- Email: [email protected]
Direct targeting of proteins to lipid droplets demonstrated by time-lapse live cell imaging
Journal of Bioscience and Bioengineering VOL. 116 No. 5, 620e623, 2013 www.elsevier.com/locate/jbiosc
NOTE
Direct targeting of proteins to lipid droplets demonstrated by time-lapse live cell imaging Torahiko Tanaka,1, * Kazumichi Kuroda,2 Masanori Ikeda,3 Nobuyuki Kato,3 Kazufumi Shimizu,4 and Makoto Makishima1 Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan,1 Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan,2 Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan,3 and Division and Department of Obstetrics and Gynecology, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan4 Received 19 March 2013; accepted 2 May 2013 Available online 4 June 2013
A protein that specifically targets lipid droplets (LDs) was created by connecting two domains of nonstructural protein 4B containing amphipathic helices from hepatitis C virus. We demonstrated its direct targeting and accumulation to the LD surface by time-lapse live cell imaging, comparable to those observed with adipose differentiation-related protein. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Adipose differentiation-related protein; Hepatitis C virus; Lipid droplets; Nonstructural protein 4B; Time-lapse imaging]
Lipid droplets (LDs) are the organelles regulating storage and dynamics of lipids (1,2). The processes involving LDs are also linked to the progression of metabolic diseases. LDs store neutral lipids mainly triglycerides and cholesteryl esters in their core. Surrounding membrane of LDs is a phospholipid monolayer, which contains many LD proteins, such as perilipin family proteins (previously named as PAT proteins), lipid synthetic enzymes, signaling proteins, and proteins involved in membrane trafficking and protein degradation (3). LD-targeting sequences or motifs of LD proteins are complex and not universal. Targeting sequences that are currently known include amphipathic helices, hairpin loops, and a variety of rather undefined hydrophobic sequences (1). Lipid anchors are also known for LD targeting (1). LDs are thought to be generated by accumulation of lipids between the two membrane leaflets of endoplasmic reticulum (ER), and then released into cytosol by budding (1,2). Vesicular trafficking to isolated LDs may also be important (4). Proteins can be targeted to LDs by several different manners, such as those via ER or not, and those involving vesicular transport or not (1). LD proteins harboring potential sequences that associate with ER often show dual localization to ER and LDs (5,6). On the other hand, LD proteins that lack sequences for ER localizations are assumed to target LDs directly from cytosol as a major targeting pathway (1). However, such direct targeting pathway has not been experimentally shown. In the current study, we created a new LD-targeting protein with two amphipathic helices and followed its expression by using time-lapse imaging in * Corresponding author. Tel.: þ81 3 3972 8111x2241; fax: þ81 3 3972 8199. E-mail address: [email protected] (T. Tanaka).
living cells for a prolonged duration. We report visualization of the LD proteins targeted directly to LDs. We recently reported that hepatitis C virus (HCV) nonstructural protein 4B (NS4B) targets LDs: this interaction is critical for infectious virion production of HCV (7). NS4B consists of three major domains: the N- and C-terminal cytosolic domains and the central membrane domain integrated into ER membranes (8). Amphipathic helices within the N- and C-terminal domains are the determinant for LD targeting in NS4B (7). These findings led us to hypothesize that a chimeric protein made with the N- (amino acid 1e73) and C-terminal (amino acid 192e261) domains of NS4B (4BNC; Fig. 1A) be a highly specific LD-targeting protein. When Cherry-4BNC, the 4BNC fusion protein with mCherry, was expressed in Oc cells, which were derived from a hepatoma cell line Huh-7 (9), it demonstrated clear ring-shape localizations surrounding LDs, indicating exclusive localizations to LD membranes (Fig. 1B). Such localization patterns were observed in more than 99% of the transfectants. Similarly, 4BNC fused with EGFP (EGFP-4BNC) showed exclusive localizations