Communication Cite This: Biochemistry XXXX, XXX, XXX−XXX
pubs.acs.org/biochemistry
Monitoring Lipid Droplet Dynamics in Living Cells by Using Fluorescent Probes Yuki Tatenaka,*,† Hironori Kato,‡ Munetaka Ishiyama,† Kazumi Sasamoto,† Masanobu Shiga,† Hideki Nishitoh,‡ and Yuichiro Ueno† †
Dojindo Laboratories, Tabaru 2025-5, Mashiki-machi, Kumamoto 861-2202, Japan Laboratory of Biochemistry and Molecular Biology, Department of Medical Sciences, University of Miyazaki, 5200 Kihara Kiyotake, Miyazaki 889-1692, Japan
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‡
S Supporting Information *
single marker to measure the entire population of LDs is available. Therefore, the current approach to identify all kinds of LDs is to stain cells with fluorescent dyes that are specific with a strong affinity toward the neutral lipids of LDs.7 In order to monitor the morphological and dynamic interaction of LDs with other organelles, both the long-term observation of the LDs in living cells as well as multicolor imaging by fluorescence microscopy with common filter sets for UV B (470−490 nm) and G (520−550 nm)-excitation will be essential. As a fluorescent probe for LDs, Nile Red and BODIPY 493/503 have mostly been used, as they fluoresce in a hydrophobic environment such as in the core of an LD.8,9 However, one of their drawbacks is the fluorescence background resulting from nonspecific binding to lipophilic sites other than an LD in a cell. Additionally, since the excitation wavelength of Nile Red is largely shifted from 450 to 560 nm by solvatochromism, Nile Red cannot be used for multicolor staining with B- and G-excitation filters.10 Therefore, highly specific and retainable fluorescent probes for LDs need to be developed. Novel fluorescent probes we report here, Lipi-Blue, LipiGreen, and Lipi-Red, are all more specific to LDs, and the fluorescent backgrounds of stained cells are extremely low compared to Nile Red and BODIPY 493/503. The fluorescence properties of Lipi-Blue, Lipi-Green, and LipiRed fit to common filter sets in fluorescence microscopy. Additionally, LDs stained by Lipi-Blue and Lipi-Green can be monitored for over 48 h. Therefore, they can be applied not only for simultaneous monitoring of several different organelles but also for “time-lag staining” of LDs in a single cell, suggesting that these probes are useful to understand the mechanism of cellular events interrelated with LDs. We report herein the fluorescence imaging of LDs in living cells with Lipiprobes and the monitoring of the dynamics of LDs.
ABSTRACT: We have developed three types of lipid droplet (LD)-specific fluorescent probes for live-cell imaging, Lipi-Blue, Lipi-Green, and Lipi-Red, which exhibit fluorescence upon being incorporated into LDs both of living and of fixed cells. These Lipi-probes are LDspecific probes that contain a pyrene or perylene group as a fluorescent scaffold and can be used to observe dynamics of LD in live cells and also interrelations with other organelles by simultaneous staining with multiple organelle-specific probes. Additionally, Lipi-Blue and LipiGreen allow monitoring LDs in live cells even for 48 h after the staining. Here we show that newly formed LDs and previously existed LDs can be separately monitored in a single cell by using these probes and that intercellular transfer of whole LDs is observed in KB cells, but not in HepG2 cells under the same culturing condition. These findings indicate that newly developed LD-specific probes are useful to analyze the dynamics of LDs in live cells.
