Live Cell Analysis and Targeting of the Lipid Droplet-binding Adipocyte Differentiation-related Protein

Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes. Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes. adipocyte differentiation-related protein human ADRP mouse ADRP enhanced green fluorescent protein enhanced yellow fluorescent protein open reading frame phosphate-buffered saline reverse transcriptase hepatitis C virus fluorescent recovery after photobleaching In mammalian cells, lipid droplets serve as storage organelles, consisting primarily of cholesterol ester and triacylglyerols (reviewed in Ref. 1Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Although long considered to be inert structures, there is now increasing evidence that they play active and diverse roles in the life cycle of cells. Recently, they have been implicated in maintenance of intracellular cholesterol balance and transport of lipids through association with caveolin proteins (2Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (274) Google Scholar). Moreover, the lipid stored in droplets is utilized for specialized purposes in certain cell types. For example, steroid hormones in steroidogenic cells are produced from cholesterol ester stored in lipid droplets (3Freeman D.A. Ascoli M. J. Biol. PubMed Google cells from their surface that are derived from lipid droplets J. Biol. PubMed Scopus Google lipid droplets to be the of that are into and into in PubMed Scopus Google Scholar). is also a between human and of lipid droplets that they are of and R. Cell Res. PubMed Scopus Google D.J. J. Biol. PubMed Scopus Google Scholar). been that targeting of the protein to lipid droplets to lipid transport that could with M. R. S. S. Biol. PubMed Scopus Google Scholar). lipid droplets have in and M. S. S. A. PubMed Scopus Google J. Cell PubMed Google R.G. J. J. PubMed Scopus Google R.G. D.J. J. J. Biol. PubMed Scopus Google J. PubMed Scopus Google surface of lipid droplets a that is to fusion with analysis of the properties of surface proteins The most lipid proteins are the a of by from a J. D.J. 2001; PubMed Scopus Google and adipocyte differentiation-related protein also R. Cell Res. PubMed Scopus Google J. Lipid Res. PubMed Google J. J. PubMed Scopus Google S. A. PubMed Scopus Google Scholar). of the to and steroidogenic cells S. A. PubMed Scopus Google and D.A. J. J. J. Biol. Scopus Google in Ref. Biol. PubMed Scopus Google ADRP is in a of J. Lipid Res. PubMed Google Scholar). of a of cells also revealed ADRP as a of lipid droplets R. Cell Res. PubMed Scopus Google J. Lipid Res. PubMed Google Scholar). Moreover, enhanced of ADRP is a for that are by of lipid droplets R. Cell Res. PubMed Scopus Google M. E. M. 2001; PubMed Scopus Google J. PubMed Scopus Google S. R. J. A. Res. PubMed Scopus Google PubMed Scopus Google Scholar). analysis of ADRP into the of proteins that associate with lipid droplets. In ADRP for the of lipid this study, we have green and yellow fluorescent proteins and to the human and mouse ADRP coding sequences and the ability of the fusion proteins to associate with lipid droplets in cells. This system was employed to identify sequences in ADRP that are required for to lipid droplets. ADRP with fluorescent proteins also was to the behavior of lipid droplets in live cells and their interaction with intracellular is a of lipid droplets found in a of cells and is a protein to examine the properties of of lipid droplets and the behavior of these In study, we have employed fusion of human and mouse of ADRP with to of these of the sequences required to to lipid droplets revealed that targeting are the coding region In the of study, we targeting as the through which ADRP is to lipid droplets and a association with the targeting could from the of the proteins to to lipid droplets, through with found that of the lipid and a targeting was between and that to association with lipid droplets also were in the region from to but this of the protein was not of directing the protein to droplets. of the protein do not a targeting sequences between and be with the of lipid that of these targeting sequences but not lipid that there is in the for of sequences not been also within the sequences that were for directing to lipid droplets A. A. J. Biol. PubMed Scopus Google Scholar). Moreover, targeting for membrane proteins have not been and that targeting within such proteins J. Cell Biol. 2001; PubMed Scopus Google Scholar). be a for directing proteins to the surface of organelles such as and lipid been that ADRP be into and with lipid proteins J. D.J. 2001; PubMed Scopus Google Scholar). is considered to of the of the protein and the is between for lipid proteins is that for lipid proteins have been in J. D.J. 2001; PubMed Scopus Google Scholar). analysis that the of which are for are within the sequences that have been as for lipid association are in The region within that been as for to lipid droplets also in A. A. J. Biol. PubMed Scopus Google Scholar). This that the in between lipid proteins such as ADRP and does not targeting the sequences that these proteins to