An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics
2021; Chinese Chemical Society; Volume: 4; Issue: 2 Linguagem: Inglês
10.31635/ccschem.021.202101143
ISSN2096-5745
AutoresEngui Zhao, Chuen Kam, Mingyu Wu, Erjing Wang, Jiawei Wang, Ben Zhong Tang, Sijie Chen,
Tópico(s)Microbial Metabolic Engineering and Bioproduction
ResumoOpen AccessCCS ChemistryRESEARCH ARTICLE1 Feb 2022An Aggregation-Induced Emission Optical Highlighter for the Studies of Endoplasmic Reticulum-Lipid Droplet Content Dynamics Engui Zhao†, Chuen Kam†, Ming-Yu Wu, Erjing Wang, Jiawei Wang, Ben Zhong Tang and Sijie Chen Engui Zhao† School of Science, School of Electronics and Information Engineering, Harbin Institute of Technology, Shenzhen, HIT Campus of University Town, Shenzhen 518055 , Chuen Kam† Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong 999077 , Ming-Yu Wu Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong 999077 , Erjing Wang Overseas Expertise Introduction Center for Discipline Innovation (D18025), Collaborative Innovation Center for Advanced Organic Chemical Materials Co-Constructed by the Province and Ministry, School of Material Science and Engineering, Hubei University, Wuhan 430062 , Jiawei Wang School of Science, School of Electronics and Information Engineering, Harbin Institute of Technology, Shenzhen, HIT Campus of University Town, Shenzhen 518055 , Ben Zhong Tang Department of Chemistry, The Hong Kong Branch of Chinese National Engineering Research Center for Tissue Restoration and Reconstruction, SCUT-HKUST Joint Research Institute, Institute for Advanced Study, Department of Chemical and Biological Engineering, The Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong 999077 Shenzhen Institute of Molecular Aggregate Science and Engineering, School of Science and Engineering, Guangdong of Electronic and Information Engineering, The Chinese University of Hong Kong, Shenzhen, 2001 Longxiang Boulevard, Longgang District, Shenzhen City, Guangdong 518172 and Sijie Chen *Corresponding author: E-mail Address: [email protected] Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, Hong Kong 999077 https://doi.org/10.31635/ccschem.021.202101143 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesDownload CitationsTrack Citations ShareFacebookTwitterLinked InEmail The lipid droplet (LD) is a dynamic organelle responsible for lipid storage and metabolism that plays important roles in maintaining lipid homeostasis. However, limited strategies are available for tracking the LD content exchange. In this contribution, we report a novel fluorescent probe, TPE-AmAl, for real-time LD content dynamics tracking. TPE-AmAl is LD-specific, but emits faintly due to its intramolecular motion. Upon photoactivation, it undergoes a photocyclodehydrogenation reaction and shows a large fluorescence increment. Thus, it can be used for highlighting selected LDs with high spatial resolution. By measuring the fluorescence changes in the distal region, the lipid content exchange efficiency can be estimated. In our experiment, LD content exchange rate differences between nascent and mature LDs as well as cells with normal and deficient LD budding machinery are observed. This probe expands the fluorescence-based toolbox for LD content dynamics studies. Download figure Download PowerPoint Introduction For a long time, lipid droplets (LDs) were perceived merely as an inert form of fat storage in cells. As more regulatory proteins of LDs have been discovered, the LD has come to be regarded as an organelle with dynamic associations with other organelles.1–3 Recent investigations have revealed the important roles of LDs in cell functions and human diseases, as well as in viral infections. In the human body, LDs are responsible for maintaining the homeostasis of neutral lipids, which are enriched in adipocytes, hepatocytes, and enterocytes.4,5 By immobilizing fatty acids into lipids and encircling them with phospholipids, LDs also play a crucial protective role in preventing the