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or many years, a lipid droplet (LD) has been assumed to be a simple reservoir that stores excess lipids.1 It has been recognized, however, that an LD is also an important organelle that plays indispensable roles in, for example, membrane formation, lipoprotein formation, and intracellular signal transduction in cells.2 An LD is mainly composed of neutral lipids including triacylglycerol and cholesteryl esters, and the surface is covered by a single phospholipid monolayer, on which membrane proteins are presented. An LD is found not only in adipocytes but also ubiquitously in most cells ranging from bacterial cells to mammalian cells.3 The storage and the consumption of fatty acids are tightly regulated, and their failure in this regulation leads to metabolic disorders such as obesity and diabetes.4 Recently, an LD has also been reported to have relevance to autophagy and cellular senescence.5,6 Morphological approaches, therefore, have gained great attention as an important tool to elucidate the mechanisms of formation, growth, fusion, and retraction of LDs. In view of such expanding biology of LDs, a new method useful for the research of LD dynamics is expected to contribute to the diagnosis and/or therapy of related diseases. LD-specific fluorescent dyes are a powerful tool to monitor the morphological and dynamic relations of LDs with other organelles. Since LDs are not homogeneous in contents, no © XXXX American Chemical Society
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MATERIALS Reagents. All chemicals for synthesis were purchased from Tokyo Chemical Industries (Tokyo, Japan), Fuji Film Wako Pure Chemical Corporation (Osaka, Japan), and used as purchased unless stated otherwise. 1H NMR spectra were
Received: October 9, 2018 Revised: December 27, 2018 Published: January 10, 2019 A
DOI: 10.1021/acs.biochem.8b01071 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry recorded on a Bruker AVANCE III HD 400 MHz spectrometer. Chemicals for biological experiments were purchased from Fuji Film Wako Pure Chemical, nacalai tesque (Kyoto, Japan), TAKARA BIO (Shiga, Japan), Dojindo Laboratories (Kumamoto, Japan), Enzo Life Sciences (Farmingdale, NY, USA), and Thermo Fisher Scientific (Waltham, MA, USA) and used by following the manufacturer’s manual. Cell viabilities were measured with the Cell Counting Kit-8 (CCK-8) (Dojindo) following the product manual. Instruments. UV−visible spectra were obtained on a UV2450 UV/vis spectrophotometer (SHIMADZU, Kyoto, Japan), and fluorescence spectroscopic studies were performed on a FP-6300 fluorescence spectrophotometer (JASCO, Tokyo, Japan). Fluorescence images were obtained with a LSM710 and LSM800 confocal laser scanning microscope (ZEISS, Oberkochen, Germany) or a TSC-SP8 (Leica, Wetzler, Germany) confocal laser scanning microscope. Cell viability assays on 96-well plates were performed with an Infinite 200 pro plate reader (TECAN, Maennedorf, Switzerland).
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Table 1. Photophysical Properties of Lipi-Blue, Lipi-Green, and Lipi-Red in THF and Ethanola probe Lipi-Blue
Lipi-Green
Lipi-Red
solvent
λex (nm)
λem (nm)
FI (a.u.)
100% THF 1% THF EtOH 100% THF 1% THF EtOH 100% THF 1% THF EtOH
381 (381) 379 472 (472) 468 520 (520) 525
445 (445) 443 501 (501) 495 650 (650) 682
600.84 12.22b
QY (Φ)
0.23 830.76 0.55b 0.60 105.12 0.17b 0.12
The quantum yields were determined by using fluorescein as a reference. bMeasured by the excitation and emission wavelength indicated in the parentheses. a
The fluorescence of Lipi-Blue, Lipi-Green, and Lipi-Red is hardly observed in water, but is enhanced 49.2, 1,510, and 618 times in THF, respectively. Cytotoxicity of Lipi-Probes. We examined the cytotoxicity of Lipi-probes for HepG2 cells with CCK-8 and found that Lipi-probes were not cytotoxic up to 10 μM, which is 10− 100 times higher than the concentration we used for live-cell imaging experiments (Figure S1). Staining Profile of Lipi-Probes in Mammalian Cells. In order to assess the specificity of Lipi-probes for LDs, we costained cells with a fluorescent-labeled antibody against an adipocyte differentiation-related protein (ADRP). The ADRP is known as one of the most common membrane proteins of LDs presented on the surface.12 HepG2 cells, which contain relatively large amounts of LDs, were fixed with paraformaldehyde and then costained with Alexa Fluor 647-labeled antiADRP antibody and Lipi-probes. We also conducted a double staining experiment with Nile Red or BODIPY 493/503 and Alexa Fluor 647-labeled anti-ADRP antibody. Figure S2 indicates that the staining patterns with Lipi-probes and with anti-ADRP antibody overlap Nile Red and BODIPY 493/503, however, staining other parts of the cells. Therefore, the specificity as an LD-staining dye of Lipi-probes is better than that of BODIPY 493/503 and Nile Red. Inhibition of LD Formation by Monitoring with LipiProbes. We then treated HepG2 cells with Triacsin C (TC) to evaluate whether LD dynamics can be monitored by Lipiprobes. TC, one of the fungal metabolites, is known to block LD formation by inhibiting acyl-CoA synthase activities.13 HepG2 cells were cultured with a 5 μM TC-containing medium for 24 h and then stained with Lipi-probes. The number and the size of fluorescent spots of the HepG2 cells were clearly less and smaller than those of untreated cells (Figure S3). The amount of LDs quantified by an image software, ImageJ,14 indicated that the total fluorescent area of untreated cells was more than triple of that of TC-treated cells (Figure S4). These data also provide additional evidence supporting that Lipi-probes are LD-specific. Imaging of LDs in Brown Adipocytes. Next, we evaluated the performance of Lipi-probes with a brown adipocyte that is known to control energy metabolism of cells and has been reported to have antiobesity effects.15 All Lipi-probes clearly detect LDs, but not other organelles including the nucleus and cytoplasm, in brown adipocytes (Figure 2A−I). The line-scan analysis of Lipi-probes staining clearly show the specific fluorescence signal from the LDs
RESULTS AND DISCUSSION
Synthesis and Spectroscopic Analysis. In designing a molecule that has an affinity to LDs and increases its fluorescence upon binding, we selected pyrene and perylene structures as a fluorogenic scaffold,11 which were expected to enhance their fluorescence in a hydrophobic environment. In order to perform multicolor staining using organelle-specific fluorescent probes and fluorescent-labeled antibodies, we have developed three types of fluorescent probes, Lipi-Blue, LipiGreen, and Lipi-Red for UV B- or G-excitation, respectively, which were named as Lipi-probes. The chemical structures and their emission spectra of Lipi-probes are shown in Figure. 1, and the photophysical properties are shown in Table 1; the syntheses of these probes are described in the Supporting Information.