to the margins of LDs (data not shown). The ring-shape intracellular localizations of 4BNC were also confirmed in non-hepatic cell-line HEK293 (data not shown). We previously showed that hydrophobic residues in the amphipathic helices (43W, 46L, 50W, 57F, 61I, 64L, 242I, 246L, 249L, and 253I in NS4B) are the determinants of LD targeting (7). Alanine substitutions of those residues in Cherry-4BNC abolished its specific LD localizations, further suggesting that these residues are critical for LD targeting of Cherry-4BNC (data not shown). Together, these data confirmed that the Cherry-4BNC (EGFP-4BNC) is a highly specific LD (membrane) marker.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.006
VOL. 116, 2013
NOTE
A
N-terminal 73 74
1
Membrane
621
C-terminal 191 192
261
NS4B 4BNC Cherry4BNC
mCherry
4BNC
B Cherry-4BNC
+ Bodipy
FIG. 1. LD targeting of Cherry-4BNC. (A) Schematic representation of NS4B, 4BNC, and Cherry-4BNC. HCV sequences were obtained from type 1b HCV strain O [HCV-O, DDBJ/EMBL/ GenBank accession number AB191333; (9)] for construction of the proteins. The “N-” and “C-” fragments were amplified by PCR with a high fidelity polymerase PrimeSTAR (Takara Bio, Shiga, Japan) using a template plasmid pON/C5B/KE (9), according to the manufacturer’s protocol. The primer sets used were: for “N”, 50 -TTTAGATCTGCCTCGCACCTCCCTTAC-30 and 50 -CTCCCCCGGGCCCACGTGCCGGCGGTTCCCAGGCAGAGTGGA-30 , with Bgl II site underlined; and for “C”, 50 -CGCCGGCACGTGGGCC-30 and 50 -TTTGAATTCTTAGCATGGCGTGGAGCAGTC-30 , with Eco RI site double-underlined. The two fragments were connected by overlapping PCR, digested with Bgl II and Eco RI, and ligated with Bgl II-Eco RI-digested pmCherry-C1 vector (Clontech). (B) Exclusive localization of Cherry-4BNC to LDs. Oc cells (9), which are derived from a hepatoma cell line HuH-7, were transfected with Cherry4BNC as described previously (7). After 24 h of transfection, the cells were fixed, stained with Bodipy 493/503 and Hoechst 33342 (for nuclei), and observed with a confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan). Scale bars: 10 mm.
We next examined a potential mechanism by which 4BNC targets LDs using time-lapse confocal imaging (Fig. 2 and Supplementary Movie S1). Oc cells were transfected with Cherry4BNC followed by staining with Bodipy 493/503, and observed continuously on a confocal microscope. The observation was started before Cherry-4BNC became visible in order to clarify the initial stages of LD targeting. Images for the early stages of expression are shown in Fig. 2. A movie that covers about initial 7 h of observation period is also shown as Supplementary Movie S1. The fluorescence of Cherry-4BNC became visible at about 4e5 h post-transfection (1e2 h after the observation was started) and was gradually intensified around LDs. The fluorescence then reached a near plateau level of expression at around 9e10 h post-transfection. There were considerable differences in the increasing rates and the final levels of the fluorescence among transfectants. In some cells, Cherry-4BNC overflowed in cytosol after the accumulation was saturated around LDs (data not shown). Though LDs moved vigorously, they seemed rather stable: emerging, enlarging, or fusing LDs were not observed. Hence, we primarily detected the accumulation of Cherry-4BNC in the surface of mature, isolated LDs. LD targeting seemed not to be dependent on the sizes and locations of cytosolic LDs. Targeting to LDs that were observed in the nuclear region seemed slightly delayed (Supplementary Movie S1). Importantly, Cherry-4BNC solely localized to the surface of LDs throughout the observation period: the fluorescence of Bodipy and that of surrounding Cherry were observed at all locations and time points, although the ring-shape localizations were unclear for some LDs at slightly out-of-focus positions (Fig. 2 and Supplementary Movie S1). Also, we did not detect any retention or pool of the protein at sites other than LDs. Therefore, these results suggest that LD targeting by 4BNC occur in a direct fashion. The time-laps imaging of the transfected 4BNC also indicated that combination of the two amphipathic helices conferred a strong LDtargeting activity onto proteins, which can be used as a model protein to target LDs.
