Plasma Membrane Domains Specialized for Clathrin-mediated Endocytosis in Primary Cells

Abstract

Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells. Here we have analyzed clathrin organization in primary adipose cells isolated from mice, which are nonadherent and fully differentiated. The majority of clathrin on the plasma membrane of these cells (>60%) was found in large clathrin patches that displayed virtually no lateral mobility and persisted for many minutes, and a smaller amount was found in small spots that appeared and disappeared rapidly. Direct visualization of transferrin revealed that it bound onto large arrays of clathrin, internalizing through vesicles that emerge from these domains. High resolution imaging (50 images/s) revealed fluorescence intensity fluctuations consistent with the formation and detachment of coated vesicles from within large patches. These results reveal that large clathrin assemblies are active regions of endocytosis in mammalian cells and highlight the importance of understanding the mechanistic basis for this organization. Clathrin assembly at the plasma membrane is a fundamental process required for endocytosis. In cultured cells, most of the clathrin is localized to large patches that display little lateral mobility. The functional role of these regions is not clear, and it has been thought that they may represent artifacts of cell adhesion of cultured cells. Here we have analyzed clathrin organization in primary adipose cells isolated from mice, which are nonadherent and fully differentiated. The majority of clathrin on the plasma membrane of these cells (>60%) was found in large clathrin patches that displayed virtually no lateral mobility and persisted for many minutes, and a smaller amount was found in small spots that appeared and disappeared rapidly. Direct visualization of transferrin revealed that it bound onto large arrays of clathrin, internalizing through vesicles that emerge from these domains. High resolution imaging (50 images/s) revealed fluorescence intensity fluctuations consistent with the formation and detachment of coated vesicles from within large patches. These results reveal that large clathrin assemblies are active regions of endocytosis in mammalian cells and highlight the importance of understanding the mechanistic basis for this organization. Clathrin assembly at the plasma membrane is required for the endocytosis of many receptors and transmembrane proteins. Insight into the mechanism of clathrin assembly and coated vesicle formation was obtained initially from electron microscopy and biochemical studies. This information formed the basis for a well accepted model in which individual clathrin-coated pits form on the plasma membrane, grow and invaginate by progressive association of coat subunits, and detach to form free, short-lived vesicles enriched in endocytic cargo (1Schmid S.L. McNiven M.A. De Camilli P. Curr. Opin. Cell Biol. 1998; 10: 504-512Crossref PubMed Scopus (354) Google Scholar, 2Sorkin A. Curr. Opin. Cell Biol. 2004; 16: 392-399Crossref PubMed Scopus (166) Google Scholar, 3Robinson M.S. Trends Cell Biol. 2004; 14: 167-174Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar, 4Marsh M. McMahon H.T. Science. 1999; 285: 215-220Crossref PubMed Scopus (475) Google Scholar, 5Kirchhausen T. Annu. Rev. Biochem. 2000; 69: 699-727Crossref PubMed Scopus (492) Google Scholar, 6Brodsky F.M. Chen C.Y. Knuehl C. Towler M.C. Wakeham D.E. Annu. Rev. Cell Dev. Biol. 2001; 17: 517-568Crossref PubMed Scopus (529) Google Scholar). The availability of fluorescent derivatives of clathrin provided the opportunity to test this model in live cells. The first visualization of fluorescent clathrin generated several surprising findings, among which was the perception that clathrin-coated vesicles were produced recurrently from specific sites within the plasma membrane. In addition, many of these sites displayed a gradual loss of fluorescence, not a sudden catastrophic disappearance, suggesting that individual coated pits could form from within an expansive clathrin patch. Results consistent with the existence of large clathrin lattices harboring invaginated structures have also been captured by electron microscopy (4Marsh M. McMahon H.T. Science. 1999; 285: 215-220Crossref PubMed Scopus (475) Google Scholar). The use of total internal reflection fluorescence microscopy (TIRF-M) 3The abbreviations used are: TIRF-M, total internal reflection fluorescence microscopy; GFP, green fluorescent protein.3The abbreviations used are: TIRF-M, total internal reflection fluorescence microscopy; GFP, green fluorescent protein. has provided the additional technical advantage over confocal microscopy of being able to specifically see events occurring within 100 nm of the plasma membrane (7Axelrod D. Traffic. 