Lipid Droplets, Perilipins and Cytokeratins – Unravelled Liaisons in Epithelium-Derived Cells

<div><p>Lipid droplets (LDs) are spherical accumulations of apolar lipids and other hydrophobic substances and are generally surrounded by a thin cortical layer of specific amphiphilic proteins (APs). These APs segregate the LDs from the mostly polar components of the cytoplasm. We have studied LDs in epithelium-derived cell cultures and in particular characterized proteins from the perilipin (PLIN) gene family - in mammals consisting of the proteins <i>Perilipin, Adipophilin, TIP47, S3-12 and MLDP/OXPAT (PLIN 1-5)</i>. Using a large number of newly generated and highly specific mono- and polyclonal antibodies specific for individual APs, and using improved LD isolation methods, we have enriched and characterized APs in greater detail and purity. The majority of lipid-AP complexes could be obtained in the top layer fractions of density gradient centrifugation separations of cultured cells, but APs could also be detected in other fractions within such separations. The differently sized LD complexes were analyzed using various biochemical methods and mass spectrometry as well as immunofluorescence and electron– in particular immunoelectron-microscopy. Moreover, by immunoprecipitation, protein-protein binding assays and by immunoelectron microscopy we identified a direct linkage between LD-binding proteins and the intermediate-sized filaments (IF) cytokeratins 8 and 18 (also designated as keratins K8 and K18). Specifically, in gradient fractions of higher density supposedly containing small LDs, we received as co-precipitations cytidylyl-, palmitoyl- and cholesterol transferases and other specific enzymes involved in lipid metabolism. So far, common proteomic studies have used LDs from top layer fractions only and did not report on these transferases and other enzymes. In addition to findings of short alternating hydrophobic/hydrophilic segments within the PLIN protein family, we propose and discuss a model for the interaction of LD-coating APs with IF proteins.</p></div

Summary scheme and brief description of stimulation methods used for adipose conversion and for the generation of different, distinct types of lipid droplets.

<p>Major treatments and involved PLIN proteins are shown. Preadipocytes containing many small LDs are <u>not</u> differentiated for several weeks with AIM containing media as conventionally described (top row; cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386.s001" target="_blank">Fig. S1</a>), but <u>only</u> very briefly (1–3 days), giving rise to “Adipocytes” (boxed area). Additional, short OA-treatment leads to “OA-Adipocytes” (boxed area, right side). Treatment with OA only - without AIM stimulation - leads to “OA-Preadipocytes” (bottom). Note the huge heterogeneity of sizes and colors of LDs seen within “OA-Adipocytes”. LDs are endogenously generated at the endoplasmic reticulum and stained positively for perilipin (“Endogenous-LDs”, red). Other LDs are obtained from the exogenous uptake of OA and stained positively for adipophilin, TIP47 and S3-12 (“Exogenous-LDs”, green). Merged LDs by fusion and mixed-type expression are seen by yellow color. The backway arrows indicate possible routes of LDs during lipolysis.</p

Immunoelectron microscopic localization of adipophilin in briefly AIM-stimulated and OA-treated human preadipocytes.

<p>(a) Cells reveal besides perilipin-positive also adipophilin-positive LDs. Localization was performed with mab AP125. A few positive, mostly smaller LDs are seen as well as plenty of non-labeled, larger LDs. Almost all LDs are closely associated and anchored with IF bundles. (b) A small, strongly immunolabeled LD is seen approaching a big, scarcely labeled LD. These LDs are obviously at the rim of combining. (c,d) Enlargements with details of filament attachment and immunolabeling sites which were highlighted by arrows (vimentin IFs) and arrowheads (adipophilin immunoreaction). Bars: a,b: 1 µm; c,d: 0.50 µm.</p

<i>Immunoelectron microscopic localization of perilipin in briefly AIM-stimulated human preadipocytes.</i>

<p>(<b>a</b>) Shown are groups of LDs positive for perilipin by nanogold-label and silver enhancement. Localization was with mab Peri112.17. The LDs are seen closely associated and anchored with IF bundles. (<b>b</b>) Enlarged perilipin labeling with two small LDs (approximately 300–400 nm in diameter) approaching a big LD (approximately 2.0–2.5 µm in diameter) for combining and coalescence. Bundles of intermediate-sized filaments are associated to grains of the immunolabeled perilipin (arrows). Bars: 0.50 µm.</p

Laser scanning microscopy showing LD labeling of perilipin proteins in briefly AIM-stimulated human preadipocytes which were additionally treated shortly with oleic acid (OA).