Figure 1. Chemical structures and fluorescent spectra of Lipi-probes emission spectra of Lipi-probes in 100% THF and 1% THF in water. Concentration of Lipi-probes: 10 μM. Excitation wavelength: (A) Lipi-Blue λex= 381 nm, (B) Lipi-Green λex = 472 nm, (C) Lipi-Red λex = 520 nm. B
DOI: 10.1021/acs.biochem.8b01071 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry
Figure 2. Fluorescence imaging of brown adipocytes stained with Lipi-probes. Mature brown adipocytes were grown on glass-bottom dishes and stained with 0.5 μM Lipi-Blue (A−C), 0.5 μM Lipi-Green (D−F), or 2.5 μM Lipi-Red (G−I) for 30 min at 37 °C. Images of brown adipocytes were taken by confocal laser microscopy (Leica TSC-SP8). (A, D, and G) Images of differential interference contrast. (B, E, and H) Images of fluorescence. (C, F, and I) Images of overlay. The graphs on the right are results of the line-scan analysis: B, Lipi-Blue; E, Lipi-Green; or H, LipiRed. (J, K, and L) Fluorescence intensity of the range indicated by the arrows shown in parts B, E, and H, respectively. Scale bar is 10 μm.
(Figure 2J−L). These results strongly suggest that all Lipiprobes are useful for the detection and the analysis of LDs. Stability of Lipi-probes in LDs. We then examined the stability of Lipi-probes in LDs. The HepG2 cells were stained with Lipi-probes, and with conventional fluorescent probes, Nile Red and BODIPY 493/503 as references. The LDs stained with Lipi-Blue and Lipi-Green can be monitored even after 24 h culturing. However, no clear fluorescent signals of Nile Red, BODIPY 493/503, and Lipi-Red were observed (Figure S5). This result suggests that long-term monitoring of LDs is possible, and therefore, “time-lag staining” of LDs in a single cell with Lipi-Blue and Lipi-Green is technically feasible, which will be a big advantage to investigate the mechanism of LD formation, growth, and retraction. Monitoring of Intracellular Dynamics of LDs. In order to investigate whether LDs move intercellularly, we used KB cells and HepG2 cells. We prepared two dishes of each cell: one dish stained with Lipi-Blue and the other dish with LipiGreen. Lipi-Blue-stained cells and Lipi-Green-stained cells were then co-cultured for 48 h. As shown in Figure 3, LDs in KB cells were transferred as previously reported (Figure 3A).16 However, no LDs of HepG2 cells were transferred (Figure 3B). Although the mechanism of intercellular LD-transferring of KB cells is unknown, we suspect that the transferring through tunneling nanotubes (TNTs) might be one of the possibilities.17 TNT-like structures were shown in the image of confocal microscopy (Figure S6-2, A1), but no LDs were found in the structures. Fractional Staining of LDs in a Single Cell. “Time-lag staining” of LDs in individual cells by using two Lipi-probes, Lipi-Blue and Lipi-Green, was attempted. First, HepG2 cells were stained with Lipi-Green for 30 min and cultured with a medium containing OA overnight, and then the cells were stained with Lipi-Blue. We expected that, if the fusion and
Figure 3. Co-culturing of cells separately stained with Lipi-Blue and Lipi-Green. (A) KB cells were stained with 1 μM Lipi-Blue or with Lipi-Green. Lipi-Blue- and Lipi-Green-stained KB cells were mixed and co-cultured for 48 h. (B) HepG2 cells were stained with 1 μM Lipi-Blue or with Lipi-Green. Lipi-Blue- and Lipi-Green-stained HepG2 cells were mixed and co-cultured for 48 h. Images were taken with a confocal laser scanning microscope. All images used to prepare Figure 3A,B are shown in the Supporting Information (Figures S6-1 and S6-2). Scale bar is 20 μm.
