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. 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. Adipocytes play a major role in whole body fuel homeostasis. They represent a major site of lipid storage and in turn a major energy source for other tissues. 3T3-L1 adipocytes have been widely used as a model system to study adipogenesis (1Spiegelman B.M. Choy L. Hotamisligil G.S. Graves R.A. Tontonoz P. J. Biol. Chem. 1993; 268: 6823-6826Abstract Full Text PDF PubMed Google Scholar). Originally isolated from disaggregated mouse embryos by selecting clones that had the potential to differentiate into adipocytes, they share many of the properties ofbona fide mature adipocytes in vivo (2Green H. Kehinde O. Cell. 1975; 5: 19-27Abstract Full Text PDF PubMed Scopus (1086) Google Scholar, 3Green H. Meuth M. Cell. 1974; 3: 127-133Abstract Full Text PDF PubMed Scopus (811) Google Scholar, 4Green H. Kehinde O. Cell. 1976; 7: 105-113Abstract Full Text PDF PubMed Scopus (615) Google Scholar, 5Russell T.R. Ho R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 4516-4520Crossref PubMed Scopus (154) Google Scholar, 6Williams I.H. Polakis S.E. Biochem. Biophys. Res. Commun. 1977; 77: 175-186Crossref PubMed Scopus (84) Google Scholar). Shortly after the initiation of differentiation, the expression of a number of gene products that regulate lipogenesis and insulin action are markedly increased (7Kuri-Harcuch W. Green H. J. Biol. Chem. 1977; 252: 2158-2160Abstract Full Text PDF PubMed Google Scholar, 8Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Abstract Full Text PDF PubMed Google Scholar). The cells then round up, presumably due to a rearrangement of the cytoskeleton, and electron-lucent lipid droplets can be seen scattered throughout the cell cytoplasm (9Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (262) Google Scholar). If the cells are cultured for long periods of time, the lipid droplets tend to fuse to form one large droplet resembling mature white adipocytes. The morphological changes that accompany adipocyte differentiation have been studied in detail (9Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (262) Google Scholar, 10Franke W.W. Hergt M. Grund C. Cell. 1987; 49: 131-141Abstract Full Text PDF PubMed Scopus (207) Google Scholar). Most notably, each rounded lipid droplet is surrounded by a single lipid layer, which is in turn ensheathed by endoplasmic reticulum. Another characteristic morphological feature of differentiating adipocytes is the increase in abundance of caveolae. Caveolae are evident as single pits at the cell surface as well as associated with surface-connected spherical structures, to form characteristic caveolae-studded features termed “rosettes” (9Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (262) Google Scholar). Caveolar rosettes are found in many eukaryotic cells (11Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (452) Google Scholar) but they are particularly abundant in adipocytes. The relationship between these organelles and adipogenesis is not clear, although it may be related to a role in cholesterol homeostasis and/or lipid transport between the lipid droplet and the cell surface. One of the metabolic processes that is acquired during 3T3-L1 adipocyte differentiation is a highly insulin-responsive glucose-transport system (12Rubin C.S. Lai E. Rosen O.M. J. Biol. Chem. 1977; 252: 3554-3557Abstract Full Text PDF PubMed Google Scholar). Insulin stimulates glucose transport in muscle and adipocytesin vivo via the translocation of a facilitative glucose transporter GLUT4 from intracellular tubulovesicular elements to the plasma membrane (13Rea S. James D.E. Diabetes. 1997; 46: 1667-1677Crossref PubMed Google Scholar). It has been shown that two separate insulin-responsive signal-transduction pathways regulate cell surface levels of GLUT4 in an additive manner (reviewed in Ref. 14Saltiel A.R. Pessin J.E. Trends Cell Biol. 2002; 12: 65-71Abstract Full Text Full Text PDF PubMed Scopus (500) Google Scholar). The first pathway involves the lipid kinase, phosphatidylinositol 3′-kinase, and the serine/threonine protein kinase Akt/PKB. The second pathway is independent of phosphatidylinositol 3′-kinase and involves the recruitment of a multimeric complex that includes c-Cbl and CAP to a specialized microdomain of the plasma membrane (15Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (556) Google Scholar, 16Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 1-13Crossref Scopus (147) Google Scholar). This specialized domain has been characterized as a lipid raft domain that is highly enriched in the lipid raft marker proteins caveolin and flotillin. Very recently, these structures have been defined as a specialized form of caveolae associated with the filamentous actin cytoskeleton, referred to as Cav-actin (17Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). We have also observed the presence of large ring-like structures associated with the plasma membrane of 3T3-L1 adipocytes. In the present study, we have further characterized these rings