oxidation of polyunsaturated fatty acids and protecting cells against damages from reactive oxygen species.6,7 Pathologically, abnormal accumulation of lipids in LDs is associated with metabolic diseases8,9 and unusually more or larger LDs in patients are indicative of steatosis, which is associated with impaired homeostasis of triacylglycerol (TAG).10 Besides, LDs are associated with the production and assembly of virus particles.11,12 In this sense, understanding the regulation of LD biogenesis and lipolysis will expand our knowledge of the pathophysiology of LD-related diseases. With the continuous endeavor of scientists worldwide, the following LD biogenesis process has been revealed. In eukaryotes, LDs originate from the endoplasmic reticulum (ER). Early steps of LD biogenesis involve the local synthesis and deposition of TAG between the leaflets of the ER bilayer, resulting in the formation of a lens-like structure. Subsequently, the formed lens structure grows asymmetrically toward the cytoplasmic side with the help of seipin.13,14 Afterward, nascent LDs bud from the ER, facilitated by the fat storage-inducing transmembrane 1/2 (FITM1/2) proteins, which are evolutionarily conserved integral ER membrane proteins.15 During LD biogenesis, the nascent LD phospholipid monolayer remains continuous with the cytoplasmic leaflet of the ER membrane, which allows the translocation of ER membrane proteins to LDs.16 These membrane proteins, such as glycerol-3-phosphate acyltransferase 4 (GPAT4) and diacylglycerol O-acyltransferase 2 (DGAT2), are crucial enzymes for the local biosynthesis of TAG, thereby facilitating the subsequent storage of neutral lipids in LDs.4,17 Although the general picture of LD biogenesis is known and some critical proteins involved in LD biogenesis have been identified, the role of these proteins in regulating LD biogenesis and how LDs dynamically interact with other organelles remain unknown. One critical hurdle for the investigation of LDs is that limited tools and techniques are available for LD dynamic studies. Fluorescence is a powerful tool for tracking biological processes. To stain and study LDs in cells, conventional fluorescent probes (e.g., Nile Red18 and oil red O19), BODIPY-conjugated lipids,20 or genetically encoded fluorescent LD membrane proteins21 are widely used. Other types of emerging fluorescent probes possessing the aggregation-induced emission (AIE) property are also potential candidates to label LDs,22–27 given that they are not quenched at high concentrations.28 While these fluorescent probes are informative in quantitative studies of the size, number, and subcellular localization of LDs, further insight into the content exchange between LDs themselves and the ER requires a reporter reflecting the content dynamics. Optical highlighters which are a class of fluorescent probes possessing the photoactivatable, photoconvertible, or photoswitchable property are versatile tools in this regard.29 Upon laser irradiation in a region of interest, the “turned-on” fluorescent probe can be chased under fluorescence microscopy to study the dynamics of interested targets.30 Recently, we have reported that an AIE-active fluorescent probe, named TPE-AmAl, stained LDs in live cells and green algae.22 In further investigation of the photophysical property of TPE-AmAl, we unexpectedly discovered its photoactivation (PA) process during imaging. TPE-AmAl can dissolve in common solvents and fatty acids, and thus it fluoresces weakly. Upon irradiation with light, it undergoes a photocyclodehydrogenation reaction to produce another luminogen with more rigid structure, named photoactivated TPE-AmAl (PA-TPE-AmAl). PA-TPE-AmAl emits more intensely than TPE-AmAl in solution states, thus achieving a PA effect. We then verified the photoactivatable property of TPE-AmAl in LDs in cells under 405 nm irradiation. By selectively photoactivating TPE-AmAl in LDs of endothelial cells, we found that nascent and mature LDs had different degrees of content