Autophagy is a central mechanism by which hepatocytes catabolize lipid droplets (LDs). Currently, the regulatory mechanisms that control this important process are poorly defined. The small GTPase Rab7 has been implicated in the late endocytic pathway and is known to associate with LDs, although its role in LD breakdown has not been tested. In this study, we demonstrate that Rab7 is indispensable for LD breakdown (“lipophagy”) in hepatocytes subjected to nutrient deprivation. Importantly, Rab7 is dramatically activated in cells placed under nutrient stress; this activation is required for the trafficking of both multivesicular bodies (MVBs) and lysosomes to the LD surface during lipophagy, resulting in the formation of a lipophagic “synapse.” Depletion of Rab7 leads to gross morphological changes of MVBs, lysosomes, and autophagosomes, consequently leading to attenuation of hepatocellular lipophagy. Conclusion These findings provide additional support for the role of autophagy in hepatocellular LD catabolism while implicating the small GTPase Rab7 as a key regulatory component of this essential process.

Cells form transient, circular dorsal ruffles or ''waves'' in response to stimulation of receptor tyrosine kinases, including epidermal growth factor receptor (EGFR) or platelet-derived growth factor receptor. These dynamic structures progress inward on the dorsal surface and disappear, occurring concomitantly with a marked reorganization of F-actin. The cellular function of these structures is largely unknown. Here we show that EGF-induced waves selectively sequester and internalize f50% of ligand-bound EGFR from the cell surface. This process requires receptor phosphorylation, active phosphatidylinositol 3-kinase, and dynamin 2, although clathrincoated pits or caveolae are not required. Epithelial and fibroblast cells stimulated with EGF sequestered EGFR rapidly into waves that subsequently generated numerous receptorpositive tubular-vesicular structures. Electron microscopy confirmed that waves formed along the dorsal membrane surface and extended numerous tubules into the cytoplasm. These findings characterize a structure that selectively sequesters large numbers of activated EGFR for their subsequent internalization, independent of traditional endocytic mechanisms such as clathrin pits or caveolae. (Cancer Res 2006; 66(7): 3603-10)

The actin cytoskeleton is believed to contribute to the formation of clathrin-coated pits, although the specific components that connect actin filaments with the endocytic machinery are unclear. Cortactin is an F-actinassociated protein, localizes within membrane ruffles in cultured cells, and is a direct binding partner of the large GTPase dynamin. This direct interaction with a component of the endocytic machinery suggests that cortactin may participate in one or several endocytic processes. Therefore, the goal of this study was to test whether cortactin associates with clathrin-coated pits and participates in receptor-mediated endocytosis. Morphological experiments with either anti-cortactin antibodies or expressed red fluorescence protein-tagged cortactin revealed a striking colocalization of cortactin and clathrin puncta at the ventral plasma membrane. Consistent with these observations, cells microinjected with these antibodies exhibited a marked decrease in the uptake of labeled transferrin and low-density lipoprotein while internalization of the fluid marker dextran was unchanged. Cells expressing the cortactin Src homology three domain also exhibited markedly reduced endocytosis. These findings suggest that cortactin is an important component of the receptor-mediated endocytic machinery, where, together with actin and dynamin, it regulates the scission of clathrin pits from the plasma membrane. Thus, cortactin provides a direct link between the dynamic actin cytoskeleton and the membrane pinchase dynamin that supports vesicle formation during receptor-mediated endocytosis.The actin cytoskeleton, with its associated proteins, has been implicated in endocytic processes in both yeast and mammalian cells (5,25,41). How this cytoskeletal network might interact with the endocytic machinery to regulate vesicle formation and scission remains unclear. Recently, Pelkmans and colleagues reported that actin and dynamin accumulated at simian virus 40-docked caveolae concomitant with internalization, thus demonstrating that actin functions during the endocytic process (24). Similarly, Merrifield et al. demonstrated that clathrin, dynamin, and actin are sequentially recruited to form clathrin pits, marked by an accumulation of actin and movement of the vesicles away from the plasma membrane (20). Both of these studies demonstrated an accumulation of dynamin and actin at the forming endocytic membranes but did not directly link the endocytic and cytoskeletal machineries. Recently, it was demonstrated that the actin-binding protein cortactin associates directly with the large GTPase dynamin (Dyn2) via their Src homology three (SH3) and prolinerich domains (PRD), respectively (19). As Dyn2 participates in the liberation of clathrin-coated pits (CCPs) from the plasma membrane, a potential link exists between the endocytic machinery and the actin cytoskeleton through cortactin (18).Cortactin is an 80-and 85-kDa protein that localizes within membrane ruffles in cultured cells (19,43) and was originally identified as a subst...

Cortactin is an actin-binding protein that has recently been implicated in endocytosis. It binds directly to dynamin-2 (Dyn2), a large GTPase that mediates the formation of vesicles from the plasma membrane and the Golgi. Here we show that cortactin associates with the Golgi to regulate the actin- and Dyn2-dependent transport of cargo. Cortactin antibodies stain the Golgi apparatus, labelling peripheral buds and vesicles that are associated with the cisternae. Notably, in vitro or intact-cell experiments show that activation of Arf1 mediates the recruitment of actin, cortactin and Dyn2 to Golgi membranes. Furthermore, selective disruption of the cortactin-Dyn2 interaction significantly reduces the levels of Dyn2 at the Golgi and blocks the transit of nascent proteins from the trans-Golgi network, resulting in swollen and distended cisternae. These findings support the idea of an Arf1-activated recruitment of an actin, cortactin and Dyn2 complex that is essential for Golgi function.

Lipid droplet (LD) catabolism in hepatocytes is mediated by a combination of lipolysis and a selective autophagic mechanism called lipophagy, but the relative contributions of these seemingly distinct pathways remain unclear. We find that inhibition of lipolysis, lipophagy, or both resulted in similar overall LD content but dramatic differences in LD morphology. Inhibition of the lipolysis enzyme adipose triglyceride lipase (ATGL) resulted in large cytoplasmic LDs, whereas lysosomal inhibition caused the accumulation of numerous small LDs within the cytoplasm and degradative acidic vesicles. Combined inhibition of ATGL and LAL resulted in large LDs, suggesting that lipolysis targets these LDs upstream of lipophagy. Consistent with this, ATGL was enriched in larger-sized LDs, whereas lipophagic vesicles were restricted to small LDs as revealed by immunofluorescence, electron microscopy, and Western blot of size-separated LDs. These findings provide new evidence indicating a synergistic relationship whereby lipolysis targets larger-sized LDs to produce both size-reduced and nascently synthesized small LDs that are amenable for lipophagic internalization.