lipid droplets be also been that a region in the of ADRP with a in the membrane Biol. PubMed Scopus Google M. PubMed Scopus Google Scholar). show that this from ADRP lipid association to that with this is not to targeting of discontinuous of the targeting sequences in ADRP from the of lipid proteins from of protein with lipid droplets a of that is also in a but not in and that the as R.G. J. J. PubMed Scopus Google R.G. D.J. J. J. Biol. PubMed Scopus Google Scholar). from a of or of the of the lipid In have a region of that of the sequences required for in mammalian and this region is by that to be for lipid association R.G. D.J. J. J. Biol. PubMed Scopus Google PubMed Scopus Google Scholar). Moreover, the targeting sequences in the and proteins are segments with are found in either human or mouse This that which are from for the and of ADRP to lipid droplets. In the of of the protein to lipid droplets targeting and of a protein at the and is found at the membrane as as at the surface of lipid droplets J. R.G. J. PubMed Scopus Google Scholar). is that of between the organelles is by the of lipid droplets to the which we from live cell for of the is found by either or live cell The that of ADRP is a of between either or of lipid droplets and the that interaction with lipid droplets. In the that ADRP is and not Biol. PubMed Scopus Google Scholar). been demonstrated for the J. Biol. PubMed Scopus Google Scholar). be that association of ADRP and with the is not to fusion of lipid droplets with the after their droplets as in a number of cell types. This that they a in which of between droplets could is to the of GFP-tagged proteins in organelles with such as the and M. M. J. PubMed Scopus Google S. J. E. J. Cell Biol. PubMed Scopus Google J. Cell Biol. PubMed Scopus Google in Ref. Cell Biol. 2001; PubMed Scopus Google Scholar). revealed that of are and J. PubMed Scopus Google Scholar). ADRP does not rapidly diffuse between lipid droplets that are in with This that droplets as and that there is or of surface proteins between a there be of ADRP at the surface of lipid droplets after their of live cells for the of lipid droplets. Lipid droplets are in between cell and even in cells. The that their are not but could be their is also that lipid is after either through from the or from lipid droplets. In HuH-7 cells, lipid droplets were in in live cells as a and with to lipid droplets with their to not that this from fusion of closely droplets and the in cells, which are is not have a the of lipid in cells that of lipid and in conditions where lipid storage is For example, of the be as either or the of lipid droplets A. Google Scholar). is that the of lipid droplets with does not their in and is of the of lipid droplets is considered to by a at the D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google D.A. Biol. 2001; PubMed Scopus Google Scholar). live cell their droplets to to the lipid droplets are a their close association with the be for maintenance and of the the cell association also to a storage conditions where lipid is either or by cells. lipid droplets be to lipid and of the in where lipid is either or in In HuH-7 cells, we that a of lipid droplets that were with transient to the was from the of droplets that were that association of lipid droplets with movement from the to a the that lipid association with are not did not movement in cells. This cell lipid droplets with HuH-7 cells the of lipid droplets that move rapidly in HuH-7 cells is is that are to identify movement of lipid droplets in cells. could be cell as as a of lipid for lipid droplets have specialized in certain cell which their with cellular processes. For example, by cells are derived from lipid droplets from the surface and is that movement of droplets to of PubMed Scopus Google Scholar). In are a for the of and stored in lipid droplets of the lipid in these PubMed Scopus Google Scholar). this is but of lipid droplets to of the where are the cells for the human from which HuH-7 cells are derived and for the that lipid droplets. the association of lipid droplets with in HuH-7 cells not be found in cell we have sequences within ADRP that are for association with lipid droplets and have demonstrated that the protein be utilized to examine the properties of these This analysis of the interaction between lipid droplets and cellular processes. and where association with R. Cell Res. PubMed Scopus Google M. PubMed Scopus Google and M. J. 2001; PubMed Scopus Google have been that lipid droplets are not inert of lipid storage but with the of these to the of the of lipid droplets to cell In mammalian cells, lipid droplets serve as storage organelles, consisting primarily of cholesterol ester and triacylglyerols (reviewed in Ref. 1Murphy D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google Scholar). Although long considered to be inert structures, there is now increasing evidence that they play active and diverse roles in the life cycle of cells. Recently, they have been implicated in maintenance of intracellular cholesterol balance and transport of lipids through association with caveolin