COPII vesicle biogenesis and secretion
The trafficking of most secretory cargoes, including proteins and lipids, begins with their export from the endoplasmic reticulum (ER). In metazoans, cargoes are packaged into transport carriers that emerge at defined sites on the ER and ultimately fuse with the ER-Golgi intermediate compartment (ERGIC). This process relies on the efficient recruitment of a set of soluble factors known as the COPII coat, which is composed of two multimeric protein complexes (Sec23/24 and Sec13/31).
Although COPII coated vesicle formation has been reconstituted with purified coat proteins on synthetic membranes, regulators of COPII assembly in vivo remain largely unexplored. Of the known COPII interacting proteins, two membrane-associated proteins, the small GTPase Sar1 and the putative scaffolding protein Sec16, are among the best characterized. In its GTP bound state, an amphipathic α-helix within Sar1 is exposed and inserts into the lipid bilayer to induce membrane curvature. Additionally, Sar1-GTP recruits the Sec23/24 complex, which forms an adaptor layer for Sec13/31 lattice assembly, completing the COPII coat. Sec23 also functions as a Sar1 GAP, which is ultimately stimulated by Sec31, leading to coat disassembly following vesicle budding.
In contrast to Sar1, which has only been shown to associate directly with Sec23, Sec16 interacts with multiple components of the COPII coat, potentially serving as a scaffold for their recruitment. Furthermore, Sec16 has been postulated to stabilize the COPII coat to prevent premature disassembly following activation of the Sec23 GAP. In vitro, Sec16 is dispensable for the generation of COPII coated vesicles. However, it can stimulate the function of COPII proteins, suggesting an important regulatory function. Consistent with this idea, multiple studies using a variety of model systems have demonstrated that Sec16 is essential for the exit of secretory proteins from the ER.
In addition to Sar1 and Sec16, few other proteins have been implicated in the regulation of COPII recruitment. Considering the necessity for controlling secretory flux during cell differentiation and development, additional factors that govern this process likely exist, and some may function via direct regulation of Sar1 or Sec16. To identify new regulators of ER export, we conduct biochemical and genetics screens to identify new components that control COPII-mediated vesicle biogenesis, vesicle budding, and vesicle fusion with post-ER compartments. Our identification of TFG (Trk-fused gene) at the ER/ERGIC interface has fundamentally altered our understanding of the regulatory mechanisms that govern early secretory pathway trafficking.
Clathrin-mediated endocytosis
The clathrin coat functions at multiple intracellular locations to package a wide range of cargoes for transport. However, clathrin does not engage substrates directly nor does it exhibit the ability to associate with membranes. Instead, recruitment of clathrin is mediated by specific adaptor proteins found on different organelles, which interact with cargoes and lipid bilayers. At the plasma membrane, several endocytic adaptors coordinately regulate clathrin accumulation. The best characterized is AP-2, a
heterotetrameric adaptor complex that can simultaneously bind to clathrin, sorting signals on cargoes and phosphatidylinositol 4,5-bisphosphate, a lipid specifically enriched on the cell surface. Additionally, AP-2 associates directly with other endocytic adaptors including the Eps15 homology (EH) domain containing proteins Eps15 and intersectin, members of the muniscin family such as FCHO1, and the cargo-binding factors Dab2 and Epsin. Thus, the AP-2 complex is a multifunctional scaffold that promotes the formation of cargo-laden, clathrin-coated subdomains on the plasma membrane.
Despite its central role in clathrin-mediated endocytosis, the AP-2 complex is unlikely to be the sole initiator of the process. Studies in C. elegans have demonstrated that cells lacking components of AP-2 continue to perform clathrin-mediated endocytosis, indicating that alternative pathways exist to recruit clathrin to the cell surface. In particular, the FCHO proteins, Eps15 and intersectin exhibit highly similar recruitment kinetics as compared to AP-2 and clathrin and may function independently of AP-2. Current models suggest that the FCHO proteins initiate membrane bending and cargo clustering via their F-BAR and μ-homology domains, respectively. Simultaneously, Eps15 and intersectin recruit downstream adaptors and accessory proteins, which collectively drive maturation of the nascent endocytic pit.
