Reconstitution of Microtubule-Dependent Organelle Transport

Barak, Pradeep; Rai, Ashim; Dubey, Alok Kumar; Rai, Priyanka; Mallik, Roop

doi:10.1016/b978-0-12-397924-7.00013-3

Cited by 13 publications

(25 citation statements)

References 41 publications

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“…We therefore performed LC-MS–based quantitative lipid profiling of LDs purified from liver of normally fed rats (NLDs) and 16-h-fasted rats (FLDs). The procedures for purifying LDs and LC-MS analysis have been described (Barak et al, 2014; Rai et al, 2017). Lipidomics data showed that multiple species of PA are elevated on NLDs compared with FLDs (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Insulin activates intracellular transport of lipid droplets to release triglycerides from the liver

Kumar

Ojha

Rai

et al. 2019

Journal of Cell Biology

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Triglyceride-rich lipid droplets (LDs) are catabolized with high efficiency in hepatocytes to supply fatty acids for producing lipoprotein particles. Fasting causes a massive influx of adipose-derived fatty acids into the liver. The liver in the fasted state is therefore bloated with LDs but, remarkably, still continues to secrete triglycerides at a constant rate. Here we show that insulin signaling elevates phosphatidic acid (PA) dramatically on LDs in the fed state. PA then signals to recruit kinesin-1 motors, which transport LDs to the peripherally located smooth ER inside hepatocytes, where LDs are catabolized to produce lipoproteins. This pathway is down-regulated homeostatically when fasting causes insulin levels to drop, thus preventing dangerous elevation of triglycerides in the blood. Further, we show that a specific peptide against kinesin-1 blocks triglyceride secretion without any apparent deleterious effects on cells. Our work therefore reveals fundamental mechanisms that maintain lipid homeostasis across metabolic states and leverages this knowledge to propose a molecular target against hyperlipidemia.

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Section: Resultsmentioning

confidence: 99%

“…LDs for biochemical assays were purified from adherent McA-RH777 cells using protocols previously reported (Barak et al, 2014; Rai et al, 2017), with minor modifications. Briefly, naive cells and KIF5B-GFP– and KIF5B(PA − )-GFP–overexpressing stable McA-RH7777 cells were cultured in ≥6 plates of 150 mm size each.…”

Section: Methodsmentioning

confidence: 99%

Insulin activates intracellular transport of lipid droplets to release triglycerides from the liver

Kumar

Ojha

Rai

et al. 2019

Journal of Cell Biology

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“…We have described a detailed protocol (also see methods ) for the purification of LDs from rat liver by density gradient ultracentrifugation [ 33 , 34 ]. LDs purified from liver of normally fed rats will be called N ormal LD s (NLDs) .…”

Section: Resultsmentioning

confidence: 99%

Feeding-fasting dependent recruitment of membrane microdomain proteins to lipid droplets purified from the liver

2017

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Lipid droplets (LDs) are cellular stores of neutral fat that facilitate lipid and protein trafficking in response to metabolic cues. Unlike other vesicles, the phospholipid membrane on the LD is a monolayer. Interestingly, this monolayer membrane has free cholesterol, and may therefore contain lipid microdomains that serve as a platform for assembling proteins involved in signal transduction, cell polarity, pathogen entry etc. In support of this, cell culture studies have detected microdomain-associated “raftophilic” proteins on LDs. However, the physiological significance of this observation has been unclear. Here we show that two proteins (Flotillin-1 and SNAP23) that bind to membrane microdomains associate differently with LDs purified from rat liver depending on the feeding/fasting state of the animal. Flotillin-1 increases on LDs in the fed state, possibly because LDs interact with the endoplasmic reticulum (ER), facilitating supply of flotillin-1 from ER to LDs. Interestingly, this increase in flotillin-1 is correlated with an increase in free cholesterol on the LDs in fed state. In opposite behaviour to flotillin-1, SNAP23 increases on LDs in the fasted state and this appears to mediate LD-mitochondria interactions. Such LD-mitochondria interactions may provide fatty acids to mitochondria for promoting beta-oxidation in hepatocytes in response to fasting. Our work brings out physiologically relevant aspects of lipid droplet biology that are different from, and may not be entirely possible to replicate and study in cell culture.

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“…LDs are ideally suited for optical trapping experiments [215], and this property makes it possible to measure the forces with which LDs move, both in vitro and in vivo [154, 216, 217]. Such force measurements can be used, for example, to determine the number of motor molecules moving a single droplet [218, 219], to determine how opposing motors interact [220], and how the activity of motors and their force production is regulated [221-224].…”

Section: 0 Other Roles Of Lipid Dropletsmentioning

confidence: 99%

Lipid droplet functions beyond energy storage

Welte

Gould

2017

Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biolog

458

358

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Lipid droplets are cytoplasmic organelles that store neutral lipids and are critically important for energy metabolism. Their function in energy storage is firmly established and increasingly well characterized. However, emerging evidence indicates that lipid droplets also play important and diverse roles in the cellular handling of lipids and proteins that may not be directly related to energy homeostasis. Lipid handling roles of droplets include the storage of hydrophobic vitamin and signaling precursors, and the management of endoplasmic reticulum and oxidative stress. Roles of lipid droplets in protein handling encompass functions in the maturation, storage, and turnover of cellular and viral polypeptides. Other potential roles of lipid droplets may be connected with their intracellular motility and, in some cases, their nuclear localization. This diversity highlights that lipid droplets are very adaptable organelles, performing different functions in different biological contexts.

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Reconstitution of Microtubule-Dependent Organelle Transport

Cited by 13 publications

References 41 publications

Insulin activates intracellular transport of lipid droplets to release triglycerides from the liver

Insulin activates intracellular transport of lipid droplets to release triglycerides from the liver

Feeding-fasting dependent recruitment of membrane microdomain proteins to lipid droplets purified from the liver

Lipid droplet functions beyond energy storage

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