Similar time-lapse imaging was performed for adipose differentiation-related protein (ADRP), one of the most abundant cellular LD proteins, fused with mCherry (Cherry-ADRP) (7) (Fig. S1 and Supplementary Movie S2). The determinant of LD-targeting for ADRP is not thought to be a contiguous, indispensable single element, but rather assumed as three or more diffused elements (10). Cherry-ADRP also accumulated on the surface of LDs with increasing time without retention or pool at sites other than LDs, suggesting direct LD targeting of ADRP. The fluorescence began to overflow into cytosol, after its signal intensity at the LD surface reached a substantial level (Supplementary Movie S2). Even in such stages, LD targeting of Cherry-ADRP continued. Reticular staining patterns that are suggestive of ER localizations were not observed throughout the observation periods. These results also suggested a direct fashion of targeting. In the present study, we demonstrated a simple, direct targeting pathway of two distinct LD-targeting proteins. Since both proteins heavily accumulated on LDs, although their absolute amounts were not determined, surface of pre-existing LDs appear to have a large capacity to accept LD proteins. Such free space on the LD surface may enable acute responses by association and dissociation of functional LD proteins upon stimuli. At present, mechanisms for such specific targeting of LD proteins are unknown. In particular, it is of interest that although both amphipathic helices of NS4B reportedly have membrane-association activity, presumably to ER membranes (11,12), a chimeric protein of the two helices exerted no ER association but an exclusive LD-targeting activity. If targeting simply depends on hydrophobicity of the determinants in LD proteins, they could also target other membrane structures in cells: which was not the case in our live image observation of the transfected 4BNC. Thus, yet unknown mechanisms must contribute to a tight regulation of LD protein localization. Typically, targeting sequences or regions of LD proteins, such as amphipathic helices and hairpin loops, would fit physicochemically to the environments at the LD surface including phospholipid monolayers and core
622
TANAKA ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. Time-lapse imaging following the expression of Cherry-4BNC in living cells. Oc cells (2.5 105) were seeded on a glass-base dish (35 mm diameter, Iwaki, Japan). After 16 h, the medium was changed into a phenol red-free MEM (Invitrogen) supplemented with 10% FBS, and the cells were transfected with 10 mg of plasmid with the aid of PLUS reagent (Invitrogen, 5 ml) and Lipofectamine LTX (Invitrogen, 25 ml) according to the manufacturer’s protocol followed by the addition of Bodipy 493/503. The dish was then set on the X, Y, and Z axis-controlled electrical stage of FV1000 encased in a Perspex box in which temperature was maintained at 37 C in 100% humidity and 5% CO2 atmosphere. Starting at 3 h post-transfection, fluorescent and Nomarski differential interference contrast images were acquired at about 16 min intervals. The obtained images were processed by Fluoview software (Olympus). The panels represent three time frames in an early stage (time 0 corresponds to 3 h post-transfection). Scale bars: 10 mm. Inset, magnified image.
lipids underneath of the monolayers. However, presence of specific proteinous receptor(s) on the surface of LDs is not clarified except for few examples (1). If such molecules are required for ADRP or amphipathic helices in NS4B, a substantial amount of the putative receptors must be present on the LD surface, to support heavy localization of the LD proteins as our observation suggested. If, on the other hand, such receptors do not exist, transfected LD proteins might be self-associating to account for their heavy accumulation to LDs. When an LD protein has such propensity, the protein would target LDs to a large extent till saturated and stay on the surface of LDs by interacting each other. We showed that 4BNC can be used as an LD membrane marker. In addition, 4BNC could also be applied for a wide variety of studies on LDs and lipid dynamics in vitro and in vivo, by fusing with various proteins, for example, enzymes involved in lipid metabolisms. Cherry-ADRP may also be used as an LD membrane marker unless it does not affect on the experiments as a functional ADRP protein. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.006. Technical assistance by K. Toyosawa is gratefully acknowledged. We thank Drs. M. Moriyama and T. Hayashida for valuable comments. This work was supported by Strategic Research Base Development Program for Private Universities subsidized by the Ministry of Education, Culture, Sports, Science and
Technology, Japan (since 2010), the Nihon University Multidisciplinary Research Grant (2010e2011), and grants-in-aid for research on hepatitis from the Ministry of Health, Labour and Welfare of Japan.