A large number of cell surface molecules undergo internalization in a clathrin-dependent fashion. This process requires multiple endocytic adaptors to recognize largely distinct cargoes in a manner that relies on short signal sequences or post-translational modifications found within substrates. To understand how such an array of macromolecules can be efficiently internalized, we take advantage of genetic, biochemical, and microscopy-based approaches to dissect the mechanisms underlying clathrin recruitment to the plasma membrane.
The ESCRT machinery
Endosomal compartments undergo a wide variety of remodeling events to efficiently sort cargoes to multiple, unique destinations. Membrane tubulation at endosomes, mediated by BAR domain proteins and members of the EHD family of ATPases, has been shown to promote endocytic recycling to the cell surface. In contrast, components of the ESCRT (Endosomal Sorting Complex Required for Transport) machinery have been implicated in the formation of intralumenal vesicles (ILVs), which exhibit negative membrane curvature
and bud away from the cytoplasm toward the endosome interior. In topologically similar processes, the ESCRT machinery also participates in membrane abscission during cytokinesis, the formation of retroviral particles that bud from the cell surface during infection, plasma membrane repair, and nuclear envelope resealing after mitosis.
The ESCRT machinery is composed of five multi-subunit complexes (ESCRT-0, ESCRT-I, ESCRT-II, ESCRT-III, and the Vps4 complex) that each exhibits unique membrane binding properties. At the endosome, early acting components of the machinery function to select and concentrate cargo molecules for deposition into ILVs. This sorting event depends on the presence of ubiquitin, which is conjugated to membrane proteins destined for turnover. Subsequently, downstream ESCRT complexes maintain cargo within endosomal subdomains, while simultaneously initiating inward membrane bending. Ultimately, assembly of ESCRT-III filaments at the vesicle bud neck drives vesicle scission, thereby sequestering cargo molecules within the lumen of the endosome. The ATPase activity of the Vps4 complex is necessary to remodel and disassemble the ESCRT-III complex and recycle its
components for future rounds of vesicle biogenesis. Using a wide range of biochemical, biophysical, genetic, and structural approaches, we aim to define mechanisms by which the ESCRT machinery regulates cargo trafficking and degradation in the endosomal system. Furthermore, we are extending our studies to determine whether similar principles apply to ESCRT function during cytokinesis, nuclear envelope sealing, and viral budding.
In a particularly exciting line of investigation, we identified the first curvature sensitive component of the ESCRT machinery, which may play a key role in targeting ESCRT function during lumenal vesicle formation at endosomes. Specifically, in collaboration with investigators in the UK, we have shown that a complex of ESCRT-II bound to the ESCRT-III component Vps20 binds selectively to membranes of high curvature, similar to that found at a vesicle bud neck. Our data further suggest that the ESCRT-II/Vps20 complex is mechanosensitive, binding more tightly to membranes as they become increasingly bent, which may stabilize curvature sensitive ESCRT-III filaments within the vesicle bud neck as the membrane deforms. Ongoing studies aim to define the contributions of individual ESCRT-III subunits to ILV biogenesis.
Lipid signaling
Cellular membranes are composed of numerous lipid species that function together to maintain subcellular compartmentalization and recruit downstream effector proteins. In particular, acidic phospholipids, including phosphorylated derivatives of phosphatidylinositol (PIPs) and phosphatidylserine (PS), are ideally suited to bind positively charged peptide sequences within peripheral membrane proteins, often activating these effectors to carry out their specific function(s). Using domains that uniquely recognize particular lipid head groups, a distribution map of PIPs and PS has evolved, highlighting important roles for PI3P at early endosomes, PI4P at the Golgi, and PI4,5P2 and PS at the plasma membrane of C. elegans embryos and human tissue culture cells. Using a variety of biochemical and genetic approaches, we are studying the roles of lipid signaling in regulating key steps of membrane reorganization, which is necessary for vesicle formation throughout the endomembrane system.