2001; 2: 764-774Crossref PubMed Scopus (718) Google Scholar, 8Beaumont V. Biochem. Soc. Trans. 2003; 31: 819-823Crossref PubMed Google Scholar). Images derived from TIRF-M of clathrin are consistent among many groups using either dsRed-clathrin or enhanced green fluorescent protein (EGFP)-clathrin, in stable or transiently transfected cells, and also have provided unexpected results. Foremost among these findings is that the vast majority (80%) of the clathrin signal on the plasma membrane is present as a population that is static during the average period of observation, typically 60 s. Only a small population of clathrin (10–20%) is present as small, rapidly disappearing structures, which would be the ones expected to predominate if the earlier model of clathrin-mediated endocytosis were occurring in live cells (9Rappoport J.Z. Benmerah A. Simon S.M. Traffic. 2005; 6: 539-547Crossref PubMed Scopus (42) Google Scholar, 10Rappoport J.Z. Simon S.M. Benmerah A. Traffic. 2004; 5: 327-337Crossref PubMed Scopus (80) Google Scholar, 11Rappoport J.Z. Taha B.W. Lemeer S. Benmerah A. Simon S.M. J. Biol. Chem. 2003; 278: 47357-47360Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Rappoport J.Z. Taha B.W. Simon S.M. Traffic. 2003; 4: 460-467Crossref PubMed Scopus (43) Google Scholar, 13Rappoport J.Z. Simon S.M. J. Cell Sci. 2003; 116: 847-855Crossref PubMed Scopus (144) Google Scholar, 14Moskowitz H.S. Yokoyama C.T. Ryan T.A. Mol. Biol. Cell. 2005; 16: 1769-1776Crossref PubMed Scopus (37) Google Scholar, 15Merrifield C.J. Perrais D. Zenisek D. Cell. 2005; 121: 593-606Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 16Keyel P.A. Watkins S.C. Traub L.M. J. Biol. Chem. 2004; 279: 13190-13204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). Although these observations have been reported repeatedly, the significance of this organization has remained unclear, and the possibility has been suggested that it may represent an artifact of cell adherence in culture (17Ehrlich M. Boll W. Van Oijen A. Hariharan R. Chandran K. Nibert M.L. Kirchhausen T. Cell. 2004; 118: 591-605Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). To better define the physiological significance of these observations, we have analyzed the dynamics of clathrin assembly using three novel approaches. First, we visualized clathrin dynamics in primary adipose cells isolated from mouse epididymal fat. Isolated adipocytes float in culture medium because of the buoyancy conferred by their lipid content, and therefore artifacts due to adherence onto glass or plastic are eliminated. Moreover, this cell type has been used in studies of endocytic trafficking of GLUT4, a transmembrane transporter known to internalize through a clathrin-mediated mechanism (18Lizunov V.A. Matsumoto H. Zimmerberg J. Cushman S.W. Frolov V.A. J. Cell Biol. 2005; 169: 481-489Crossref PubMed Scopus (141) Google Scholar, 19Robinson L.J. Pang S. Harris D.S. Heuser J. James D.E. J. Cell Biol. 1992; 117: 1181-1196Crossref PubMed Scopus (254) Google Scholar). Second, we have used algorithms that incorporate the signal detected throughout the whole adherent surface of the cells, avoiding the potential bias introduced by the selection of single events for analysis. Third, we have developed an imaging platform consisting of a TIRF microscope equipped with two lasers and a highly sensitive, high speed camera that allows the capture of images at rates up to 50 Hz (50 images/s). Our results indicate that apparently static clathrin patches display small subregional intensity changes consistent with the formation of clathrin-coated pits from within these patches. Moreover, simultaneous imaging of transferrin and clathrin demonstrates that ligands are internalized from large clathrin patches. These results suggest a model in which defined regions of the plasma membrane are specialized for the assembly of large clathrin assemblies that functionally support endocytosis. Optical System—Coherent Innova 70C lasers were used (argon ion and an argon-krypton ion) to produce the 488 and 568 nm light, respectively. The combined beams were coupled into a single mode fiber using a KineFLEX fiber coupler (Point Source). A modified Olympus IX81 inverted microscope, TIRF fiber illuminator, and Plan APO 60× objective with a numerical aperture of 1.45 were used. TIRF illumination was introduced through the edge of the objective at an angle set between 65 and 68 degrees, giving a penetration depth of 90–121 nm at 488 nm and 105–141 nm at 568 nm. Light was collimated through the objective and a layer of immersion oil onto the coverslip. The quality of the collimation was set halfway between the best qualities for 488 and 568 nm. Light from the fluorophores was collected and relayed onto a 640 × 