References 1. Walther, T. C. and Farese, R. V., Jr.: Lipid droplets and cellular lipid metabolism, Annu. Rev. Biochem., 81, 687e714 (2012). 2. Ohsaki, Y., Cheng, J., Suzuki, M., Shinohara, Y., Fujita, A., and Fujimoto, T.: Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure, Biochim. Biophys. Acta, 1791, 399e407 (2009). 3. Yang, L., Ding, Y., Chen, Y., Zhang, S., Huo, C., Wang, Y., Yu, J., Zhang, P., Na, H., Zhang, H., Ma, Y., and Liu, P.: The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans, J. Lipid Res., 53, 1245e1253 (2012). 4. Hashimoto, T., Segawa, H., Okuno, M., Kano, H., Hamaguchi, H. O., Haraguchi, T., Hiraoka, Y., Hasui, S., Yamaguchi, T., Hirose, F., and Osumi, T.: Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes, J. Cell Sci., 125, 6127e6136 (2012). 5. Zehmer, J. K., Bartz, R., Bisel, B., Liu, P., Seemann, J., and Anderson, R. G.: Targeting sequences of UBXD8 and AAM-B reveal that the ER has a direct role in the emergence and regression of lipid droplets, J. Cell Sci., 122, 3694e3702 (2009). 6. Horiguchi, Y., Araki, M., and Motojima, K.: Identification and characterization of the ER/lipid droplet-targeting sequence in 17beta-hydroxysteroid dehydrogenase type 11, Arch. Biochem. Biophys., 479, 121e130 (2008). 7. Tanaka, T., Kuroda, K., Ikeda, M., Wakita, T., Kato, N., and Makishima, M.: Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices, J. Lipid Res., 54, 881e892 (2013).
VOL. 116, 2013 8. Gouttenoire, J., Penin, F., and Moradpour, D.: Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory, Rev. Med. Virol., 20, 117e129 (2010). 9. Ikeda, M., Abe, K., Dansako, H., Nakamura, T., Naka, K., and Kato, N.: Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system, Biochem. Biophys. Res. Commun., 329, 1350e1359 (2005). 10. Targett-Adams, P., Chambers, D., Gledhill, S., Hope, R. G., Coy, J. F., Girod, A., and McLauchlan, J.: Live cell analysis and targeting of the lipid droplet-binding
NOTE
623
adipocyte differentiation-related protein, J. Biol. Chem., 278, 15998e16007 (2003). 11. Gouttenoire, J., Montserret, R., Kennel, A., Penin, F., and Moradpour, D.: An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association, J. Virol., 83, 11378e11384 (2009). 12. Gouttenoire, J., Castet, V., Montserret, R., Arora, N., Raussens, V., Ruysschaert, J. M., Diesis, E., Blum, H. E., Penin, F., and Moradpour, D.: Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B, J. Virol., 83, 6257e6268 (2009).
