Staying in touch with the endocytic network: The importance of contacts for cholesterol transport

Abstract Cholesterol homeostasis is critical for cell function and human health. Cholesterol is heterogeneously distributed among cellular membranes, with the redistribution of endocytosed dietary cholesterol playing a pivotal role in the regulation of cholesterol homeostasis. While gaps remain in our understanding of intracellular dietary cholesterol transport, a highly complex network of pathways is starting to emerge, often involving inter‐dependent vesicular and non‐vesicular transport mechanisms. The last decade has seen a surge in interest in non‐vesicular transport and inter‐organellar communication at membrane contact sites. By providing platforms for protein interactions, signalling events, lipid exchange and calcium flux, membrane contact sites (MCS) are now appreciated as controlling the fate of large amounts of lipid and play central roles in the regulation and co‐ordination of endocytic trafficking. Here, we review the role of MCS in multiple pathways for cholesterol export from the endocytic pathway and highlight the intriguing interplay between vesicular and non‐vesicular transport mechanisms and relationship with neurodegenerative disease.

on the endo/lysosomal limiting membrane. 4 Loss of NPC1 or NPC2 activity results in an accumulation of free cholesterol in endocytic organelles and a corresponding reduction in re-esterification by the ER-resident enzyme ACAT, indicating impaired endosome to ER cholesterol transport. Allthough present throughout the endocytic pathway, acid lipases are more active in the increasingly acidic environment of late endosomes (LE) and lysosomes (Lys). 5 Taken together with the localization of NPC proteins in LE and Lys, this suggests that late endocytic organelles represent a major site of cholesterol egress.
Cholesterol sensing in the ER by Insig retains SCAP-SREBP complexes in the ER. When cholesterol transport to the ER is low, however, SCAP-SREBP is transported to the Golgi. Proteolytic cleavage by Golgi-resident proteases releases a soluble SREBP fragment that relocalizes to the nucleus to upregulate transcription of cholesterol biosynthesis and uptake genes. 6 Thus, delivery of dietary cholesterol to the ER is a key step in cholesterol homeostasis, necessary for the feedback loop that regulates de novo cholesterol biosynthesis as well as LDLR gene expression and therefore uptake of dietary cholesterol.
Given its importance, it is not surprising that the cell has evolved intricate mechanisms and multiple pathways for the traffic of LDL-derived cholesterol from the endocytic pathway to the ER. Defective cholesterol transport from the endocytic pathway to the ER is associated with the lysosomal storage disorder Niemann Pick disease type-C (NPC), where loss of function mutations in NPC1 or NPC2 prevent the egress of dietary cholesterol to the ER. This manifests as a devastating progressive neurodegenerative disease often presenting in early childhood. At a cellular level it is characterized by a marked accumulation of cholesterol and multiple sphingolipids in the late endocytic system. 7 Delivery of dietary cholesterol to target cellular membranes involves a complex relationship between both vesicular and nonvesicular transport mechanisms. The role of vesicular traffic in delivering material from one organelle to another has been widely studied, but only recently have we begun to appreciate the importance of non-vesicular transport in inter-organellar communication. Nonvesicular lipid transport is mediated by membrane contact sites (MCS), regions of close membrane apposition (5-30 nm) between neighbouring organelles. MCS are stabilized by tethering complexes that maintain close proximity between the opposing membranes, but without membrane fusion. These tethers can often be discerned by electron microscopy, as multiple strands between the opposing membranes of the two organelles. 8 Since their identification only a decade ago, 9,10 there has been an explosion of interest in the field of ER-endosome/lysosome MCS, leading to major advances in our understanding of their regulation and function. [11][12][13][14][15] The endocytic pathway introduces large amounts of proteins and lipids into the cell and we are starting to recognize the importance of regulatory events that take place at the interface of endocytic organelles and the ER. However, endocytic organelles form contact sites not just with the ER but also with a variety of functionally distinct organelles. Roles for these MCS in orchestrating coordinated and highly regulated signalling and trafficking events along the endocytic pathway are rapidly emerging. Here, we will briefly review our current understanding of the regulation and molecular architecture of these MCS and go on to discuss their role in the redistribution of dietary cholesterol from the endocytic pathway to other cellular membranes and the implications of these MCS in neurodegenerative disease.