<p>(a, a, a) AIM treatment for 1–3 days reveals many newly generated small and medium-sized LDs positively staining with perilipin antibodies (red). Additional OA-treatment for 2 hours leads to the appearance of LDs positive for adipophilin (green) in higher numbers and bigger sizes when compared to non-stimulated or AIM-stimulated cells solely (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone-0090386-g001" target="_blank">Fig. 1</a>). (a) Enlarged part taken from double staining seen in (a). (a) Enlarged part from (a) showing single color and mosaic double-stained droplets, obviously in the midst of a fusion process with involvement of different types of LD-binding PLIN proteins and with yellow, partly mixed colored LDs for co-localization. (b) A different perilipin/adipophilin antibody combination reveals a similar heterogeneous staining of LDs as seen in a-a. (c) Perilipin (red) vs. S3-12 (green) double staining. Several smaller S3-12-positive droplets are seen attaching and combining with larger perilipin-positive droplets. Many newly generated, very small and middle-sized rings of S3-12-positive droplet staining with punctate patterns can be seen by single green color staining only (examples marked by arrowheads). (d) Perilipin (red) vs. TIP47 (green) double staining. Some of the many newly appearing TIP47-positive small droplets adhere occasionally to the much bigger perilipin positive droplets. (e) Adipophilin (red) vs. TIP47 (green) double staining. Most of the droplets are visible with sizes estimated smaller than 1 µm in diameter. Some heterogeneously colored droplets are marked by arrowheads. Note, OA-treatment leads to the re-appearance of a tremendous number of small droplets positive for adipophilin, TIP47 and S3-12. Note in addition, the complexity of the PLIN staining of LDs. Different LD-binding proteins are expressed and different types of LDs appear in single cells by specific “endogenous” and “exogenous” stimulations. DAPI (blue). Bar in a: 5 µm; all other Bars: 20 µm.</p

PLIN proteins and vimentin detected in different density gradient centrifugation fractions of briefly AIM-stimulated preadipocytes.

<p>(a) Fractions obtained by cell disintegration using nitrogen cavitation, iodixanol gradient centrifugation followed by SDS-PAGE separations and Coomassie blue staining are shown (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Heid1" target="_blank">[13]</a>). Separated fractions were tested with Western blotting (WB) using pabs TIP47-hCT(b), S3-12-hNT (c,c), Adipo-hCT (d,d) and mabs Peri112.17 (e,é) and Vim3B4 (f,f´). Positions of molecular weight markers are given on the left margin of (a) and fraction numbers on top of (a,b). (c,d,é,f´) represent prolonged exposures of reactions shown in (c,d,e,f) respectively. The higher molecular band reactions of adipophilin and perilipin seen in LD1 fractions are unknown modifications of PLIN proteins (asterisks in d,e). Note, the separation shown here is given with AIM-stimulated preadipocytes and not with AIM-stimulated <u>plus</u> OA-treated preadipocytes. Therefore only the few small (i.e. freshly endocytosed) LDs are detected. These are mostly coalesced with the bigger endogenously generated LDs and therefore preferentially found here within the LD1 fraction. Importantly PLIN proteins can be detected also in these non OA-treated preadipocytes together with vimentin within <u>all</u> three major LD gradient areas LD1, LD2, LD3 - including the top layer LD1.</p

The LD-PLIN-vimentin model.

<p>(<b>a</b>) Specific hydrophobic interactions are thought to be responsible for the general binding of PLIN proteins to surfaces of LDs (cp. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Heid1" target="_blank">[13]</a>). (<b>b</b>) Perilipin exhibits these LD targeting sites too. In contrast and uniquely compared to the other PLIN proteins, perilipin owns no helical domains at the C-terminus, but an additional acidic <b>E-rich</b> domain (see alignments given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Heid1" target="_blank">[13]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Hickenbottom1" target="_blank">[43]</a>). The possible interaction based on differently charged domains of perilipin and vimentin is presented. For details and for the implications of the proposed binding with the basic <b>R-rich</b> head region of vimentin see results and discussion. (<b>c</b>) A schematic summary of immunolocalization and of spatial arrangement in the proximity of emerging nascent LDs is depicted (not true-to-scale). Perilipin binds to the surface of LDs due to hydrophobic – hydrophilic short sequence domains. In a next layer, vimentin is attracted by perilipin and wraps the LDs tightly in “cage-like” spherical structures, followed by multiple concentric layers of smooth ER cisternae. This arrangement is delineated in a simplified bar-illustrated transversal section (bottom). (<b>c</b>) The schematic scenario shown in (<b>c</b>) is illustrated with a corresponding EM picture showing sheaths of non-fenestrated ER cisternae, regularly spaced dots of transversal sectioned vimentin IFs and a small rim of PLIN proteins directly bound to the LD surface. Abbreviations: E  =  glutamic acid; R  =  arginine; NT  =  N-terminal; CT  =  C-terminal; P  =  perilipin; Vim  =  vimentin; ER  =  endoplasmic reticulum. Bar in (<b>c</b>): 0.20 µm.</p

