] [Order article via Infotrieve]
  49. Klinge, L., Laval, S., Keers, S., Haldane, F., Straub, V., Barresi, R., and Bushby, K. (2007) FASEB J. 21, 1768-1776[Abstract/Free Full Text]
  50. Carozzi, A. J., Roy, S., Morrow, I. C., Pol, A., Wyse, B., Clyde-Smith, J., Prior, I. A., Nixon, S. J., Hancock, J. F., and Parton, R. G. (2002) J. Biol. Chem. 277, 17944-17949[Abstract/Free Full Text]
  51. Morrow, I. C., Rea, S., Martin, S., Prior, I. A., Prohaska, R., Hancock, J. F., James, D. E., and Parton, R. G. (2002) J. Biol. Chem. 277, 48834-48841[Abstract/Free Full Text]
  52. Razani, B., Engelman, J. A., Wang, X. B., Schubert, W., Zhang, X. L., Marks, C. B., Macaluso, F., Russell, R. G., Li, M., Pestell, R. G., Di Vizio, D., Hou, H., Jr., Kneitz, B., Lagaud, G., Christ, G. J., Edelmann, W., and Lisanti, M. P. (2001) J. Biol. Chem. 276, 38121-38138[Abstract/Free Full Text]
  53. Sotgia, F., Razani, B., Bonuccelli, G., Schubert, W., Battista, M., Lee, H., Capozza, F., Schubert, A. L., Minetti, C., Buckley, J. T., and Lisanti, M. P. (2002) Mol. Cell. Biol. 22, 3905-3926[Abstract/Free Full Text]
  54. Kirkham, M., Fujita, A., Chadda, R., Nixon, S. J., Kurzchalia, T. V., Sharma, D. K., Pagano, R. E., Hancock, J. F., Mayor, S., and Parton, R. G. (2005) J. Cell Biol. 168, 465-476[Abstract/Free Full Text]
  55. Naslavsky, N., Weigert, R., and Donaldson, J. G. (2004) Mol. Biol. Cell 15, 3542-3552[Abstract/Free Full Text]
  56. Sabharanjak, S., Sharma, P., Parton, R. G., and Mayor, S. (2002) Dev. Cell 2, 411-423[CrossRef][Medline] [Order article via Infotrieve]
  57. Macia, E., Ehrlich, M., Massol, R., Boucrot, E., Brunner, C., and Kirchhausen, T. (2006) Dev. Cell 10, 839-850[CrossRef][Medline] [Order article via Infotrieve]
  58. Newton, A. J., Kirchhausen, T., and Murthy, V. N. (2006) Proc. Natl. Acad. Sci. U. S. A. 103, 17955-17960[Abstract/Free Full Text]
  59. Wyse, B. D., Prior, I. A., Qian, H., Morrow, I. C., Nixon, S., Muncke, C., Kurzchalia, T. V., Thomas, W. G., Parton, R. G., and Hancock, J. F. (2003) J. Biol. Chem. 278, 23738-23746[Abstract/Free Full Text]
  60. Ren, X., Ostermeyer, A. G., Ramcharan, L. T., Zeng, Y., Lublin, D. M., and Brown, D. A. (2004) Mol. Biol. Cell 15, 4556-4567[Abstract/Free Full Text]
  61. Sotgia, F., Woodman, S. E., Bonuccelli, G., Capozza, F., Minetti, C., Scherer, P. E., and Lisanti, M. P. (2003) Am. J. Physiol. 285, C1150-C1160
  62. Galbiati, F., Volonte, D., Minetti, C., Chu, J. B., and Lisanti, M. P. (1999) J. Biol. Chem. 274, 25632-25641[Abstract/Free Full Text]
  63. Tagawa, A., Mezzacasa, A., Hayer, A., Longatti, A., Pelkmans, L., and Helenius, A. (2005) J. Cell Biol. 170, 769-779[Abstract/Free Full Text]
  64. Parton, R. G., and Simons, K. (2007) Nat. Rev. 8, 185-194[CrossRef]
  65. Cohen, A. W., Razani, B., Wang, X. B., Combs, T. P., Williams, T. M., Scherer, P. E., and Lisanti, M. P. (2003) Am. J. Physiol. 285, C222-C235
  66. Brazer, S. C., Singh, B. B., Liu, X., Swaim, W., and Ambudkar, I. S. (2003) J. Biol. Chem. 278, 27208-27215[Abstract/Free Full Text]
  67. Huang, Y., Laval, S. H., van Remoortere, A., Baudier, J., Benaud, C., Anderson, L. V., Straub, V., Deelder, A., Frants, R. R., den Dunnen, J. T., Bushby, K., and van der Maarel, S. M. (2007) FASEB J. 21, 732-742[Abstract/Free Full Text]
  68. Borgonovo, B., Cocucci, E., Racchetti, G., Podini, P., Bachi, A., and Meldolesi, J. (2002) Nat. Cell Biol. 4, 955-962[CrossRef][Medline] [Order article via Infotrieve]
  69. Cocucci, E., Racchetti, G., Podini, P., Rupnik, M., and Meldolesi, J. (2004) Mol. Biol. Cell 15, 5356-5368[Abstract/Free Full Text]
  70. Le, P. U., Guay, G., Altschuler, Y., and Nabi, I. R. (2002) J. Biol. Chem. 277, 3371-3379[Abstract/Free Full Text]
  71. Damm, E. M., Pelkmans, L., Kartenbeck, J., Mezzacasa, A., Kurzchalia, T., and Helenius, A. (2005) J. Cell Biol. 168, 477-488[Abstract/Free Full Text]
  72. Nabi, I. R., and Le, P. U. (2003) J. Cell Biol. 161, 673-677[Abstract/Free Full Text]
  73. Sharma, D. K., Brown, J. C., Choudhury, A., Peterson, T. E., Holicky, E., Marks, D. L., Simari, R., Parton, R. G., and Pagano, R. E. (2004) Mol. Biol. Cell 15, 3114-3122[Abstract/Free Full Text]
  74. Di Guglielmo, G. M., Le Roy, C., Goodfellow, A. F., and Wrana, J. L. (2003) Nat. Cell Biol. 5, 410-421[CrossRef][Medline] [Order article via Infotrieve]
  75. Pelkmans, L., Kartenbeck, J., and Helenius, A. (2001) Nat. Cell Biol. 3, 473-483[CrossRef][Medline] [Order article via Infotrieve]
  76. de Paula, F., Vainzof, M., Bernardino, A. L., McNally, E., Kunkel, L. M., and Zatz, M. (2001) Am. J. Med. Genet. 99, 303-307[CrossRef][Medline] [Order article via Infotrieve]
  77. Vatta, M., Ackerman, M. J., Ye, B., Makielski, J. C., Ughanze, E. E., Taylor, E. W., Tester, D. J., Balijepalli, R. C., Foell, J. D., Li, Z., Kamp, T. J., and Towbin, J. A. (2006) Circulation 114, 2104-2112[Abstract/Free Full Text]
  78. Fanin, M., Duggan, D. J., Mostacciuolo, M. L., Martinello, F., Freda, M. P., Soraru, G., Trevisan, C. P., Hoffman, E. P., and Angelini, C. (1997) J. Med. Genet. 34, 973-977[Abstract/Free Full Text]
  79. McNally, E. M., Yoshida, M., Mizuno, Y., Ozawa, E., and Kunkel, L. M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 9690-9694[Abstract/Free Full Text]
  80. Draviam, R. A., Wang, B., Shand, S. H., Xiao, X., and Watkins, S. C. (2006) Traffic 7, 793-810[CrossRef][Medline] [Order article via Infotrieve]

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