proteins (2Pol A. Luetterforst R. Lindsay M. Heino S. Ikonen E. Parton R.G. J. Cell Biol. 2001; 152: 1057-1070Crossref PubMed Scopus (274) Google Scholar). Moreover, the lipid stored in droplets is utilized for specialized purposes in certain cell types. For example, steroid hormones in steroidogenic cells are produced from cholesterol ester stored in lipid droplets (3Freeman D.A. Ascoli M. J. Biol. PubMed Google cells from their surface that are derived from lipid droplets J. Biol. PubMed Scopus Google lipid droplets to be the of that are into and into in PubMed Scopus Google Scholar). is also a between human and of lipid droplets that they are of and R. Cell Res. PubMed Scopus Google D.J. J. Biol. PubMed Scopus Google Scholar). been that targeting of the protein to lipid droplets to lipid transport that could with M. R. S. S. Biol. PubMed Scopus Google Scholar). lipid droplets have in and M. S. S. A. PubMed Scopus Google J. Cell PubMed Google R.G. J. J. PubMed Scopus Google R.G. D.J. J. J. Biol. PubMed Scopus Google J. PubMed Scopus Google Scholar). The surface of lipid droplets a that is to fusion with analysis of the properties of surface proteins The most lipid proteins are the a of by from a J. D.J. 2001; PubMed Scopus Google and adipocyte differentiation-related protein also R. Cell Res. PubMed Scopus Google J. Lipid Res. PubMed Google J. J. PubMed Scopus Google S. A. PubMed Scopus Google Scholar). of the to and steroidogenic cells S. A. PubMed Scopus Google and D.A. J. J. J. Biol. Scopus Google in Ref. Biol. PubMed Scopus Google ADRP is in a of J. Lipid Res. PubMed Google Scholar). of a of cells also revealed ADRP as a of lipid droplets R. Cell Res. PubMed Scopus Google J. Lipid Res. PubMed Google Scholar). Moreover, enhanced of ADRP is a for that are by of lipid droplets R. Cell Res. PubMed Scopus Google M. E. M. 2001; PubMed Scopus Google J. PubMed Scopus Google S. R. J. A. Res. PubMed Scopus Google PubMed Scopus Google Scholar). analysis of ADRP into the of proteins that associate with lipid droplets. In ADRP for the of lipid droplets. In this study, we have green and yellow fluorescent proteins and to the human and mouse ADRP coding sequences and the ability of the fusion proteins to associate with lipid droplets in cells. This system was employed to identify sequences in ADRP that are required for to lipid droplets. ADRP with fluorescent proteins also was to the behavior of lipid droplets in live cells and their interaction with intracellular is a of lipid droplets found in a of cells and is a protein to examine the properties of of lipid droplets and the behavior of these In study, we have employed fusion of human and mouse of ADRP with to of these of the sequences required to to lipid droplets revealed that targeting are the coding region In the of study, we targeting as the through which ADRP is to lipid droplets and a association with the targeting could from the of the proteins to to lipid droplets, through with found that of the lipid and a targeting was between and that to association with lipid droplets also were in the region from to but this of the protein was not of directing the protein to droplets. of the protein do not a targeting sequences between and be with the of lipid that of these targeting sequences but not lipid that there is in the for of sequences not been also within the sequences that were for directing to lipid droplets A. A. J. Biol. PubMed Scopus Google Scholar). Moreover, targeting for membrane proteins have not been and that targeting within such proteins J. Cell Biol. 2001; PubMed Scopus Google Scholar). be a for directing proteins to the surface of organelles such as and lipid been that ADRP be into and with lipid proteins J. D.J. 2001; PubMed Scopus Google Scholar). is considered to of the of the protein and the is between for lipid proteins is that for lipid proteins have been in J. D.J. 2001; PubMed Scopus Google Scholar). analysis that the of which are for are within the sequences that have been as for lipid association are in The region within that been as for to lipid droplets also in A. A. J. Biol. PubMed Scopus Google Scholar). This that the in between lipid proteins such as ADRP and does not targeting the sequences that these proteins to lipid droplets be also been that a region in the of ADRP with a in the membrane Biol. PubMed Scopus Google M. PubMed Scopus Google Scholar). show that this from ADRP lipid association to that with this is not to targeting of discontinuous of the targeting sequences in ADRP from the of lipid proteins from of protein with lipid droplets a of that is also in a but not in and that the as R.G. J. J. PubMed Scopus Google R.G. D.J. J. J. Biol. PubMed Scopus Google Scholar). from a of or of the of the lipid In have a region of that of the sequences required for in mammalian and this region is by that to be for lipid association R.G. D.J. J. J. Biol. PubMed Scopus Google PubMed Scopus Google Scholar). Moreover, the targeting sequences in the and proteins are segments with are found in either human or mouse This that which are from for the and of ADRP to lipid droplets. In