dynamics in an ER-dependent manner. With this discovery, we also developed a simple method to induce nascent and mature LDs in cells. This study provides a new optical highlighter tool for the LD study to understand the mechanism and physiological importance of LD content exchange through the ER. Experimental Methods Synthesis Photocyclodehydrogenation reaction of TPE-AmAl to prepare PA-TPE-AmAl: Into a 250 mL round-bottom flask placed in a photoreaction vessel were added TPE-AmAl (75.8 mg, 0.17 mmol), I2 (50 mg, 0.197 mmol), propylene oxide (PO) (987 mg, 17 mmol), and toluene (170 mL). The solution was bubbled with N2 for 30 min prior to photoreaction to completely remove the oxygen. Afterward, the reaction mixture was irradiated with UV light from a 500 W high-pressure mercury vapor lamp placed in the immersion quartz well under condensation for 6.5 h. After completion of the photoreaction, the solvent was evaporated under reduced pressure with rotatory evaporator, and the crude product was purified with silica-gel column chromatography by using hexane and dichloromethane (DCM) as eluent (v/v, 3/1) to give PA-TPE-AmAl as yellow powder in 10.6% yield (8 mg). 1H NMR (400 MHz, CDCl3, δ): 10.26 (s, 1H), 9.19 (s, 1H), 7.96 (s, 1H), 7.87 (dd, 1H, J = 0.8 Hz, J = 6.8 Hz), 7.62 (d, 1H, J = 6.8 Hz), 7.57–7.54 (m, 1H), 7.22–7.19 (m, 2H), 7.14 (d, 2H, J = 6.4 Hz), 7.09–7.01 (m, 4H), 6.67 (br, 2H), 3.20 (s, 6H), 2.94 (s, 6H). 13C NMR (100 MHz, CDCl3, δ):192.8, 139.9, 137.0, 133.2, 133.0, 132.4, 132.0, 131.7, 131.5, 129.6, 129.3, 129.0, 128.6, 127.9, 126.9, 126.5, 125.1, 122.8, 115.2, 102.8, 72.0, 41.0. High-resolution mass spectrometry (HRMS) [matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF)] (m/z): M+ calcd for C31H28N2O, 444.2202; found, 444.2256. 1H and 13C NMR analyses 1H and 13C NMR spectra were measured on a Bruker ARX 400 NMR spectrometer using chloroform-d as solvent and tetramethylsilane (TMS) as internal reference. HRMS analyses HRMS were recorded on a Finnigan MAT TSQ 7000 mass spectrometer system operating in a MALDI-TOF mode. X-ray diffraction to determine the single-crystal structure Single-crystal X-ray diffraction (XRD) intensity data collections were performed on a Bruker D8 VENTURE diffractometer at 298 K with Mo K-alpha radiation. PA of TPE-AmAl TPE-AmAl was dissolved in dimethyl sulfoxide (DMSO) to prepare a 100 μM stock solution. To examine the PA process, 0.5 μL of 100 μM TPE-AmAl stock solution was mixed with 30 μL of the corresponding solution (DMSO, glycerol, water, oleic acid, or arachidonic acid), and dropped on a coverslip and photoactivated by an inverted wide-field fluorescence microscope (ECLIPSE Ts2R, Nikon, Tokyo, Japan). In the time-lapse imaging, specimens were continuously UV-irradiated using a white-light light-emitting diode (LED) source (X-Cite 110LED, Excelitas Technologies) and an UV-2A filter cube (excitation filter: 330–380 nm; dichromic mirror: 400 nm; emission filter: LP 410 nm) for 120 s. Images were acquired every 1 s using a color camera (DS-Fi3, Nikon, Tokyo, Japan). In cells, TPE-AmAl was photoactivated by a laser scanning confocal microscope (LSM 880, Zeiss, BW, Germany) using the photobleaching mode. A region of interest for PA was selected by the region tool, and the parameters used were: iterations: 25; power for 405 nm laser: 20%, equivalent to 60 kW/cm2; dwell time: 0.26 μs/pixel. Density functional theory calculations Based on the crystal structure of PA-TPE-AmAl, the B3LYP functional and a split valence plus polarization basis set 6-31G(d) were used to obtain equilibrium geometry, from which the molecular orbital amplitude plots of highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) energy levels of PA-TPE-AmAl were obtained. Cell culture Human embryonic kidney (HEK) 293T cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, MA, USA; 11965092) supplemented with 10% (v/v) fetal bovine serum (Gibco, MA, USA; 10270106). Human umbilical vein endothelial cells (HUVECs) were cultured in vascular