using both light and electron microscopy. These structures are detected in differentiated adipocytes but not in preadipocytes. They represent large surface-connected invaginations of the plasma membrane and hence have been named caves. In contrast to previous studies (15Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (556) Google Scholar, 16Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 1-13Crossref Scopus (147) Google Scholar, 17Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), we show that although these structures are studded with caveolae, they simply represent an extension of the cell surface, also containing clathrin-coated pits and non-caveolar markers (raft and non-raft markers). By light microscopy we observe an enrichment of actin staining around caves and by electron microscopy we observe a filamentous network associated with these structures resembling actin filaments. Thus, caves represent an adipocyte-specific specialization of the cell surface. 3T3-L1 fibroblasts, from the American Type Culture Collection, were cultured in Dulbecco's modified Eagle's medium (DMEM) 1The abbreviations used for: DMEM, Dulbecco's modified Eagle's medium; BSA, bovine serum albumin; PBS, phosphate-buffered saline; SNAP, soluble NSF attachment protein; CT-B, cholera toxin binding subunit B; HRP, horseradish peroxidase. supplemented with 10% (v/v) newborn calf serum (CSL Limited, Parkville, Victoria, Australia), 2 mml-glutamine, 100 units/liter penicillin and 100 μg/liter streptomycin at 37 °C in 10% CO2, and passaged at ∼70% confluence. Confluent fibroblasts were induced to differentiate 1 d after reaching confluence by the addition of DMEM containing 10% (v/v) heat-inactivated fetal calf serum (FCS; Invitrogen), 4 mg/ml insulin, 0.25 mm dexamethasone, 0.5 mm3-isobutyl-1-methylxanthine, and 100 ng/ml d-biotin. After 72 h, the differentiation medium was replaced with fresh FCS/DMEM containing insulin. Adipocytes were re-fed with FCS/DMEM every 72 h and utilized for experiments at 8–10 days post differentiation. All tissue culture media were purchased from Invitrogen, except fetal calf serum, which was obtained from Trace Biosciences (Clayton, Australia). Insulin was obtained fromCalbiochem. Bovine serum albumin (BSA) was purchased from ICN (Costa Mesa, CA). The GLUT4 polyclonal antibody (R017) was raised against a synthetic peptide corresponding to the COOH-terminal 20 amino acids of rat GLUT4. Monoclonal anti-tubulin antibody (DM10) was purchased from Sigma. Polyclonal anti-caveolin antibody was obtained from Transduction Laboratories. Transferrin receptor monoclonal antibody was obtained from Zymed Laboratories Inc. Syntaxin4, GLUT1, and SNAP 23 polyclonal antibodies are described elsewhere (18Rea S. Martin L.B. McIntosh S. Macaulay S.L. Ramsdale T. Baldini G. James D.E. J. Biol. Chem. 1998; 273: 18784-18792Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar, 19Piper R.C. Hess L.J. James D.E. Am. J. Physiol. 1991; 260: C570-C580Crossref PubMed Google Scholar, 20Tellam J.T. Macaulay S.L. McIntosh S. Hewish D.R. Ward C.W. James D.E. J. Biol. Chem. 1997; 272: 6179-6186Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). The insulin-regulated aminopeptidase (IRAP) polyclonal antibody was kindly provided by Dr. Susanna Keller. Clathrin monoclonal antibody X22 was a kind gift of Dr. Frances Brodsky. Phalloidin-FITC and all the fluorophore-tagged secondary antibodies were obtained from Molecular Probes (Eugene, Oregon). Cells cultured on coverslips in 6- or 12-well dishes were starved for 2 h in DMEM/0.2%BSA or Krebs Ringer Phosphate buffer (KPP)/0.2% BSA buffers and incubated with the appropriate treatments. Cells were fixed in acetone for 5 min, washed with PBS, washed again with PBS/0.15 mglycine, and incubated with 1% BSA/PBS for 30 min. Cells were incubated in primary antibodies diluted in PBS/1% BSA for 1 h. Cells were then washed with PBS/0.1% BSA and incubated for 30 min with phalloidin-FITC or the corresponding FITC or Texas Red-conjugated secondary antibody diluted in PBS/1% BSA. Normal rabbit serum was used as a negative control. The coverslips were washed with PBS, mounted onto glass microscope slides, and viewed using a X63/1.4 Zeiss oil immersion objective on a Zeiss Axiovert fluorescence microscope equipped with a Bio-Rad MRC-600 laser confocal imaging system. In experiments designed for staining of cytoskeletal structures, cells were fixed and permeabilized simultaneously in cytoskeleton-stabilizing buffer CSB (10 mm PIPES pH 6.9, 100 mm NaCl, 300 mm sucrose, 3 mm MgCl2, 1 mm as described in Ref. C. R. U. S. C. J. Biol. 1997; PubMed Scopus Google membrane were from basal and adipocytes as described L.J. S. J. James D.E. J. Cell Biol. PubMed Scopus Google Scholar, L.J. James D.E. Am. J. Physiol. Google Scholar). adipocytes were on glass coverslips and using a to a of plasma membrane that to the These were then with the corresponding antibodies as in the were and using a confocal laser 3T3-L1 fibroblasts or differentiated adipocytes on dishes were incubated with for 30 min at 4 °C in DMEM containing BSA as described R.G. J. Cell Biol. 1994; PubMed Scopus Google Scholar). They were then and for cells were fixed in containing 1 mg/ml The cell was in a of in were to the culture from the of the In of containing the cells were with fresh between and after were to the culture of plasma membrane by electron fibroblasts were cultured on in a After differentiating for were to a well of a containing of were fixed with on the of the and the was then for using a with a 5 mm the at 1 the to The plasma membrane were then with polyclonal antibodies against and protein 5 by and staining as described elsewhere A. J. J. R.G. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). We have described a that we to as the plasma membrane for the of GLUT4 to the plasma membrane L.J. S. J. James D.E. J. Cell Biol. PubMed Scopus Google Scholar). highly plasma membrane termed plasma membrane are by of whole cells to glass of these with antibodies specific for GLUT4 in the of insulin, with insulin is a increase in The GLUT4 the plane of the membrane but in many large structures that we have in the not been to These structures are shown in from 3T3-L1 adipocytes were with antibodies to GLUT4 1 or 1 and for proteins a of the ring-like structures in many plasma membrane that both proteins such as caveolin and non-raft proteins such as GLUT4 James D.E. 1997; PubMed Scopus Google Scholar) are present in these caveolin and GLUT4 were both found the basal surface of the than that in rings and in a more plane not We then the of GLUT4 in cells or using of confocal the basal of the In the of insulin, GLUT4 was found in intracellular structures but after insulin was a increase in the cell surface GLUT4 and in of the rings 1 The that these ring-like structures to the plasma membrane during of plasma membrane that they were we adipocytes with cholera toxin subunit at 4 these the the cell surface and is not shown in the structures were by cholera toxin and with the caveolae We also the relationship of the rings to the cortical actin The structures were surrounded by of We other plasma membrane markers are associated with the membrane were from adipocytes and for We first with antibodies to the shown in 3 a plane to the the surface with a of a plane the staining of the characteristic in the of plasma membrane that the membrane of the cell is also evident in presumably of the plasma membrane the after We other plasma membrane markers are present in the shown in and the insulin-regulated was evident the caves of and the the glucose transporter GLUT1, and the proteins 4 and were all evident the rings of basal Thus, markers of caveolae, clathrin-coated pits and other surface markers GLUT1, and are all associated with the further the surface of differentiated 3T3-L1 adipocytes, we used an cholera toxin and electron microscopy. Cells were with at 4 °C and then for and in were to the culture or were for to the culture of the surface and of associated caveolae 4 both single caveolae as well as the characteristic of caveolae around a spherical surface termed which have been well characterized in both 3T3-L1 cells (9Novikoff A.B. Novikoff P.M. Rosen O.M. Rubin C.S. J. Cell Biol. 1980; 87: 180-196Crossref PubMed Scopus (262) Google Scholar) and other cell (11Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (452) Google Scholar). in other cell (11Parton R.G. J. Histochem. Cytochem. 1994; 42: 155-166Crossref PubMed Scopus (452) Google Scholar), clathrin-coated pits were also associated with these structures in adipocytes not that they are not simply an caveolar to the also long invaginations of the surface of caveolae to the cell surface 4 These surface-connected invaginations appear to for the presence of small of caveolae the cell A of is shown in in which a caveolae can be observed an area of cytoplasm surrounded by the a cell surface marker these structures be to be caveolae, or L. J. A. Cell Biol. 2001; 3: PubMed Scopus Google Scholar). The in these studies was the presence of structures the cell from cell to these structures were in to the culture after and cells were at 4 these structures are surface-connected and with the structures with and seen by light microscopy. is evident in at these structures be for lipid to large electron-lucent and in the cell the of these structures with as well as the presence of associated caveolae that these structures are We also used a surface the plasma membrane marker also of the surface-connected structures in differentiated adipocytes. these structures in 3T3-L1 adipocytes not that they are specific to the differentiated We termed these structures to presence in the differentiated cells and surface of adipocyte caves. 3T3-L1 adipocytes were and as described in the to 4 and or were to the culture The large caves are micrometers in and the The caves numerous caveolae as shown at in The in that are large invaginations of the basal surface to the culture caves. of adipocyte caves. 