compared to quiescent cells. LDs were abundantly present in the many (newly formed) cell extensions. Quantification of LD size and number by Image J analysis confirmed that the size of the LDs decreased during the first days of activation, and remained similar between day 4 and 7 after plating, whereas the number of LDs continually increased (Fig. 1B). From these data, it can be calculated that the total volume of neutral lipids per cell did not decrease during the first 7 days. In the successive period the LDs were completely degraded, as 2 weeks after isolation hardly any LDs could be observed in the myofibroblastic HSCs (Fig. 1A- day14, 1B). To investigate the fate of individual lipids in the LDs upon HSC activation in more detail, freshly isolated HSCs were incubated for 5 hr with 25 mM Bodipy-C12, a fluorescent fatty acid which is rapidly incorporated into TAG. After the incubation period, fluorescent fatty acid was accumulated in perinuclear LDs (Fig. S1). HSCs were subsequently cultured in the absence of exogenous Bodipy-C12 to follow the incorporated lipid in time. At day 4, fluorescently labelled LDs were detected at the cell extensions (Fig. S1). At day 7 fluorescent LDs were still observed at the growing cell tips (not shown), suggesting that the small LDs were at least partly derived from the larger perinuclear droplets rather than completely de novo formed from (unlabeled) lipids imported from the medium. The notion that small peripheral LDs are derived from the large perinuclear LDs predicts that the LDs migrate to the cell extensions of activated HSCs. We therefore performed time-lapse live cell imaging on HSCs around day 3–4 after plating. At this time point the most dramatic alterations were observed in LD localization and cellular reshaping. As shown in Fig. 2A and Movie S1, migration of perinuclear LDs towards the growing cell extensions was apparent. LD redistribution could be disturbed by treatment with nocodazole, an agent affecting microtubule organisation, and LDs were scattered through the cytoplasm (Fig. 2B). This suggests that redistribution of the LDs towards the cell extensions is mediated by the microtubule system. In addition, nocodazole treated cells had less clear cell extensions in comparison with the control cells. Lipidome of Activated Hepatic Stellate Cells In order to obtain insight in the mechanism of the observed changes in LD number and size, we determined the level of a number of proteins implicated in LD formation in HSCs at day 1 and after activation at day 7 (See Fig. S2). We found minor changes in the level of diacylglycerol acyltransferase 2 (DGAT2), implicated in TAG synthesis. In contrast, LRAT, an enzyme responsible for the synthesis of RE, was down regulated during HSC activation. The level of a key enzyme in phosphatidylcholine (PC) synthesis, CTP:phosphocholine cytidylyltransferase alpha (CCTa) was somewhat lower in activated HSCs. The subcellular localization of CCTa as determined by immunofluorescence microscopy, was not different between day 1 and day 7 and was mostly nuclear (data not shown), Raman spectrum of retinyl esters is changed in activated HSC During the activation process the localisation and size of the LDs altered dramatically. To address whether the LD composition changed as well, confocal Raman microspectroscopy was used to investigate the LD composition of HSCs in the quiescent and activated state. Due to its conjugated structure, retinoids display a specific Raman spectrum. As seen in Fig. 3 (upper right panel) RE are abundantly present in quiescent HSCs, with a characteristic main peak at 1595 cm21 and a number of smaller peaks in the region between 900 and 1300 cm21 (marked with asterisks). Also the acyl chains of fatty acid containing lipids like TAG could be well detected by their characteristic peak at 1440 cm21 (C-H bending; marked with #) and a peak around 1660 cm21, which is partly obscured in the presence of the main retinyl peak, if present. By determining the ratio between the main retinyl peak (1595 cm21) and the acyl peak at 1440 cm21 both in HSC samples and in different mixtures of trioleylglycerol and retinyl palmitate standards, we could make an estimate of the retinyl content of the LDs. By this method, we found that approximately 10% of the LD content of quiescent HSCs consisted of retinylesters. However upon activation of the HSCs, a drastic decrease in the level of RE relative to triacylglycerol was seen Figure 2. LD redistribution during HSC activation is microtubule dependent. A. Frames from time lapse life cell analysis revealing redistribution of dynamic LDs towards growing cellular extensions. Tracking shows the typical movements of a dynamic LD from 72 h to 84 h of HSC activation. Arrows indicate other regions of dynamic LDs (trackings not shown). B. To examine microtubule involvement in the LD redistribution process, freshly isolated HSCs were after 24 h in culture either treated with 10 mM nocodazole or vehicle (control) for 72 h at 37uC and after fixation, morphology and LD localization were analyzed by differential interference contrast microscopy (DIC) and fluorescence microscopy after Bodipy staining. doi:10.1371/journal.pone.0034945.g002 PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34945