the of of the protein to lipid droplets targeting and of a protein at the and is found at the membrane as as at the surface of lipid droplets J. R.G. J. PubMed Scopus Google Scholar). is that of between the organelles is by the of lipid droplets to the which we from live cell for of the is found by either or live cell The that of ADRP is a of between either or of lipid droplets and the that interaction with lipid droplets. In the that ADRP is and not Biol. PubMed Scopus Google Scholar). been demonstrated for the J. Biol. PubMed Scopus Google Scholar). be that association of ADRP and with the is not to fusion of lipid droplets with the after their droplets as in a number of cell types. This that they a in which of between droplets could is to the of GFP-tagged proteins in organelles with such as the and M. M. J. PubMed Scopus Google S. J. E. J. Cell Biol. PubMed Scopus Google J. Cell Biol. PubMed Scopus Google in Ref. Cell Biol. 2001; PubMed Scopus Google Scholar). revealed that of are and J. PubMed Scopus Google Scholar). ADRP does not rapidly diffuse between lipid droplets that are in with This that droplets as and that there is or of surface proteins between a there be of ADRP at the surface of lipid droplets after their of live cells for the of lipid droplets. Lipid droplets are in between cell and even in cells. The that their are not but could be their is also that lipid is after either through from the or from lipid droplets. In HuH-7 cells, lipid droplets were in in live cells as a and with to lipid droplets with their to not that this from fusion of closely droplets and the in cells, which are is not have a the of lipid in cells that of lipid and in conditions where lipid storage is For example, of the be as either or the of lipid droplets A. Google Scholar). is that the of lipid droplets with does not their in and is of the of lipid droplets is considered to by a at the D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google D.A. Biol. 2001; PubMed Scopus Google Scholar). live cell their droplets to to the lipid droplets are a their close association with the be for maintenance and of the the cell association also to a storage conditions where lipid is either or by cells. lipid droplets be to lipid and of the in where lipid is either or in In HuH-7 cells, we that a of lipid droplets that were with transient to the was from the of droplets that were that association of lipid droplets with movement from the to a the that lipid association with are not did not movement in cells. This cell lipid droplets with HuH-7 cells the of lipid droplets that move rapidly in HuH-7 cells is is that are to identify movement of lipid droplets in cells. could be cell as as a of lipid for lipid droplets have specialized in certain cell which their with cellular processes. For example, by cells are derived from lipid droplets from the surface and is that movement of droplets to of PubMed Scopus Google Scholar). In are a for the of and stored in lipid droplets of the lipid in these PubMed Scopus Google Scholar). this is but of lipid droplets to of the where are the cells for the human from which HuH-7 cells are derived and for the that lipid droplets. the association of lipid droplets with in HuH-7 cells not be found in cell we have sequences within ADRP that are for association with lipid droplets and have demonstrated that the protein be utilized to examine the properties of these This analysis of the interaction between lipid droplets and cellular processes. and where association with R. Cell Res. PubMed Scopus Google M. PubMed Scopus Google and M. J. 2001; PubMed Scopus Google have been that lipid droplets are not inert of lipid storage but with the of these to the of the of lipid droplets to cell ADRP is a of lipid droplets found in a of cells and is a protein to examine the properties of of lipid droplets and the behavior of these In study, we have employed fusion of human and mouse of ADRP with to of these of the sequences required to to lipid droplets revealed that targeting are the coding region In the of study, we targeting as the through which ADRP is to lipid droplets and a association with the targeting could from the of the proteins to to lipid droplets, through with found that of the lipid and a targeting was between and that to association with lipid droplets also were in the region from to but this of the protein was not of directing the protein to droplets. of the protein do not a targeting sequences between and be with the of lipid that of these targeting sequences but not lipid that there is in the for of sequences not been also within the sequences that were for directing to lipid droplets A. A. J. Biol. PubMed Scopus Google Scholar). Moreover, targeting for membrane proteins have not been and that targeting within such proteins J. Cell Biol. 2001; PubMed Scopus Google Scholar). be a for directing proteins to the surface of organelles such as and lipid droplets. been that ADRP be into and with lipid proteins J. D.J. 2001; PubMed Scopus Google Scholar). is considered to of the of the protein and the is between for lipid proteins is that for