3T3-L1 adipocytes were as described in to were by from the of the are The first as shown in contain numerous caveolae with In and show large surface-connected with and with of caveolae. and to the of the and to lipid the which between of the caves. of adipocyte caves using 3T3-L1 adipocytes were fixed in the presence of Caves are with which the surface-connected domain and associated caveolae area in A shown at area in at in that are both caveolae and non-caveolar the lipid surface the caves were found to the and lipid in the area of the cell of the caves to be of both caveolar membrane and non-caveolar membrane and The density of caveolae associated with the membrane was but in the as that observed at the plasma pits were also associated with the caves 5 as well as on the we represent actin A We used to further the relationship of the caves to the basal plasma In we observed large invaginations at the of the cell that the that the caves are pits in the basal plasma We also of a of from the of the cell for The first as that the basal cell surface was in caveolae structures were in and in and that lipid and were in the plane as the that the caves from the of the cell into the with of that the caves are large in the plane to but in the plane or membrane have been characterized by light microscopy. of the seen by light we to the structures seen in plasma membrane at the by electron microscopy. 3T3-L1 adipocytes were cultured on and then to plasma membrane to the The were then using antibodies to shown in caves were with as or structures the of the with anti-caveolin and clathrin-coated pits by characteristic were abundant around many of the caves caves to have been to the plane of the plasma membrane and evident of caveolae or pits with of these that of markers caves simply the of plasma membrane with the caves after The abundance of the rings was cells in the and we were to significant between abundance and other such as density of lipid cell and insulin on we have that the of these structures to the plasma membrane area may be as as in cells and these structures may play an role in or the cell surface. This study the first of a novel plasma membrane specialization in differentiated 3T3-L1 cells and has It has been that adipocytes a novel signal-transduction pathway in insulin action that is to surface-connected caveolae (15Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (556) Google Scholar, 16Watson R.T. Shigematsu S. Chiang S.H. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 1-13Crossref Scopus (147) Google Scholar, 17Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). the structures in which caveolin and the signaling proteins are to the caves are the structures observed in plasma membrane they have than the plasma membrane it can appear that proteins in these by light microscopy. of such as with caveolin in the ring-like structures has been as of caveolae (15Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Crossref PubMed Scopus (556) Google Scholar). in of the here it is to at the such proteins are enriched in these as with the bulk plasma It is that is to show caveolae on the or caves as we are not simply of caveolae but represent large plasma membrane with associated caveolae. a well characterized membrane protein that not show raft James D.E. 1997; PubMed Scopus Google Scholar), is also associated with These also appear to as for We have observed the presence of in these structures and at after insulin we have observed the of GLUT4 in both caves and the of the membrane not In we have shown the of clathrin-coated pits and with caves. of plasma membrane by electron as described that in the which from the plane of the membrane, may associated with the in other they may be presumably during the This on we observe large in the by microscopy not In the membrane is it may appear that marker molecules are enriched in these structures with the plasma membrane by light microscopy due to This is well for in which the abundance of marker in the of the the caves on the plane is Thus, further is to caveolae, or raft of specific signaling The caves that we have described here appear to form during the of adipocyte differentiation, because we were to in 3T3-L1 preadipocytes. caves were not observed in all adipocytes in the culture that they may have a rather specialized study be to the and of these structures in adipocytes. The caves represent of plasma membrane from the was the presence of filamentous which we the of each for a of molecules in of the plasma membrane, or of these the of the caves is After of adipocytes, structures be detected but in a of cells not It also to be caves are structures that are of actin at the of two structures be with although previous studies that caveolae are not by actin (17Kanzaki M. Pessin J.E. J. Biol. Chem. 2002; 277: 25867-25869Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). In we have shown that in a model adipocyte system large are to the Rather than simply of caveolae, these have the of a plasma membrane they are of caveolae, pits, raft and non-raft and have an associated cortical actin We for the 3T3-L1 cells and and for of the experiments and for on the The for Molecular is a of the