retraction speed of LDs were slow enough, LDs that were present before the LD induction with OA would be stained by Lipi-Green and Lipi-Blue, whereas newly formed and grown LDs would be stained by Lipi-Blue alone. Figure 4 shows two staining patterns observed: one stained with both Lipi-Green and Lipi-Blue and the other with Lipi-Blue only. It is likely that LDs with the yellow arrows already existed before the induction of LD with OA and that the white arrows are newly formed and grown LDs during the incubation with OA (Figure 4G). Most of the old LDs are surrounded with the newly formed LDs, with some of the old LDs being diluted by the incorporation of OA. Marked with a solid white line and broken white line in Figure 4A are a healthy cell and a damaged cell, respectively, which indicates that the LDs were formed and grown in a healthy cell but not in a damaged cell. C
DOI: 10.1021/acs.biochem.8b01071 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry
Figure 4. Multicolor staining of HepG2 cells. HepG2 cells were stained with 1 μM Lipi-Green for 30 min and then incubated for 24 h with 200 μM OA. The cells were then stained with 1 μM Lipi-Blue and 1 μM ER-Tracker Red for 30 min. Fluorescent images were taken with a confocal laser scanning microscope. (A) Image of HepG2 cells with confocal laser scanning microscopy. (B) Stained with Lipi-Green. (C) Stained with Lipi-Blue. (D) Merged image of panels B and C. (E) Merged image of panels A, B, C, and F. (F) Stained with ER tracker. (G) Magnified image of D. Scale bar is 20 μm.
think that close observations of LDs using these fluorescent probes will disclose unknown functions of LDs.
Figure 4E shows that the LDs are on the stained area with ERTracker Red. The result indicates that Lipi-probes enable a separate staining of LDs in individual cells as well as monitoring the formation and growth of LDs in a living cell. As we expected, newly formed LDs and previously existing LDs in individual cells can be separately stained by using both LipiGreen and Lipi-Blue.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.biochem.8b01071.
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CONCLUSIONS The LDs stained with Lipi-probes in mammalian cells can clearly be identified with a background fluorescence level that is extremely low compared to Nile Red and BODIPY 493/503. Since Lipi-Blue and Lipi-Green were retained in LDs after the staining and the toxicities of these probes are very low, longterm monitoring of the LD dynamics in a single cell was successfully achieved. On the basis of these features, we were able to identify previously existing LDs from newly formed and grown ones by “time-lag staining” by using both Lipi-Blue and Lipi-Green. Lipi-probes, in combination with other fluorescent probes for various organelles, will be also useful to investigate the interaction of LDs with other organelles in living cells. Additionally, the intercellular transfer of a whole LD was observed in KB cells during 48 h culturing, but not in HepG2 cells. The intercellular transfer of LDs may be an important mechanism to survive or to communicate for cells under certain conditions. To investigate the role of the LDs, tagging and tracing of LDs are indispensable methods. We therefore
Experimental details, syntheses, spectra, procedures, cell viability vs concentration, fluorescence imaging, effects of Triacsin C (TC) on the formation of lipid droplets in HepG2 cells, quantitative image analysis, merging of a fluorescent image and a confocal image of HepG2 cells stained with fluorescent probes, and co-culturing images (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Yuki Tatenaka: 0000-0003-1900-9032 Funding
This work was partly supported by JSPS KAKENHI grant no. JP17H06419 (to H.N.). D
DOI: 10.1021/acs.biochem.8b01071 Biochemistry XXXX, XXX, XXX−XXX
Communication
Biochemistry Notes
The authors declare the following competing financial interest(s): Y. Tatenaka, M. Ishiyama, K. Sasamoto, M. Shiga, and Y. Ueno are employees of Dojindo Laboratories. Dojindo Laboratories commercialized Lipi-probes.
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ABBREVIATIONS LD, lipid droplet; ADRP, adipocyte differentiation-related protein; OA, oleic acid; TC, Triacsin C REFERENCES
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DOI: 10.1021/acs.biochem.8b01071 Biochemistry XXXX, XXX, XXX−XXX
