scholarly journals Topography of Lipid Droplet-Associated Proteins: Insights from Freeze-Fracture Replica Immunogold Labeling

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Horst Robenek ◽  
Insa Buers ◽  
Mirko J. Robenek ◽  
Oliver Hofnagel ◽  
Anneke Ruebel ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Droplets ◽  
Spatial Organization ◽  
Freeze Fracture ◽  
Protein Composition ◽  
Immunogold Labeling ◽  
Intracellular Lipids ◽  
Associated Proteins ◽  
Developing Area ◽  
And Function

Lipid droplets are not merely storage depots for superfluous intracellular lipids in times of hyperlipidemic stress, but metabolically active organelles involved in cellular homeostasis. Our concepts on the metabolic functions of lipid droplets have come from studies on lipid droplet-associated proteins. This realization has made the study of proteins, such as PAT family proteins, caveolins, and several others that are targeted to lipid droplets, an intriguing and rapidly developing area of intensive inquiry. Our existing understanding of the structure, protein organization, and biogenesis of the lipid droplet has relied heavily on microscopical techniques that lack resolution and the ability to preserve native cellular and protein composition. Freeze-fracture replica immunogold labeling overcomes these disadvantages and can be used to define at high resolution the precise location of lipid droplet-associated proteins. In this paper illustrative examples of how freeze-fracture immunocytochemistry has contributed to our understanding of the spatial organization in the membrane plane and function of PAT family proteins and caveolin-1 are presented. By revisiting the lipid droplet with freeze-fracture immunocytochemistry, new perspectives have emerged which challenge prevailing concepts of lipid droplet biology and may hopefully provide a timely impulse for many ongoing studies.

scholarly journals Fixation Methods for the Study of Lipid Droplets by Immunofluorescence Microscopy

2003 ◽  
Vol 51 (6) ◽  
pp. 773-780 ◽  
Author(s):  
Deanna DiDonato ◽  
Dawn L. Brasaemle
Keyword(s):  
Light Microscopy ◽  
Lipid Droplet ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Cell Structure ◽  
Associated Proteins ◽  
Cellular Lipids ◽  
Cold Acetone ◽  
Developing Area ◽  
Cytoskeletal Elements

The study of proteins associated with lipid droplets in adipocytes and many other cells is a rapidly developing area of inquiry. Although lipid droplets are easily visible by light microscopy, few standardized microscopy methods have been developed. Several methods of chemical fixation have recently been used to preserve cell structure before visualization of lipid droplets by light microscopy. We tested the most commonly used methods to compare the effects of the fixatives on cellular lipid content and lipid droplet structure. Cold methanol fixation has traditionally been used before visualization of cytoskeletal elements. We found this method unacceptable for study of lipid droplets because it extracted the majority of cellular phospholipids and promoted fusion of lipid droplets. Cold acetone fixation is similarly unacceptable because the total cellular lipids are extracted, causing collapse of the shell of lipid droplet-associated proteins. Fixation of cells with paraformaldehyde is the method of choice, because the cells retain their lipid content and lipid droplet structure is unaffected. As more lipid droplet-associated proteins are discovered and studied, it is critical to use appropriate methods to avoid studying artifacts.

Identification of Perilipin-2 as a lipid droplet protein regulated in oocytes during maturation

2010 ◽  
Vol 22 (8) ◽  
pp. 1262 ◽  
Author(s):  
Xing Yang ◽  
Kylie R. Dunning ◽  
Linda L.-Y. Wu ◽  
Theresa E. Hickey ◽  
Robert J. Norman ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Content ◽  
Lipid Droplets ◽  
Intracellular Lipids ◽  
Epidermal Growth ◽  
Novel Method ◽  
Perilipin 2 ◽  
Lipid Droplet Protein