NOTE
Direct targeting of proteins to lipid droplets demonstrated by time-lapse live cell imaging Torahiko Tanaka,1, * Kazumichi Kuroda,2 Masanori Ikeda,3 Nobuyuki Kato,3 Kazufumi Shimizu,4 and Makoto Makishima1 Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan,1 Division of Microbiology, Department of Pathology and Microbiology, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan,2 Department of Tumor Virology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, 2-5-1 Shikata-cho, Okayama 700-8558, Japan,3 and Division and Department of Obstetrics and Gynecology, Nihon University School of Medicine, 30-1 Oyaguchi-kamimachi, Itabashi-ku, Tokyo 173-8610, Japan4 Received 19 March 2013; accepted 2 May 2013 Available online 4 June 2013
A protein that specifically targets lipid droplets (LDs) was created by connecting two domains of nonstructural protein 4B containing amphipathic helices from hepatitis C virus. We demonstrated its direct targeting and accumulation to the LD surface by time-lapse live cell imaging, comparable to those observed with adipose differentiation-related protein. Ó 2013, The Society for Biotechnology, Japan. All rights reserved. [Key words: Adipose differentiation-related protein; Hepatitis C virus; Lipid droplets; Nonstructural protein 4B; Time-lapse imaging]
Lipid droplets (LDs) are the organelles regulating storage and dynamics of lipids (1,2). The processes involving LDs are also linked to the progression of metabolic diseases. LDs store neutral lipids mainly triglycerides and cholesteryl esters in their core. Surrounding membrane of LDs is a phospholipid monolayer, which contains many LD proteins, such as perilipin family proteins (previously named as PAT proteins), lipid synthetic enzymes, signaling proteins, and proteins involved in membrane trafficking and protein degradation (3). LD-targeting sequences or motifs of LD proteins are complex and not universal. Targeting sequences that are currently known include amphipathic helices, hairpin loops, and a variety of rather undefined hydrophobic sequences (1). Lipid anchors are also known for LD targeting (1). LDs are thought to be generated by accumulation of lipids between the two membrane leaflets of endoplasmic reticulum (ER), and then released into cytosol by budding (1,2). Vesicular trafficking to isolated LDs may also be important (4). Proteins can be targeted to LDs by several different manners, such as those via ER or not, and those involving vesicular transport or not (1). LD proteins harboring potential sequences that associate with ER often show dual localization to ER and LDs (5,6). On the other hand, LD proteins that lack sequences for ER localizations are assumed to target LDs directly from cytosol as a major targeting pathway (1). However, such direct targeting pathway has not been experimentally shown. In the current study, we created a new LD-targeting protein with two amphipathic helices and followed its expression by using time-lapse imaging in * Corresponding author. Tel.: þ81 3 3972 8111x2241; fax: þ81 3 3972 8199. E-mail address: [email protected] (T. Tanaka).
living cells for a prolonged duration. We report visualization of the LD proteins targeted directly to LDs. We recently reported that hepatitis C virus (HCV) nonstructural protein 4B (NS4B) targets LDs: this interaction is critical for infectious virion production of HCV (7). NS4B consists of three major domains: the N- and C-terminal cytosolic domains and the central membrane domain integrated into ER membranes (8). Amphipathic helices within the N- and C-terminal domains are the determinant for LD targeting in NS4B (7). These findings led us to hypothesize that a chimeric protein made with the N- (amino acid 1e73) and C-terminal (amino acid 192e261) domains of NS4B (4BNC; Fig. 1A) be a highly specific LD-targeting protein. When Cherry-4BNC, the 4BNC fusion protein with mCherry, was expressed in Oc cells, which were derived from a hepatoma cell line Huh-7 (9), it demonstrated clear ring-shape localizations surrounding LDs, indicating exclusive localizations to LD membranes (Fig. 1B). Such localization patterns were observed in more than 99% of the transfectants. Similarly, 4BNC fused with EGFP (EGFP-4BNC) showed exclusive localizations to the margins of LDs (data not shown). The ring-shape intracellular localizations of 4BNC were also confirmed in non-hepatic cell-line HEK293 (data not shown). We previously showed that hydrophobic residues in the amphipathic helices (43W, 46L, 50W, 57F, 61I, 64L, 242I, 246L, 249L, and 253I in NS4B) are the determinants of LD targeting (7). Alanine substitutions of those residues in Cherry-4BNC abolished its specific LD localizations, further suggesting that these residues are critical for LD targeting of Cherry-4BNC (data not shown). Together, these data confirmed that the Cherry-4BNC (EGFP-4BNC) is a highly specific LD (membrane) marker.