cell basal medium (ATCC, PCS-100-030) supplemented with endothelial cell growth kit-BBE (ATCC, PCS-100-040). All cell lines were maintained in the presence of 5% CO2 in a humidified incubator at 37 °C. Leakage of fluorescent dyes in cells HEK-293T cells were treated with 400 μM of oleic acid (Meryer, Shanghai, China; M02615) overnight and loaded with 10 μM of TPE-AmAl or 1× LipidSpot 610 (Biotium, CA, USA; 70069) for 1 h. After that cells were washed with phosphate-buffered saline (PBS) twice and incubated with phenol red-free complete medium (Gibco, MA, USA; 21063029) supplemented with 400 μM of oleic acid at 37 °C for the designated period of time. To determine the leakage of TPE-AmAl, TPE-AmAl was photoactivated to the maximum level at the indicated time-point followed by imaging. For PA-TPE-AmAl, TPE-AmAl in cells was photoactivated to the maximum level in the beginning before incubation and cells were imaged at the indicated time point. For LipidSpot 610, cells were imaged at the indicated time point. Images were taken using wide-field fluorescence microscopy. Knockdown experiments In the knockdown experiment, cells were seeded in a 35-mm glass-bottom dish (NEST, Jiangsu, China; 801002) and transfected with 30 pmol of Silencer Select predesigned siRNA targeting human FITM2 (Ambion, MA, USA; 4427037; ID: s225599; sequence 5′-GAGGUGAAGACGGACCGAAtt-3′) for 3 days. Silencer eGFP siRNA (Ambion, MA, USA; AM4626) was used as a negative control. Transfection was performed using Lipofectamine RNAiMAX transfection reagent (Invitrogen, MA, USA; 13778100) according to the manufacturer’s instruction and expressed for 3 days. Before fluorescence imaging, HUVECs were treated with 100 μM of oleic acid overnight and loaded with 10 μM TPE-AmAl and 1× LipidSpot 610 (Biotum, CA, USA) in medium for 1 h. After washing with PBS twice, cells were cultured in the complete medium supplemented with 100 μM of oleic acid for the subsequent live-cell imaging. Fluorescence imaging Cells were seeded in 35-mm glass-bottom dishes and cultured for at least overnight. Cells were then incubated with 10 μM of TPE-AmAl or 1× LipidSpot 610 for 1 h. After that cells were washed with PBS twice and incubated with the complete medium. Wide-field imaging was performed using a Nikon ECLIPSE Ts2R microscope equipped with a Plan-Apochromat 100×/1.45 NA oil-immersion objective lens (Nikon, Tokyo, Japan), a white-light LED source (X-Cite 110LED, Excelitas Technologies, MA, USA), a color camera (DS-Fi3, Nikon, Tokyo, Japan), or a monochrome camera (DS-Qi2, Nikon, Tokyo, Japan) driven by the Nikon NIS-Elements BR software (Nikon, Tokyo, Japan; version 5.01). TPE-AmAl was photoactivated and imaged using a UV-2A filter (excitation: 330–380 nm; dichromic mirror: 400 nm; emission: LP 410 nm). LipidSpot 610 was imaged using an mCherry filter (excitation: 560/40 nm; emission: 630/75 nm). Confocal imaging was performed using a Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apochromat 63×/1.40 NA oil-immersion objective lens, a photomultiplier tube, and a Gallium arsenide phosphide (GaAsP) detector driven by the Zeiss ZEN software (Zeiss, BW, Germany; version 2.1 SP1) in gray scale and pseudocolored. For TPE-AmAl, a 405-nm diode laser, a 405-nm beam splitter, and a 415–561 nm emission filter were used. For LipidSpot 610, a 561-nm diode-pumped solid-state (DPSS) laser, a 458/561 nm beam splitter, and a 568–736 nm emission filter were used. Image processing without any adjustment of contrast was done using ImageJ (National Institutes of Health, MD, USA) and Photoshop (Adobe, CA, USA). Spectral imaging Imaging was performed using the Zeiss LSM 880 laser scanning confocal microscope equipped with a Plan-Apochromat 63×/1.40 NA oil-immersion objective lens and a 32-channel GaAsP spectral detector driven by the Zeiss ZEN software (Zeiss, BW, Germany; version 2.1 SP1) in gray scale. For TPE-AmAl, a 405-nm diode laser and a 405-nm beam splitter were used. A range of 413–703 nm was detected at 10-nm resolution. For