Our laboratory investigates the molecular mechanisms by which transmembrane proteins (referred to as cargo) are sorted to different compartments of the endomembrane system in eukaryotic cells. This system comprises an array of membrane-enclosed organelles including the endoplasmic reticulum (ER), the Golgi apparatus, the trans-Golgi network (TGN), endosomes, lysosomes, lysosome-related organelles (LROs) (e.g., melanosomes and platelet dense bodies), and different domains of the plasma membrane in polarized cells (e.g., epithelial cells and neurons). Transport of cargo between these compartments is mediated by carrier vesicles or tubules that bud from a donor compartment, translocate through the cytoplasm, and eventually fuse with an acceptor compartment. Work in our laboratory focuses on the molecular machineries that mediate these processes, including (1) sorting signals and adaptor proteins that select cargo proteins for packaging into the transport carriers, (2) microtubule motors that drive movement of the transport carriers and other organelles through the cytoplasm, and (3) tethering factors that promote fusion of the transport carriers to acceptor compartments. These machineries are studied in the context of different intracellular transport pathways, including endocytosis, recycling to the plasma membrane, retrograde transport from endosomes to the TGN, biogenesis of lysosomes and LROs, and polarized sorting in epithelial cells and neurons. Knowledge gained from this research is applied to the elucidation of disease mechanisms, including congenital disorders of protein traffic such as the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) and various neurodevelopmental disorders.
An AP-1/clathrin pathway for the sorting of transmembrane receptors to the somatodendritic domain of hippocampal neurons
Figure 2. Sorting of somatodendritic and axonal
vesicles at the pre-axonal exclusion zone (PAEZ)
A major focus of research in our laboratory is on processes mediated by recognition of sorting signals in the cytosolic tails of transmembrane proteins by adaptor proteins that are components of protein coats (e.g., clathrin coats). Two types of sorting signal referred to as tyrosine-based and dileucine-based participate in various sorting events, including endocytosis, transport to lysosomes and melanosomes, and sorting to the basolateral surface of polarized epithelial cells (Traub and Bonifacino, Cold Spring Harb. Perspect. Biol. 5:a016790, 2013). In previous work, we found that tyrosine-based signals bind to a conserved site on the mu1, mu2 and mu3 subunits of three hetero-tetrameric adaptor protein (AP) complexes, AP-1, AP-2 and AP-3, respectively. Dileucine-based signals, on the other hand, bind to a different site on the surface of two subunits, gamma-sigma1, alpha-sigma2 and delta-sigma3, of the corresponding AP-1, AP-2 and AP-3 complexes. In recent years, we extended our studies to the role of signal-adaptor interactions in the process of polarized sorting in neurons. Neurons are highly polarized cells with distinct somatodendritic and axonal domains. The plasma membrane of each of these domains possesses a distinct set of transmembrane proteins, including receptors, channels, transporters and adhesion molecules. We found that several transmembrane proteins, including the transferrin receptor (TfR), the Coxsackie virus and adenovirus receptor (CAR), and the Nipah virus fusion glycoprotein (NiV-F), are sorted to the somatodendritic domain by interaction of tyrosine-based signals with the mu1A subunit of AP-1 (Fig. 1) (Farías et al., Neuron 75:810, 2012; Mattera et al., PLoS Path. 10:e1004107, 2014) More recently, we discovered that a different set of proteins, including the copper transporter ATP7B and the SNARE VAMP4, also undergo sorting to the somatodendritic domain, but in this case through recognition of dileucine-based signals by the gamma1-sigma1 subunits of AP-1 (Jain et al., Mol. Biol. Cell. 26:218, 2015). Together with previous work on epithelial cells, these findings establish the AP-1 complex as a global regulator of polarized sorting in different cell types. Defects in polarized sorting likely underlie the pathogenesis of several neurocutaneous disorders caused by mutation in sigma1 subunit isoforms, such as the MEDNIK syndrome (sigma1A), Fried/Pettigrew syndrome (sigma1B) and pustular psoriasis (sigma1C).