1389-1723/$ e see front matter Ó 2013, The Society for Biotechnology, Japan. All rights reserved. http://dx.doi.org/10.1016/j.jbiosc.2013.05.006
VOL. 116, 2013
NOTE
A
N-terminal 73 74
1
Membrane
621
C-terminal 191 192
261
NS4B 4BNC Cherry4BNC
mCherry
4BNC
B Cherry-4BNC
+ Bodipy
FIG. 1. LD targeting of Cherry-4BNC. (A) Schematic representation of NS4B, 4BNC, and Cherry-4BNC. HCV sequences were obtained from type 1b HCV strain O [HCV-O, DDBJ/EMBL/ GenBank accession number AB191333; (9)] for construction of the proteins. The “N-” and “C-” fragments were amplified by PCR with a high fidelity polymerase PrimeSTAR (Takara Bio, Shiga, Japan) using a template plasmid pON/C5B/KE (9), according to the manufacturer’s protocol. The primer sets used were: for “N”, 50 -TTTAGATCTGCCTCGCACCTCCCTTAC-30 and 50 -CTCCCCCGGGCCCACGTGCCGGCGGTTCCCAGGCAGAGTGGA-30 , with Bgl II site underlined; and for “C”, 50 -CGCCGGCACGTGGGCC-30 and 50 -TTTGAATTCTTAGCATGGCGTGGAGCAGTC-30 , with Eco RI site double-underlined. The two fragments were connected by overlapping PCR, digested with Bgl II and Eco RI, and ligated with Bgl II-Eco RI-digested pmCherry-C1 vector (Clontech). (B) Exclusive localization of Cherry-4BNC to LDs. Oc cells (9), which are derived from a hepatoma cell line HuH-7, were transfected with Cherry4BNC as described previously (7). After 24 h of transfection, the cells were fixed, stained with Bodipy 493/503 and Hoechst 33342 (for nuclei), and observed with a confocal laser scanning microscope FV1000 (Olympus, Tokyo, Japan). Scale bars: 10 mm.
We next examined a potential mechanism by which 4BNC targets LDs using time-lapse confocal imaging (Fig. 2 and Supplementary Movie S1). Oc cells were transfected with Cherry4BNC followed by staining with Bodipy 493/503, and observed continuously on a confocal microscope. The observation was started before Cherry-4BNC became visible in order to clarify the initial stages of LD targeting. Images for the early stages of expression are shown in Fig. 2. A movie that covers about initial 7 h of observation period is also shown as Supplementary Movie S1. The fluorescence of Cherry-4BNC became visible at about 4e5 h post-transfection (1e2 h after the observation was started) and was gradually intensified around LDs. The fluorescence then reached a near plateau level of expression at around 9e10 h post-transfection. There were considerable differences in the increasing rates and the final levels of the fluorescence among transfectants. In some cells, Cherry-4BNC overflowed in cytosol after the accumulation was saturated around LDs (data not shown). Though LDs moved vigorously, they seemed rather stable: emerging, enlarging, or fusing LDs were not observed. Hence, we primarily detected the accumulation of Cherry-4BNC in the surface of mature, isolated LDs. LD targeting seemed not to be dependent on the sizes and locations of cytosolic LDs. Targeting to LDs that were observed in the nuclear region seemed slightly delayed (Supplementary Movie S1). Importantly, Cherry-4BNC solely localized to the surface of LDs throughout the observation period: the fluorescence of Bodipy and that of surrounding Cherry were observed at all locations and time points, although the ring-shape localizations were unclear for some LDs at slightly out-of-focus positions (Fig. 2 and Supplementary Movie S1). Also, we did not detect any retention or pool of the protein at sites other than LDs. Therefore, these results suggest that LD targeting by 4BNC occur in a direct fashion. The time-laps imaging of the transfected 4BNC also indicated that combination of the two amphipathic helices conferred a strong LDtargeting activity onto proteins, which can be used as a model protein to target LDs.