Laser scanning single- and double-label immunofluorescence microscopy showing lipid droplet (LD) labeling of perilipin proteins in untreated, non-stimulated and in briefly AIM-stimulated human preadipocytes.

<p>(a-c) Non-stimulated cells. (a) Adipophilin monoclonal antibody (mab) staining reveals many small LDs (green). (b) Positive TIP47 polyclonal antibody (pab; green) vs. negative perilipin (mab; red) staining. (c) Pab S3-12 shows many very small LDs – some can be seen like arranged and placed along rows of filaments. (d-g) Adipocyte differentiation of cells with AIM medium. 1-3 days of AIM treatment newly generates many small and medium-sized LDs, which stain with antibodies for perilipin (red). In contrast after AIM treatment, LDs positive for other perilipin (PLIN) family members are reduced in numbers and size (green). Cells still appear fibroblast-like elongated, not roundish. (d) Perilipin vs. adipophilin staining. (e) Perilipin and TIP47 double staining. (f) Perilipin localization at surface of LDs (g) Perilipin comparison with S3-12. Note, whereas antibodies specific for adipophilin, S3-12 and TIP47 show plenty of small LDs in non-stimulated cells and staining for perilipin is negative, the situation changes completely with the start of AIM stimulation. (For additional examples of small LD staining conspicuously arranged along rows of filaments, see e.g. TIP47 staining patterns obtained with PLC epithelial cells given in Fig. S5 by Heid et al., 2013 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Heid1" target="_blank">[13]</a>). Nuclear staining was with DAPI (blue). Bars: 20 µm.</p

Electron micrographs showing special associations of LDs and ER within briefly AIM-stimulated human preadipocytes.

<p>(<b>a</b>) Small and characteristic LDs can be found by short-time adipose conversion showing droplets with several flat layers and cisternae of smooth endoplasmic reticulum (LD_ER, white double arrows; simultaneous fixation method). Occasionally, very regularly spaced dots of vimentin filaments can be seen at the surface of such droplets sandwiched between droplets and ER (black arrowheads). In addition, smaller LDs of 0.2– 0.5 µm in diameter without such surrounding ER layers - but with interaction of vimentin IFs (marked by black arrows; left side) – and larger, fully accomplished LDs of several µm in diameter with mitochondria (M) in the neighborhood can be seen. (<b>b</b>) The regular pattern of vimentin arrays at the surface of LDs can be recognized best where fewer and less densely packed ER sheaths surround the droplets (black arrowheads), which might constitute a specific stage of adipogenesis. In this situation LDs seem to be matured, i.e. grown enough in size and the multiple layers of ER cisternae are just about to be released. Note, such EM pictures suggest that different stages and types of LDs in differentiating adipocytes can be pictured and characterized. Bars: 0.50 µm.</p

Electron micrographs comparing briefly AIM-stimulated versus briefly AIM-stimulated plus OA-treated human preadipocytes.

<p>(a) Short AIM stimulation and simultaneous fixation method, often lead to the appearance of small LDs (approximately 1 µm in diameter) which are completely surrounded by multiple cisternae and layers of smooth ER (white double arrows). Smaller LDs of 0.2– 0.5 µm in diameter without such ER layers can also be seen (right side). Embedded in a meshwork of filaments (black arrows) are mitochondria (M) in the neighborhood of these ER layers with no direct contact to LDs. (b) Addition of OA to the cell media for one day leads to the decay of the droplet surrounding ER layers (here shown with sequential fixation method). Arrowheads mark periodically arranged vimentin cage-like structures seen directly in contact with the LD surface. These regular arrays of anchored IFs have been described already by Franke et al. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0090386#pone.0090386-Franke1" target="_blank">[15]</a>. Several IF bundles within the cytoplasma and with connections to LDs are marked by arrows. Note, with both treatments and fixation methods, mitochondria (M) are not seen in direct contact to the LDs. Bars: 0.50 µm.</p