Lipid droplet proteins regulate the storage and utilisation of intracellular lipids. Evidence is emerging that oocyte lipid utilisation impacts embryo development, but lipid droplet proteins have not been studied in oocytes. The aim of the present study was to characterise the size and localisation of lipid droplets in mouse oocytes during the periovulatory period and to identify lipid droplet proteins as potential biomarkers of oocyte lipid content. Oocyte lipid droplets, visualised using a novel method of staining cumulus–oocyte complexes (COCs) with BODIPY 493/503, were small and diffuse in oocytes of preovulatory COCs, but larger and more centrally located after maturation in response to ovulatory human chorionic gonadotrophin (hCG) in vivo, or FSH + epidermal growth factor in vitro. Lipid droplet proteins Perilipin, Perilipin-2, cell death-inducing DNA fragmentation factor 45-like effector (CIDE)-A and CIDE-B were detected in the mouse ovary by immunohistochemistry, but only Perilipin-2 was associated with lipid droplets in the oocyte. In COCs, Perilipin-2 mRNA and protein increased in response to ovulatory hCG. IVM failed to induce Perilipin-2 mRNA, yet oocyte lipid content was increased in this context, indicating that Perilipin-2 is not necessarily reflective of relative oocyte lipid content. Thus, Perilipin-2 is a lipid droplet protein in oocytes and its induction in the COC concurrent with dynamic reorganisation of lipid droplets suggests marked changes in lipid utilisation during oocyte maturation.

scholarly journals HSD17B13: A Potential Therapeutic Target for NAFLD

2022 ◽  
Vol 8 ◽  
Author(s):  
Hai-bo Zhang ◽  
Wen Su ◽  
Hu Xu ◽  
Xiao-yan Zhang ◽  
You-fei Guan
Keyword(s):  
Liver Disease ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Critical Role ◽  
Chronic Liver ◽  
Nucleotide Polymorphisms ◽  
Single Nucleotide ◽  
Nonalcoholic Fatty Liver ◽  
Promising Target ◽  
Associated Proteins

Nonalcoholic fatty liver disease (NAFLD), especially in its inflammatory form (steatohepatitis, NASH), is closely related to the pathogenesis of chronic liver disease. Despite substantial advances in the management of NAFLD/NASH in recent years, there are currently no efficacious therapies for its treatment. The biogenesis and expansion of lipid droplets (LDs) are critical pathophysiological processes in the development of NAFLD/NASH. In the past decade, increasing evidence has demonstrated that lipid droplet-associated proteins may represent potential therapeutic targets for the treatment of NAFLD/NASH given the critical role they play in regulating the biogenesis and metabolism of lipid droplets. Recently, HSD17B13, a newly identified liver-enriched, hepatocyte-specific, lipid droplet-associated protein, has been reported to be strongly associated with the development and progression of NAFLD/NASH in both mice and humans. Notably, human genetic studies have repeatedly reported a robust association of HSD17B13 single nucleotide polymorphisms (SNPs) with the occurrence and severity of NAFLD/NASH and other chronic liver diseases (CLDs). Here we briefly overview the discovery, tissue distribution, and subcellular localization of HSD17B13 and highlight its important role in promoting the pathogenesis of NAFLD/NASH in both experimental animal models and patients. We also discuss the potential of HSD17B13 as a promising target for the development of novel therapeutic agents for NAFLD/NASH.

The ryanodine receptor provides high throughput Ca2+-release but is precisely regulated by networks of associated proteins: a focus on proteins relevant to phosphorylation

2015 ◽  
Vol 43 (3) ◽  
pp. 426-433 ◽  
Author(s):  
Fiona O'Brien ◽  
Elisa Venturi ◽  
Rebecca Sitsapesan
Keyword(s):  
Spatial Organization ◽  
Ryanodine Receptors ◽  
Integrated System ◽  
Fine Tuning ◽  
Control Mechanisms ◽  
Fk 506 ◽  
High Resolution Structure ◽  
Associated Proteins ◽  
And Function ◽  
Resolution Structure

Once opened, ryanodine receptors (RyR) are efficient pathways for the release of Ca2+ from the endoplasmic/sarcoplasmic reticulum (ER/SR). The precise nature of the Ca2+-release event, however, requires fine-tuning for the specific process and type of cell involved. For example, the spatial organization of RyRs, the luminal [Ca2+] and the influence of soluble regulators that fluctuate under physiological and pathophysiological control mechanisms, all affect the amplitude and duration of RyR Ca2+ fluxes. Various proteins are docked tightly to the huge bulky structure of RyR and there is growing evidence that, together, they provide a sophisticated and integrated system for regulating RyR channel gating. This review focuses on those proteins that are relevant to phosphorylation of RyR channels with particular reference to the cardiac isoform of RyR (RyR2). How phosphorylation of RyR affects channel activity and whether proteins such as the FK-506 binding proteins (FKBP12 and FKBP12.6) are involved, have been highly controversial subjects for more than a decade. But that is expected given the large number of participating proteins, the relevance of phosphorylation in heart failure and inherited arrhythmic diseases, and the frustrations of predicting relationships between structure and function before the advent of a high resolution structure of RyR.