Similar time-lapse imaging was performed for adipose differentiation-related protein (ADRP), one of the most abundant cellular LD proteins, fused with mCherry (Cherry-ADRP) (7) (Fig. S1 and Supplementary Movie S2). The determinant of LD-targeting for ADRP is not thought to be a contiguous, indispensable single element, but rather assumed as three or more diffused elements (10). Cherry-ADRP also accumulated on the surface of LDs with increasing time without retention or pool at sites other than LDs, suggesting direct LD targeting of ADRP. The fluorescence began to overflow into cytosol, after its signal intensity at the LD surface reached a substantial level (Supplementary Movie S2). Even in such stages, LD targeting of Cherry-ADRP continued. Reticular staining patterns that are suggestive of ER localizations were not observed throughout the observation periods. These results also suggested a direct fashion of targeting. In the present study, we demonstrated a simple, direct targeting pathway of two distinct LD-targeting proteins. Since both proteins heavily accumulated on LDs, although their absolute amounts were not determined, surface of pre-existing LDs appear to have a large capacity to accept LD proteins. Such free space on the LD surface may enable acute responses by association and dissociation of functional LD proteins upon stimuli. At present, mechanisms for such specific targeting of LD proteins are unknown. In particular, it is of interest that although both amphipathic helices of NS4B reportedly have membrane-association activity, presumably to ER membranes (11,12), a chimeric protein of the two helices exerted no ER association but an exclusive LD-targeting activity. If targeting simply depends on hydrophobicity of the determinants in LD proteins, they could also target other membrane structures in cells: which was not the case in our live image observation of the transfected 4BNC. Thus, yet unknown mechanisms must contribute to a tight regulation of LD protein localization. Typically, targeting sequences or regions of LD proteins, such as amphipathic helices and hairpin loops, would fit physicochemically to the environments at the LD surface including phospholipid monolayers and core
622
TANAKA ET AL.
J. BIOSCI. BIOENG.,
FIG. 2. Time-lapse imaging following the expression of Cherry-4BNC in living cells. Oc cells (2.5 105) were seeded on a glass-base dish (35 mm diameter, Iwaki, Japan). After 16 h, the medium was changed into a phenol red-free MEM (Invitrogen) supplemented with 10% FBS, and the cells were transfected with 10 mg of plasmid with the aid of PLUS reagent (Invitrogen, 5 ml) and Lipofectamine LTX (Invitrogen, 25 ml) according to the manufacturer’s protocol followed by the addition of Bodipy 493/503. The dish was then set on the X, Y, and Z axis-controlled electrical stage of FV1000 encased in a Perspex box in which temperature was maintained at 37 C in 100% humidity and 5% CO2 atmosphere. Starting at 3 h post-transfection, fluorescent and Nomarski differential interference contrast images were acquired at about 16 min intervals. The obtained images were processed by Fluoview software (Olympus). The panels represent three time frames in an early stage (time 0 corresponds to 3 h post-transfection). Scale bars: 10 mm. Inset, magnified image.
lipids underneath of the monolayers. However, presence of specific proteinous receptor(s) on the surface of LDs is not clarified except for few examples (1). If such molecules are required for ADRP or amphipathic helices in NS4B, a substantial amount of the putative receptors must be present on the LD surface, to support heavy localization of the LD proteins as our observation suggested. If, on the other hand, such receptors do not exist, transfected LD proteins might be self-associating to account for their heavy accumulation to LDs. When an LD protein has such propensity, the protein would target LDs to a large extent till saturated and stay on the surface of LDs by interacting each other. We showed that 4BNC can be used as an LD membrane marker. In addition, 4BNC could also be applied for a wide variety of studies on LDs and lipid dynamics in vitro and in vivo, by fusing with various proteins, for example, enzymes involved in lipid metabolisms. Cherry-ADRP may also be used as an LD membrane marker unless it does not affect on the experiments as a functional ADRP protein. Supplementary data to this article can be found online at http:// dx.doi.org/10.1016/j.jbiosc.2013.05.006. Technical assistance by K. Toyosawa is gratefully acknowledged. We thank Drs. M. Moriyama and T. Hayashida for valuable comments. This work was supported by Strategic Research Base Development Program for Private Universities subsidized by the Ministry of Education, Culture, Sports, Science and
Technology, Japan (since 2010), the Nihon University Multidisciplinary Research Grant (2010e2011), and grants-in-aid for research on hepatitis from the Ministry of Health, Labour and Welfare of Japan.