448-pixel charge-coupled device camera developed by Lincoln Laboratory (MIT). A Physik Instruments PIFOC was used for fine focus control. The entire microscope was contained in a heated chamber held at 35 °C. Imaging Hardware and Software—The acquisition system uses the following components: Intel L440GX+ server motherboard with dual peripheral component interconnect (PCI) buses; two 800-MHz Pentium III processors with 2 gigabytes of RAM; Instrutech ITC-18 data acquisition system; Bitflow Roadrunner differential camera interface; ATTO UL3D Ultra160 SCSI Raid controller and four Seagate Cheetah 18-gigabyte Ultra 160 SCSI drives (model ST318351LW). The Bitflow Roadrunner uses a 66-mHz PCI slot. The other components use the 33-Mhz PCI bus. The four Seagate Cheetah drives are in a Raid-0 configuration. Two drives are attached to each of the two controllers on the ATTO UL3D. Sustained read and write speeds are over 100 megabytes/s. The acquisition software, called ScratchNG, is a custom multi-threaded Windows application designed for sustained image capture to RAM, a RAID disk, or both. The Roadrunner streams incoming images into a primary double buffer in system memory. Although one buffer is being addressed by the Roadrunner, the image from the other buffer is copied into a secondary buffer. The secondary buffer can vary in size, depending upon the number of images that need to be captured or whether the images are to be transferred directly to the RAID device. The software image capture capability is only limited by the size of the RAID device, which is 72 gigabytes currently. ScratchNG uses the Instrutech ITC-18 to generate camera read pulses, shutter openings, filter wheel changes, and focus changes. Interactive three-dimensional and four-dimensional (3D+time) visualization and co-localization analysis was performed using Data Analysis and Visualization Environment software (21Lifshitz L.M. Collins J. Moore E. Gauch J. Proceedings of the IEEE Workshop on Biomedical Image Analysis. IEEE Computer Society Press, Los Alamitos, CA1994: 166-175Google Scholar). Curve fitting was done using Origin (Origin Labs Corp., Northampton, MA). Fluorescent Probes and Cells—Alexa568-transferrin (Molecular Probes) was applied to the cells at 20 μg/ml. GFP-clathrin represents a N-terminal fusion of GFP with clathrin light chain A, generated and generously provided by J. H. Cell Biol. 1999; PubMed Scopus Google Scholar). In the cells were transfected with of using primary cell epididymal from were and into with and in 2 and The were for at in an with at 100 The were through and the isolated cells and three in and transfected by were in modified medium with and visualized Isolated adipocytes transfected with GFP-clathrin were visualized by and To between the adipocytes and the a was over a of the cell the cells in the between the two GFP-clathrin was in as a the single large lipid the objective was a small adherent could be visualized visualized by TIRF-M the appeared smaller in and because of the illumination of only 100 nm from the surface of the coverslip. Images were at Hz image 2 for a of GFP-clathrin was in regions of size and To the of clathrin within these we used the following First, images were by the fluorescence the cell Second, a average of three in each a was which due to camera a on the data Third, to generate images in which fluorescence pits and could be analyzed from fluorescence, images were by with a small, 160 which the In to be able to the clathrin spots from the on their the was by with a and this from the this we generated a image by the intensity of to and other to the by the image to display in the image with intensity the average The images could be used to the size and intensity of clathrin pits or patches over the entire adherent surface of the could also be used to the average of these regions over as well as their co-localization with cargo To the size of clathrin of images from for four cells were in A small, regions nm in was of total fluorescence was derived from regions smaller nm in from regions nm in and from regions nm in The resolution of the TIRF system was by imaging a fluorescent green three with to the best and to The of the image obtained was nm. single pits would be expected to have a of nm and be visualized in TIRF as regions of we that in primary cells, the majority of clathrin is in assemblies to individual clathrin-coated This of clathrin regions is to that by in either or transiently transfected cell J.Z. Simon S.M. Benmerah A. Traffic. 