Sorting of dendritic and axonal vesicles at the pre-axonal exclusion zone (PAEZ)
After packaging of cargo proteins into distinct vesicular carriers, the carriers themselves must be sorted to the axonal and somatodendritic domains. We recently found that sorting of these carriers occurs at a region in the axon hillock named the pre-axonal exclusion zone (PAEZ) (Fig. 2). The PAEZ precedes the axon initial segment (AIS) both spatially and temporally. Axonal carriers freely traverse the PAEZ en route to the distal axon, while most somatodendritic carriers are unable to enter the PAEZ. This different behavior of axonal and somatodendritic carriers depends on their ability to acquire an appropriately directed microtubule motor (Farías et al., Cell Reports 13:1221, 2015).
BORC: a multisubunit complex that regulates lysosome positioning and motility
Figure 3. BORC regulates kinesin-dependent
movement of lysosomes to the cell periphery.
In another line of research, we obtained unexpected insights into the mechanisms of lysosome positioning and motility. This research was an outgrowth of our previous work on the biogenesis of LROs such as melanosomes. Years ago, we discovered that mutations in AP-3 cause eye color defects in Drosophila and the pigmentation and bleeding disorder Hermansky-Pudlak syndrome (HPS) type 2 (HPS-2) in humans. Other types of HPS are caused by mutations in subunits of the hetero-octameric BLOC-1 and the hetero-dimeric BLOC-3 complexes. Whereas AP-3 mediates sorting of transmembrane proteins to melanosomes, the functions of BLOC-1 and BLOC-3 are less well understood. In experiments aimed at identifying proteins that interact with BLOC-1, we made the surprising discovery of a related hetero-octameric complex named BORC (for BLOC-one-related complex) (Fig. 3) (Pu et al., Dev. Cell 33:176, 2015). Biochemical analyses showed that BORC comprises three subunits shared with BLOC-1 (named BLOS1, BLOS2 and Snapin) and five additional subunits (named KXD1, MEF2B, Myrlysin, Lyspersin and Diaskedin). Further studies revealed that BORC is associated with late endosomes and lysosomes, where it functions to recruit the small GTPase Arl8. This initiates a chain of interactions that drives kinesin-dependent movement of lysosomes toward the peripheral cytoplasm. Mutations in BORC cause collapse of the lysosomal population into the pericentrosomal area. In addition, BORC-mutant cells exhibit defective autophagic flux, probably due to the inability of lysosomes to reach autophagosomes in the cell periphery. These cells also display reduced spreading and migration, likely caused by impaired lysosome-dependent delivery of adhesion and signaling molecules to the plasma membrane. Because of the critical importance of cell adhesion and motility in tumor growth, invasion, and metastasis, the BORC pathway of lysosome dispersal could be an attractive target for pharmacologic inhibition in cancer therapeutics.
GARP and EARP: multisubunit tethering complexes involved in endosomal retrieval pathways
Figure 4. multisubunit tethering complexes
involved in endosomal retrieval pathways.
Recycling of endocytic receptors to the cell surface involves passage through a series of membrane-bound compartments by mechanisms that are poorly understood. In the course of studies on the Golgi-associated retrograde protein (GARP) complex, we discovered a related complex named endosome-associated recycling protein (EARP) (Fig. 4) (Schindler et al., Nat. Cell Biol. 17:639, 2015). The two complexes share the Ang2 (also known as Vps51), Vps52 and Vps53 subunits, but whereas GARP comprises a fourth subunit named Vps54, EARP contains a previously uncharacterized protein named Syndetin. This change determines differential localization of GARP to the TGN and EARP to recycling endosomes. Importantly, we found that EARP is involved in recycling of internalized proteins to the plasma membrane. These findings contribute to the understanding of the pathogenesis of progressive cerebello-cerebral atrophy type 2, a neurodegenerative disorder caused by mutations in Vps53, which in light of our results could result from impairment of both GARP-mediated retrograde transport to the TGN and EARP-mediated recycling to the plasma membrane.