References 1. Walther, T. C. and Farese, R. V., Jr.: Lipid droplets and cellular lipid metabolism, Annu. Rev. Biochem., 81, 687e714 (2012). 2. Ohsaki, Y., Cheng, J., Suzuki, M., Shinohara, Y., Fujita, A., and Fujimoto, T.: Biogenesis of cytoplasmic lipid droplets: from the lipid ester globule in the membrane to the visible structure, Biochim. Biophys. Acta, 1791, 399e407 (2009). 3. Yang, L., Ding, Y., Chen, Y., Zhang, S., Huo, C., Wang, Y., Yu, J., Zhang, P., Na, H., Zhang, H., Ma, Y., and Liu, P.: The proteomics of lipid droplets: structure, dynamics, and functions of the organelle conserved from bacteria to humans, J. Lipid Res., 53, 1245e1253 (2012). 4. Hashimoto, T., Segawa, H., Okuno, M., Kano, H., Hamaguchi, H. O., Haraguchi, T., Hiraoka, Y., Hasui, S., Yamaguchi, T., Hirose, F., and Osumi, T.: Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes, J. Cell Sci., 125, 6127e6136 (2012). 5. Zehmer, J. K., Bartz, R., Bisel, B., Liu, P., Seemann, J., and Anderson, R. G.: Targeting sequences of UBXD8 and AAM-B reveal that the ER has a direct role in the emergence and regression of lipid droplets, J. Cell Sci., 122, 3694e3702 (2009). 6. Horiguchi, Y., Araki, M., and Motojima, K.: Identification and characterization of the ER/lipid droplet-targeting sequence in 17beta-hydroxysteroid dehydrogenase type 11, Arch. Biochem. Biophys., 479, 121e130 (2008). 7. Tanaka, T., Kuroda, K., Ikeda, M., Wakita, T., Kato, N., and Makishima, M.: Hepatitis C virus NS4B targets lipid droplets through hydrophobic residues in the amphipathic helices, J. Lipid Res., 54, 881e892 (2013).
VOL. 116, 2013 8. Gouttenoire, J., Penin, F., and Moradpour, D.: Hepatitis C virus nonstructural protein 4B: a journey into unexplored territory, Rev. Med. Virol., 20, 117e129 (2010). 9. Ikeda, M., Abe, K., Dansako, H., Nakamura, T., Naka, K., and Kato, N.: Efficient replication of a full-length hepatitis C virus genome, strain O, in cell culture, and development of a luciferase reporter system, Biochem. Biophys. Res. Commun., 329, 1350e1359 (2005). 10. Targett-Adams, P., Chambers, D., Gledhill, S., Hope, R. G., Coy, J. F., Girod, A., and McLauchlan, J.: Live cell analysis and targeting of the lipid droplet-binding
NOTE
623
adipocyte differentiation-related protein, J. Biol. Chem., 278, 15998e16007 (2003). 11. Gouttenoire, J., Montserret, R., Kennel, A., Penin, F., and Moradpour, D.: An amphipathic alpha-helix at the C terminus of hepatitis C virus nonstructural protein 4B mediates membrane association, J. Virol., 83, 11378e11384 (2009). 12. Gouttenoire, J., Castet, V., Montserret, R., Arora, N., Raussens, V., Ruysschaert, J. M., Diesis, E., Blum, H. E., Penin, F., and Moradpour, D.: Identification of a novel determinant for membrane association in hepatitis C virus nonstructural protein 4B, J. Virol., 83, 6257e6268 (2009).