LipidSpot 610, a 561-nm DPSS laser and a 458/561 nm beam splitter were used. A range of 573–713 nm was detected at 10-nm resolution. Quantification and statistical analyses Quantification of intensity in fluorescence images were performed by ImageJ (National Institutes of Health) with threshold selected. All data are presented as mean ± standard error of mean (SEM). Results and Discussion Photoactivatable properties of TPE-AmAl Previously, we reported the LD specificity of TPE-AmAl and its application for the selective imaging of LDs in cells, as well as in green algae.22 During our in-depth investigations of TPE-AmAl for LD imaging, we noticed the fluorescence turn-on process of TPE-AmAl upon light irradiation and conducted further investigations. TPE-AmAl was synthesized following the procedures reported previously.22 The final product of TPE-AmAl was characterized with 1H NMR and 13C NMR, from which satisfactory results corresponding to its molecular structure were obtained ( Supporting Information Figure S1). To unveil the PA process, TPE-AmAl was sprayed onto glass wafers and irradiated with a wide-field fluorescence microscope (ECLIPSE Ts2R, Nikon, excitation wavelength: 330–380 nm). Afterward, samples were collected and subjected to HRMS analysis. For comparison, TPE-AmAl without light irradiation was also examined with HRMS. The molecular ion peak of TPE-AmAl appeared at m/z 446.2332 ( Supporting Information Figure S2a). After light irradiation, a new signal at m/z 444.2256 emerged ( Supporting Information Figure S2b), which suggested the cyclodehydrogenation process of TPE-AmAl. Afterward, PA-TPE-AmAl was synthesized through oxidation reaction with iodine and PO and purified with silica-gel column chromatography (Figure 1a). The structure of PA-TPE-AmAl was characterized with 1H NMR, from which satisfactory results corresponding to its molecular structure were obtained ( Supporting Information Figure S3). The conversion from TPE-AmAl to PA-TPE-AmAl was confirmed from the 1H NMR spectra (Figure 1b and 1c). Compared with TPE-AmAl, the resonance signals of PA-TPE-AmAl protons were all shifted downfield. Specifically, the single resonance signal of Ha was shifted from δ 9.88 to δ 10.26. Hb and Hc were structurally equivalent in TPE-AmAl with a doublet resonance signal at δ 7.60, while in TPE-AmAl these resonance signals were shifted downfield to δ 9.19 and δ 7.95, respectively. The single crystal of PA-TPE-AmAl was obtained from its solution in DCM/hexane mixture by slow solvent evaporation and subjected to single-crystal XRD analysis (Figure 1d and Supporting Information Table S1), which substantially confirmed the structure of PA-TPE-AmAl (CCDC 2070269).a Interestingly, the dehydrogenation reaction mainly took place between the phenyl ring with the electron-donating dimethylamine group and the phenyl ring with the electron-withdrawing aldehyde group, and formed a phenanthrene core in the structure. The dimethylamine group was electron-donating, and the aldehyde group was electron-withdrawing, which formed a donor–acceptor structure in the chromophore. This was consistent with density functional theory (DFT) calculations. As shown in Figure 1e, the HOMO was mainly located on the dimethylamine side of the phenanthrene group, while the LUMO was mainly located on the aldehyde side of the phenanthrene group. Figure 1 | Photocyclodehydrogenation reaction of TPE-AmAl. (a) Schematic illustration of the conversion of TPE-AmAl to PA-TPE-AmAl through photocyclodehydrogenation reaction. The reaction was conducted by irradiating reaction mixture with UV light from a 500 W high-pressure mercury vapor lamp placed in the immersion quartz well under condensation for 6.5 h in the presence of iodine and PO. (b) Structure of TPE-AmAl highlighting three characteristic protons (Ha, Hb, and Hc), which existed in distinct chemical environments before and after the photoreaction. (c) Changes in the chemical shifts of the characteristic protons in TPE-AmAl and PA-TPE-AmAl. (d) ORTEP