Subcellular localization of skeletal muscle lipid droplets and PLIN family proteins OXPAT and ADRP at rest and following contraction in rat soleus muscle

2012 ◽  
Vol 302 (1) ◽  
pp. R29-R36 ◽  
Author(s):  
Rebecca E. K. MacPherson ◽  
Eric A. F. Herbst ◽  
Erica J. Reynolds ◽  
Rene Vandenboom ◽  
Brian D. Roy ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Lipid Droplet ◽  
Protein Interactions ◽  
Lipid Droplets ◽  
Droplet Surface ◽  
Protein Protein Interactions ◽  
Muscle Lipid ◽  
Droplet Distribution ◽  
Associated Proteins ◽  
Skeletal Muscle Lipid

Skeletal muscle lipid droplet-associated proteins (PLINs) are thought to regulate lipolysis through protein-protein interactions on the lipid droplet surface. In adipocytes, PLIN2 [adipocyte differentiation-related protein (ADRP)] is found only on lipid droplets, while PLIN5 (OXPAT, expressed only in oxidative tissues) is found both on and off the lipid droplet and may be recruited to lipid droplet membranes when needed. Our purpose was to determine whether PLIN5 is recruited to lipid droplets with contraction and to investigate the myocellular location and colocalization of lipid droplets, PLIN2, and PLIN5. Rat solei were isolated, and following a 30-min equilibration period, they were assigned to one of two groups: 1) 30 min of resting incubation and 2) 30 min of stimulation ( n = 10 each). Immunofluorescence microscopy was used to determine subcellular content, distribution, and colocalization of lipid droplets, PLIN2, and PLIN5. There was a main effect for lower lipid and PLIN2 content in stimulated compared with rested muscles ( P < 0.05). Lipid droplet distribution declined exponentially from the sarcolemma to the fiber center in the rested muscles ( P = 0.001, r2= 0.99) and linearly in stimulated muscles (slope = −0.0023 ± 0.0006, P < 0.001, r2= 0.93). PLIN2 distribution declined exponentially from the sarcolemma to the fiber center in both rested and stimulated muscles ( P < 0.0001, r2= 0.99 rest; P = 0.0004, r2= 0.98 stimulated), while PLIN5 distribution declined linearly (slope = −0.0085 ± 0.0009, P < 0.0001, r2= 0.94 rest; slope=−0.0078 ± 0.0010, P = 0.0003, r2= 0.91 stimulated). PLIN5-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.47; rest r2= 0.55 ± 0.02, stimulated r2= 0.58 ± 0.03). PLIN2-lipid droplets colocalized at rest with no difference poststimulation ( P = 0.48; rest r2= 0.66 ± 0.02, stimulated r2= 0.65 ± 0.02). Contrary to our hypothesis, these results show that PLIN5 is not recruited to lipid droplets with contraction in isolated skeletal muscle.

scholarly journals Direct interaction of Plin2 with lipids on the surface of lipid droplets: a live cell FRET analysis

2012 ◽  
Vol 303 (7) ◽  
pp. C728-C742 ◽  
Author(s):  
Avery L. McIntosh ◽  
Subramanian Senthivinayagam ◽  
Kenneth C. Moon ◽  
Shipra Gupta ◽  
Joel S. Lwande ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Cholesteryl Ester ◽  
Lipid Droplet ◽  
Lipid Droplets ◽  
Nile Red ◽  
Direct Interaction ◽  
Structure And Function ◽  
Live Cell ◽  
High Yield ◽  
And Function