2004; 5: 327-337Crossref PubMed Scopus (80) Google Scholar, 11Rappoport J.Z. Taha B.W. Lemeer S. Benmerah A. Simon S.M. J. Biol. Chem. 2003; 278: 47357-47360Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13Rappoport J.Z. Simon S.M. J. Cell Sci. 2003; 116: 847-855Crossref PubMed Scopus (144) Google Scholar, 15Merrifield C.J. Perrais D. Zenisek D. Cell. 2005; 121: 593-606Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 16Keyel P.A. Watkins S.C. Traub L.M. J. Biol. Chem. 2004; 279: 13190-13204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The of these regions over was by the following First, the was as a three-dimensional with the TIRF image defined by and and defined by Clathrin were defined as in this three-dimensional Second, for each in the the regions to present because were as were as Third, the total amount of fluorescence in or regions at each was This provided a of clathrin was present in rapidly disappearing regions throughout the whole adherent the was with the of two a of was which for of the A of for the of the of the clathrin regions in the cell are A of the of clathrin regions is in in which the of co-localization of an image displayed in with images displayed in is spots are displayed in and and green represent regions that appeared or disappeared the stable which the loss of co-localization was as the of with green over the whole adherent surface of the using images at over a period these were with the of two and of and for and of the were These are derived from the in because of the that this not for cell changes that would to an of the of clathrin-coated regions was to be A was in cultured cell adipocytes and cells To the size of clathrin from from three cells were in primary cells, a the of nm is which to single pits smaller nm and and nm for and of the total fluorescence, respectively. Analysis of the of in also well with that for primary cells the loss of co-localization over was and and of and for and of the regions were in primary and cultured cells, the majority of clathrin was found in large assemblies that persisted in the for of To whether these regions are static or clathrin with the we analyzed the of of fluorescence following imaging by TIRF-M, the of the cell was in mode for 2 Images were in TIRF mode for 20 additional of the signal appeared in regions that with to that these apparently static regions clathrin with the of the of of fluorescence revealed a of This of may be due to the of a of the of the may be an reflection of the of clathrin in clathrin regions To whether clathrin assemblies are for we GFP-clathrin and only the present within 100 nm of the surface is in TIRF the fluorescence from transferrin bound to receptors at the plasma membrane is high to the fluorescence from transferrin in the buffer to an signal to to the transferrin was and in regions of nm over the entire surface in in the of transferrin in the medium To the between regions of transferrin and of clathrin, we of the clathrin images on transferrin transferrin is in regions the which to large of clathrin assembly The transferrin signal these regions is and at the transferrin signal within the clathrin is also in structures to the clathrin To these observations, we total transferrin fluorescence of total and their as well as the of transferrin of and the clathrin over and Although the total amount of bound transferrin is in the the the of transferrin is the throughout the The in is within the first that transferrin is on regions clathrin is This is consistent with a of transferrin receptors in clathrin-coated regions of the membrane at a minutes, structures transferrin are the clathrin and to be from regions of clathrin of clathrin and transferrin images this process patches of clathrin rapidly upon of transferrin to the medium the of the clathrin patches co-localization of GFP-clathrin and in structures that from the clathrin patches within the cell from the plasma membrane was in images of the cells during this not transferrin was from the it disappeared from clathrin the patches remained present with These results directly that transferrin endocytosis through regions of clathrin These results suggest that clathrin-coated vesicles are generated from within these clathrin fluorescence intensity fluctuations consistent with vesicle formation and detachment within these assemblies be at and To test this possibility we clathrin patches at an resolution of 60 and at a of 50 Hz (50 images/s) for 60 To the possibility that intensity fluctuations be due to fluctuations in signal intensity or we the cells with for by to the and fluctuations in intensity within clathrin patches were in live cells that were from the detected in the A and The fluctuations in live cell patches the process of coated formation from within a large was These results are most consistent with the that individual coated vesicles can large clathrin arrays that form at specific sites on the plasma membrane. we the possibility that sites at the plasma membrane that support the assembly of of many individual coated pits could also for the data the results the existence of sites within the plasma membrane that support the formation of large clathrin assemblies for endocytosis. of clathrin assemblies have been by several groups (9Rappoport J.Z. Benmerah A. Simon S.M. Traffic. 