| Endocytic organelle-ER MCS
The most widely studied MCS population formed by endocytic organelles is the ER-endosome contact. The ER forms extensive MCS with endocytic organelles, which increase during endosome maturation, with approximately 90% of lysosomes estimated to form an ER contact site at any given time (eg, Figure 1). 16 Together with its calcium-dependent binding partner S100A11, Annexin A1 tethers MCS between the ER and a subset of endosomes that contain epidermal growth factor receptor (EGFR), providing sites for the ER-resident phosphatase, PTP1B, to mediate effects at the endosome. 18 PTP1B dephosphorylates both endocytosed EGFR and components of the endosomal sorting complex required for transport F I G U R E 1 Endoplasmic reticulum (ER)-lysosome membrane contact sites (MCS) (orange arrows). HeLa cells prepared for electron microscopy as previously described 2 were imaged on a JOEL 1400+ TEM (ESCRT) machinery to regulate endosome maturation and receptor tyrosine kinase signalling. 9 The MCS that endocytic organelles form with the ER are heavily influenced by their lipid environment. One potential mechanism for this sterol sensitivity is the conformational change that the late endosomal sterol-binding protein ORP1L is reported to undergo when its oxysterol binding domain is in a sterol-bound state. ORP1L contains a FFAT motif (two phenylalanines in an acidic tract), through which it can interact with the MSP domain of ER-localized tethering proteins VAP 10 (VAMP-associated protein) and MOSPD2. 24 Under conditions of low cholesterol in the endocytic pathway, ORP1L's FFAT motif is exposed for binding VAP (or MOSPD2) on the ER, with an associated increase in ER-endosome connections when cells are cultured with delipidated serum. 10 Another late endosomal sterol binding, FFAT motif-containing protein, STARD3, is also implicated in tethering ER-endosome contacts. However, whereas VAP or MOSPD2 silencing reduces ER-endosome MCS, depletion of STARD3 or ORP1L has little effect on MCS formation, suggesting some redundancy between these and other VAP-binding endosomal tethering proteins, including OSBP and VPS13C as well as possibly others that are yet to be identified. In addition to stabilizing the MCS, STARD3 and ORP1L can mediate the transport of newly synthesized cholesterol from the ER to endosomes to support endosome maturation. 18,27 Interestingly, both proteins have also been implicated, under certain circumstances, in traffic of LDL-derived cholesterol from LE/Lys to ER (discussed in more detail below), suggesting functionality for ER-endocytic organelle contact sites in bidirectional transport of sterols.
Unlike STARD3, which is anchored to endosomal membranes by its MENTAL domain, ORP1L is recruited to LE by the small GTPase, Rab7, which plays a number of key roles in late endocytic membrane traffic. The extended contact between Rab7-positive LE/Lys compared with earlier (Rab7-negative) endosomes 16  Rab7 cycles between an active (effector-binding), GTP-bound state on LE/Lys membranes and an inactive, GDP-bound state, which, through interaction with GDI, is retrieved from the membrane and held in the cytosol. The significance of Rab7 nucleotide binding in MCS formation is becoming increasingly apparent. Although Rab7 was found to bind ORP1L independently of GTP/GDP-binding state, both protrudin and PDZD8 specifically bind GTP-bound Rab7 and NPC1 was recently also shown to interact with GTP-bound Rab7. 28 Moreover, a recent study demonstrated reduced ER-LE/Lys contact when GTP-bound Rab7 was decreased, whereas increasing Rab7-GTP, by inhibition of the Rab7 GAP TBC1D15, expanded the MCS. 29

| MCS with other organelles
Endocytic organelles have been shown to make contact with most organelles in the cell, including the Golgi, peroxisomes and T A B L E 1 Interactions at endoplasmic reticulum (ER)-endocytic organelle membrane contact sites (MCS) Mitochondria crosstalk with the endocytic pathway, especially through the activity of AMPK, is central to the coordination of mitochondrial and lysosomal function 32 and it has been suggested that disruption of lysosome-mitochondria signalling may contribute to Alzheimer's disease. 33 In addition, a number of functions have been  Another pathway has also been recently shown to run in parallel to NPC1-dependent cholesterol export. The lysosomal integral membrane protein, LIMP-2 was found to mediate a slower pathway of cholesterol transport to ER alongside NPC1-dependent pathways. 84 Since its luminal domain extends beyond the glycocalyx, LIMP-2 can offer an alternative mechanism for cholesterol delivery to the lysosomal membrane.
As a key role of MCS in cholesterol egress continues to emerge, so too does an association between defects in MCS proteins and neurodegenerative disease (