Despite increasing awareness of the health risks associated with excess lipid storage in cells and tissues, knowledge of events governing lipid exchange at the surface of lipid droplets remains unclear. To address this issue, fluorescence resonance energy transfer (FRET) was performed to examine live cell interactions of Plin2 with lipids involved in maintaining lipid droplet structure and function. FRET efficiencies ( E) between CFP-labeled Plin2 and fluorescently labeled phosphatidylcholine, sphingomyelin, stearic acid, and cholesterol were quantitated on a pixel-by-pixel basis to generate FRET image maps that specified areas with high E (>60%) in lipid droplets. The mean E and the distance R between the probes indicated a high yield of energy transfer and demonstrated molecular distances on the order of 44–57 Å, in keeping with direct molecular contact. In contrast, FRET between CFP-Plin2 and Nile red was not detected, indicating that the CFP-Plin2/Nile red interaction was beyond FRET proximity (>100 Å). An examination of the effect of Plin2 on cellular metabolism revealed that triacylglycerol, fatty acid, and cholesteryl ester content increased while diacylglycerol remained constant in CFP-Plin2-overexpressing cells. Total phospholipids also increased, reflecting increased phosphatidylcholine and sphingomyelin. Consistent with these results, expression levels of enzymes involved in triacylglycerol, cholesteryl ester, and phospholipid synthesis were significantly upregulated in CFP-Plin2-expressing cells while those associated with lipolysis either decreased or were unaffected. Taken together, these data show for the first time that Plin2 interacts directly with lipids on the surface of lipid droplets and influences levels of key enzymes and lipids involved in maintaining lipid droplet structure and function.

scholarly journals Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulated lipolysis in adipocytes

2012 ◽  
Vol 125 (24) ◽  
pp. 6127-6136 ◽  
Author(s):  
T. Hashimoto ◽  
H. Segawa ◽  
M. Okuno ◽  
H. Kano ◽  
H.-o. Hamaguchi ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Droplets ◽  
Active Involvement ◽  
Associated Proteins

scholarly journals Microtubule Regulation and Function during Virus Infection

2017 ◽  
Vol 91 (16) ◽  
Author(s):  
Mojgan H. Naghavi ◽  
Derek Walsh
Keyword(s):  
Posttranslational Modifications ◽  
Intracellular Transport ◽  
Spatial Organization ◽  
Virus Particles ◽  
Cellular Processes ◽  
Wide Range ◽  
Associated Proteins ◽  
And Function ◽  
Induced Changes ◽  
Growth Pause

ABSTRACT Microtubules (MTs) form a rapidly adaptable network of filaments that radiate throughout the cell. These dynamic arrays facilitate a wide range of cellular processes, including the capture, transport, and spatial organization of cargos and organelles, as well as changes in cell shape, polarity, and motility. Nucleating from MT-organizing centers, including but by no means limited to the centrosome, MTs undergo rapid transitions through phases of growth, pause, and catastrophe, continuously exploring and adapting to the intracellular environment. Subsets of MTs can become stabilized in response to environmental cues, acquiring distinguishing posttranslational modifications and performing discrete functions as specialized tracks for cargo trafficking. The dynamic behavior and organization of the MT array is regulated by MT-associated proteins (MAPs), which include a subset of highly specialized plus-end-tracking proteins (+TIPs) that respond to signaling cues to alter MT behavior. As pathogenic cargos, viruses require MTs to transport to and from their intracellular sites of replication. While interactions with and functions for MT motor proteins are well characterized and extensively reviewed for many viruses, this review focuses on MT filaments themselves. Changes in the spatial organization and dynamics of the MT array, mediated by virus- or host-induced changes to MT regulatory proteins, not only play a central role in the intracellular transport of virus particles but also regulate a wider range of processes critical to the outcome of infection.

Triglyceride containing lipid droplets and lipid droplet-associated proteins

2008 ◽  
Vol 19 (5) ◽  
pp. 441-447 ◽  
Author(s):  
Sven-Olof Olofsson ◽  
Pontus Boström ◽  
Linda Andersson ◽  
Mikael Rutberg ◽  
Malin Levin ◽  
...  
Keyword(s):  
Lipid Droplet ◽  
Lipid Droplets ◽  
Associated Proteins
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