2005; 6: 539-547Crossref PubMed Scopus (42) Google Scholar, 10Rappoport J.Z. Simon S.M. Benmerah A. Traffic. 2004; 5: 327-337Crossref PubMed Scopus (80) Google Scholar, 11Rappoport J.Z. Taha B.W. Lemeer S. Benmerah A. Simon S.M. J. Biol. Chem. 2003; 278: 47357-47360Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 12Rappoport J.Z. Taha B.W. Simon S.M. Traffic. 2003; 4: 460-467Crossref PubMed Scopus (43) Google Scholar, 13Rappoport J.Z. Simon S.M. J. Cell Sci. 2003; 116: 847-855Crossref PubMed Scopus (144) Google Scholar, 14Moskowitz H.S. Yokoyama C.T. Ryan T.A. Mol. Biol. Cell. 2005; 16: 1769-1776Crossref PubMed Scopus (37) Google Scholar, 15Merrifield C.J. Perrais D. Zenisek D. Cell. 2005; 121: 593-606Abstract Full Text Full Text PDF PubMed Scopus (371) Google imaging fluorescent clathrin by TIRF-M, the functional of these assemblies whether these of assemblies in cells or are an due to cell to glass or plastic is not To this we used primary adipocytes in which the buoyancy conferred by the the cells of that the organization of clathrin on the membrane of isolated primary adipocytes is to that reported by in cultured cells J.Z. Simon S.M. Benmerah A. Traffic. 2004; 5: 327-337Crossref PubMed Scopus (80) Google Scholar, 13Rappoport J.Z. Simon S.M. J. Cell Sci. 2003; 116: 847-855Crossref PubMed Scopus (144) Google Scholar, 14Moskowitz H.S. Yokoyama C.T. Ryan T.A. Mol. Biol. Cell. 2005; 16: 1769-1776Crossref PubMed Scopus (37) Google Scholar, 15Merrifield C.J. Perrais D. Zenisek D. Cell. 2005; 121: 593-606Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 16Keyel P.A. Watkins S.C. Traub L.M. J. Biol. Chem. 2004; 279: 13190-13204Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, Cell Biol. 1999; PubMed Scopus Google and is in that a large of clathrin is found within apparently static These results two whether these large apparently static structures are for and the basis by which this organization is and indicate that large assemblies of clathrin support endocytosis. Our results are consistent with of C.J. Perrais D. Zenisek D. Cell. 2005; 121: 593-606Abstract Full Text Full Text PDF PubMed Scopus (371) Google used a transferrin to the endocytic of cell surface clathrin assemblies and the of endocytosis to Our results indicate that of clathrin assemblies for 60 or by transferrin we have been able to the of the as it from these large assemblies into the endocytic Our results that vesicles transferrin from clathrin patches that are not as transferrin is These regions are not because clathrin is being into the from a of clathrin, as by the of fluorescence High resolution imaging of clathrin patches also fluorescence intensity fluctuations of several within these these fluctuations are and consistent with vesicle formation and and fluctuations were the results suggest that clathrin patches to individual coated pits that form intensity and detach small regions of membrane that can rapidly clathrin clathrin patches a of arrays of clathrin as well as regions of coated that rapidly and be at this of this results indicate that clathrin at the plasma membrane in specific regions that are and support endocytosis. clathrin-mediated endocytic events from large assemblies (17Ehrlich M. Boll W. Van Oijen A. Hariharan R. Chandran K. Nibert M.L. Kirchhausen T. Cell. 2004; 118: 591-605Abstract Full Text Full Text PDF PubMed Scopus (662) Google Scholar). In studies using confocal microscopy in which analysis was to clathrin structures smaller that and within the imaging period of the of cargo appeared to the assemblies of These results suggested a model in which clathrin assembly at the plasma membrane on which are by the of cargo to the population of clathrin structures, and for the that TIRF-M not vesicles nm from plasma membrane assembly the analysis of data is not with that reported by (17Ehrlich M. Boll W. Van Oijen A. Hariharan R. Chandran K. Nibert M.L. Kirchhausen T. Cell. 2004; 118: 591-605Abstract Full Text Full Text PDF PubMed Scopus (662) Google with to the of and of this small of clathrin results that large clathrin which the majority of the clathrin are active the importance of of the mechanism for and of The physiological significance of the organization of clathrin into large assemblies is not possibility may be that the of clathrin and assembly with as may organization specific membrane regions enriched for these analysis of the between and the of clathrin assembly may light on this of other of the and groups for and In we the of in the focus during the of of the