drawing of PA-TPE-AmAl. (e) Molecular orbital amplitude plots of HOMO and LUMO for PA-TPE-AmAl. Download figure Download PowerPoint Afterward, we evaluated the PA properties of TEP-AmAl. Interestingly, TPE-AmAl exhibited different degrees of PA when dissolved in different solutions. In oleic acid, TPE-AmAl was substantially photoactivated under UV excitation, and its fluorescence intensity reached the plateau with the maxima (Figure 2a and Supporting Information Figure S4). The PA effect was not observed when TPE-AmAl was dispersed in aqueous solutions ( Supporting Information Figure S5). Besides, TPE-AmAl could not be effectively photoactivated in glycerol, which excluded the possibility that the PA was merely a viscosity effect. TPE-AmAl in arachidonic acid could only be photoactivated for a short period of time, and the fluorescence was gone afterward, presumably due to the low activation efficiency of TPE-AmAl and the photobleaching effect involved ( Supporting Information Figure S5). In the spectral imaging using confocal microscopy, TPE-AmAl in oleic acid showed an approximately fivefold increase in fluorescence intensity peaked at 528 nm, which was significantly higher than that of TPE-AmAl in DMSO after PA (Figure 2b). Since oleic acid was the major fatty acid found in neutral lipids stored in LDs,31 the high PA efficiency of TPE-AmAl in oleic acid over other surroundings was greatly favorable for the imaging of LDs, which ensured the specific fluorescence turn-on process of TPE-AmAl inside the LD. Figure 2 | PA of TPE-AmAl in oleic acid and in cells. (a) Quantification of relative fluorescence intensity of TPE-AmAl upon photoirradiation. (b) Quantification of fluorescence intensity of corresponding samples in the spectral imaging using confocal microscopy. PA represents samples after 405-nm PA. Photoactivated TPE-AmAl in oleic acid showed maximum emission at 528 nm. (c) Representative fluorescence image of HUVEC stained with TPE-AmAl and LipidSpot 610. Pseudocolored images were taken with 561-nm and 2% power output of 405-nm lasers. Scale bar: 20 μm. Enlarged channel represents zoom-in of the white square box in the merge channel. Scale bar: 5 μm. (d) Representative brightfield and fluorescence images of HUVEC treated with 80 μM of arachidonic acid overnight and stained with LipidSpot 610 and TPE-AmAl. Pseudocolored images were taken with 561-nm and 0.5% power output of 405-nm lasers. Dotted rectangle represents selected area with 20% power output of 405-nm PA. Scale bar: 20 μm. (e) Quantification of relative fluorescence intensity of TPE-AmAl before and after PA in the control or the photoactivated cell in panel (e). (f) Zoom-in of LDs in the photoactivated cell. Scale bar: 1 μm. (g) Quantification of fluorescence intensity along the dotted line in panel (f). Download figure Download PowerPoint TPE-AmAl could enter cells and labeled the LD. However, due to the active motions of TPE-AmAl in lipid, it fluoresced faintly in LDs. Under a regular low-power confocal laser scanning microscope, TPE-AmAl in HUVEC could be gradually turned on with consecutive iterations ( Supporting Information Figures S6a–S6c). Photoactivated TPE-AmAl in HUVEC colocalized with LipidSpot 610, a commercially available probe specific for LDs (Figure 2c). Spectral imaging revealed that the peak emission of TPE-AmAl in cells was around 508 nm, which was blue-shifted by 20 nm as compared with that of its solutions ( Supporting Information Figure S7). In HUVECs treated with oleic acid or arachidonic acid for 24 h, excess-free fatty acids induced the formation of increased number of LDs in the cytoplasm. The more neutral lipids stored in LDs increased the maximum fold-change of the fluorescence intensity of photoactivated TPE-AmAl to six- to eight-fold when compared with ∼2.5-fold in cells cultured in the basal condition ( Supporting Information Figures S6d and S6e). The photoactivatable property of TPE-AmAl enabled selective PA of LDs in individual cells by a laser-scanning