lipid proteins have been in J. D.J. 2001; PubMed Scopus Google Scholar). analysis that the of which are for are within the sequences that have been as for lipid association are in The region within that been as for to lipid droplets also in A. A. J. Biol. PubMed Scopus Google Scholar). This that the in between lipid proteins such as ADRP and does not targeting the sequences that these proteins to lipid droplets be also been that a region in the of ADRP with a in the membrane Biol. PubMed Scopus Google M. PubMed Scopus Google Scholar). show that this from ADRP lipid association to that with this is not to targeting of The discontinuous of the targeting sequences in ADRP from the of lipid proteins from of protein with lipid droplets a of that is also in a but not in and that the as R.G. J. J. PubMed Scopus Google R.G. D.J. J. J. Biol. PubMed Scopus Google Scholar). from a of or of the of the lipid In have a region of that of the sequences required for in mammalian and this region is by that to be for lipid association R.G. D.J. J. J. Biol. PubMed Scopus Google PubMed Scopus Google Scholar). Moreover, the targeting sequences in the and proteins are segments with are found in either human or mouse This that which are from for the and of ADRP to lipid droplets. In the of of the protein to lipid droplets targeting and of a protein at the and is found at the membrane as as at the surface of lipid droplets J. R.G. J. PubMed Scopus Google Scholar). is that of between the organelles is by the of lipid droplets to the which we from live cell for of the is found by either or live cell The that of ADRP is a of between either or of lipid droplets and the that interaction with lipid droplets. In the that ADRP is and not Biol. PubMed Scopus Google Scholar). been demonstrated for the J. Biol. PubMed Scopus Google Scholar). be that association of ADRP and with the is not to fusion of lipid droplets with the after their Lipid droplets as in a number of cell types. This that they a in which of between droplets could is to the of GFP-tagged proteins in organelles with such as the and M. M. J. PubMed Scopus Google S. J. E. J. Cell Biol. PubMed Scopus Google J. Cell Biol. PubMed Scopus Google in Ref. Cell Biol. 2001; PubMed Scopus Google Scholar). revealed that of are and J. PubMed Scopus Google Scholar). ADRP does not rapidly diffuse between lipid droplets that are in with This that droplets as and that there is or of surface proteins between a there be of ADRP at the surface of lipid droplets after their of live cells for the of lipid droplets. Lipid droplets are in between cell and even in cells. The that their are not but could be their is also that lipid is after either through from the or from lipid droplets. In HuH-7 cells, lipid droplets were in in live cells as a and with to lipid droplets with their to not that this from fusion of closely droplets and the in cells, which are is not have a the of lipid in cells that of lipid and in conditions where lipid storage is For example, of the be as either or the of lipid droplets A. Google Scholar). is that the of lipid droplets with does not their in and is of the of lipid droplets is considered to by a at the D.J. Prog. Lipid Res. 2001; 40: 325-438Crossref PubMed Scopus (754) Google D.A. Biol. 2001; PubMed Scopus Google Scholar). live cell their droplets to to the lipid droplets are a their close association with the be for maintenance and of the the cell association also to a storage conditions where lipid is either or by cells. lipid droplets be to lipid and of the in where lipid is either or in In HuH-7 cells, we that a of lipid droplets that were with transient to the was from the of droplets that were that association of lipid droplets with movement from the to a the that lipid association with are not did not movement in cells. This cell lipid droplets with HuH-7 cells the of lipid droplets that move rapidly in HuH-7 cells is is that are to identify movement of lipid droplets in cells. could be cell as as a of lipid for lipid droplets have specialized in certain cell which their with cellular processes. For example, by cells are derived from lipid droplets from the surface and is that movement of droplets to of PubMed Scopus Google Scholar). In are a for the of and stored in lipid droplets of the lipid in these PubMed Scopus Google Scholar). this is but of lipid droplets to of the where are the cells for the human from which HuH-7 cells are derived and for the that lipid droplets. the association of lipid droplets with in HuH-7 cells not be found in cell types. In we have sequences within ADRP that are for association with lipid droplets and have demonstrated that the protein be utilized to examine the properties of these This analysis of the interaction between lipid droplets and cellular processes. and where association with R. Cell Res. PubMed Scopus Google M. PubMed Scopus Google and M. J. 2001; PubMed Scopus Google have been that lipid droplets are not inert of lipid storage but with the of these to the of the of lipid droplets to cell and for and with live cell are to for of HuH-7 cells by cell and for the with with