confocal microscope. Under the long-term treatment of arachidonic acid, the fluorescence intensity of TPE-AmAl after PA showed up to ∼15-fold increase with a high signal-to-noise ratio, which was enough to clearly distinguish individual LD with a low background level (Figures 2d–2g). Tracing lipid content dynamics in HUVECs The photoactivatable property and LD-targeting characteristics of TPE-AmAl make it an excellent candidate for use as an optical highlighter to study the dynamics of LDs. To explore this feasibility, we selectively photoactivated a subpopulation of LDs within a region of interest in an individual cell using confocal laser-scanning microscopy and traced its subcellular localization. In a time-course experiment of 20 min in cells treated with oleic acid for 2 h, we found that LDs in the distal region lit up gradually with a ∼2.7-fold increase of its fluorescence intensity (Figures 3a and 3b). The turn-on of LDs in the distal region was significantly higher than the PA of TPE-AmAl during regular laser scanning, as the fluorescence intensity of TPE-AmAl after a total of three regular scannings only account for ∼10% or less of its maximum intensity ( Supporting Information Figure S8). The possibility of leakage for photoactivated TPE-AmAl to distal LDs was also excluded, since photoactivated TPE-AmAl showed a longer retention time in cells with a half-life of ∼7.1 h when compared with its ordinary form and LipidSpot 610 ( Supporting Information Figure S9). Thus, the leakage in 20-min tracking should be kept to a minimum. To examine whether the observed effect was physiologically relevant, we treated HUVECs with excess oleic acid for 24 h to mimic the biogenesis of mature LDs (Figures 3a–3c). For mature LDs, TPE-AmAl in the distal LDs showed ∼55% of fluorescence intensity relative to the photoactivated LDs in cells in the course of 20 min, which was lower than those nascent distal LDs showing ∼82% of fluorescence intensity (Figure 3d). These data suggest that nascent LDs have higher content exchange than mature ones. Figure 3 | Nascent LDs are more dynamic in content exchange than mature ones. (a) HUVEC treated with 100 μM oleic acid for 2 or 24 h were stained with LipidSpot 610 and TPE-AmAl. Dotted rectangles in the before PA channel represent areas with PA using 20% power output of 405 nm laser. After PA, regular fluorescence imaging was taken at different time points. Zoom-ins of LDs in the photoactivated and distal regions were enlarged from white square boarders and dotted squares, respectively, annotated in panel (a). Scale bar: 20 μm. (b and c) Quantification of net fluorescence fold change of TPE-AmAl in photoactivated and distal regions at different time points after PA in cells treated with oleic acid for (b) 2 h or (c) 24 h. (d) Percentage of fluorescence intensity in distal regions relative to photoactivated regions in the course of 20 min after PA. (b–d) Data represent mean ± SEM from four independent experiments. Student’s two-tailed t-test was used to determine statistical significance (*P < 0.05, ***P < 0.001). Download figure Download PowerPoint Differences in content exchange in nascent and mature LDs suggested their morphological or functional variations. In COS-7 cells, most of the LDs made contact with the ER through membrane contact sites.34 We then postulated that the ER served as an intermediate organelle to facilitate the content exchange between LDs in the photoactivated region and distal region. To test this possibility, we ablated LD budding from the ER and challenged the cells with long-term fatty acid treatment to examine whether LD content exchange could be restored to a higher level. In budding yeast, FITM homologues are required for the correct budding of LDs.15 In higher eukaryotes, FITM1 is primarily expressed in skeletal muscle, while FITM2 is ubiquitously expressed. We thus depleted FITM2 expression by siRNA knockdown in HUVEC. In cells depleted with FITM2, LDs accumulated in the perinucl
Referência(s)