Changes in lipid composition can alter bilayer topology and promote curvature during vesicle biogenesis and membrane tubulation. In particular, members of the phospholipase A2 (PLA2) superfamily cleave the sn-2 acyl bond of phospholipids and release two biologically active molecules, a lysophospholipid and a free fatty acid. Importantly, lysophospholipids can dramatically impact membrane architecture. More specifically, an increased local concentration of lysophospholipid density in a lipid bilayer can generate or stabilize membrane curvature by introducing cone-shaped molecules into a relatively flat surface. Independently, the released fatty acid can function as a second messenger in cell signaling. For example, arachidonic acid liberated from PLA2-mediated hydrolysis of membrane phospholipids has been shown to alter the activity of ion channels and protein kinases in neurons, which affect their excitability. Additionally, arachidonic acid is a precursor for eicosanoid production, which regulates the inflammatory response and other signal transduction events. Using biochemical and biophysical approaches, we are investigating the functions of a group of calcium-independent PLA2 enzymes that have been implicated both in organelle remodeling and membrane trafficking. Our ultimate goal is to understand how these factors function together with membrane bending proteins to orchestrate temporally and spatially regulated membrane remodeling events.
Rab-type GTPases
Entry of cells into mitosis is accompanied by a dramatic reorganization of several organelles including the ER, Golgi, and endosomal system. Previous studies have shown that members of the Rab family of small G proteins function as critical regulators of membrane organization, but their roles in organelle restructuring during development remain largely uncharacterized. We have identified the subset of essential Rab-type GTPases necessary for C. elegans embryogenesis. To explore the roles of these essential proteins upon mitotic entry, we have conducted partial depletions and focused on post-fertilization membrane reorganization. This analysis has uncovered new roles for several Rab-type GTPases that have been previously overlooked. For example, we found that depletion of the endosomal Rab-type GTPase Rab5 results in a pronounced defect in nuclear membrane disassembly during mitosis that is independent of its well-characterized roles in endocytosis. Direct analysis of post-fertilization membrane dynamics revealed a specific failure in ER reorganization that normally accompanies nuclear membrane disassembly. These observations have both provided clues to the mechanisms underlying ER remodeling during cell cycle progression and suggest a function for ER remodeling in the disassembly of the nuclear envelope. Strikingly, expression of a constitutively active form of Rab5 abnormally potentiates ER reorganization in both C. elegans and cultured HeLa cells, suggesting a direct and conserved role for Rab5 signaling in this process. To support these in vivo findings, we have also used ER enriched membranes isolated from Xenopus eggs to assemble ER tubules in vitro. Addition of Rab GDI, which specifically inhibits Rab-type GTPases, blocks ER tubule formation, while Rho GDI, an inhibitor of related Rho-type GTPases, fails to have any effect. Together these data demonstrate a new function for Rab5 in membrane reorganization, and underscore the importance of crosstalk between different organelles.
To determine relevant effectors in Rab5-mediated membrane remodeling during early development, we have initially undertaken a biochemical approach. Using comparative mass spectrometry analysis, we have identified a set of C. elegans embryonic proteins that specifically interact with Rab5-GTP, but not Rab5-GDP. These include numerous known Rab5 effectors, validating our approach, as well as three new proteins that may regulate ER and/or endosome membrane dynamics during development. We are currently using RNAi and fluorescence-based functional assays for organelle remodeling to uncover the roles of these new factors. Of particular interest, we have identified a conserved protein phosphatase important for mitotic progression that binds to active Rab5, suggesting a link between membrane remodeling and cell cycle progression. Additionally, we have successfully purified the 5 other Rab-type GTPases necessary for embryogenesis, and plan to identify new effector molecules for each using a similar mass spectrometry-based approach, followed by functional analysis in vivo. Our studies will provide a comprehensive analysis of Rab-type GTPase function in membrane dynamics during oocyte fertilization and early embryogenesis and establish a key framework that catalyzes future investigation of related events in mammalian development.
Cancer
Dysregulated cell proliferation underlies all forms of oncogenesis. In particular, chromosomal aberrations sometimes enable a subpopulation of cells to grow in an uncontrolled fashion, leading to tumor formation. Such defects are often associated with changes in cellular signal transduction pathways, such as the Ras-Raf-MEK-ERK kinase cascade, which promotes cell survival and growth. Notably, upregulated ERK activity has been implicated in numerous malignancies, including papillary thyroid carcinoma, pancreatic cancer, colorectal cancer, melanoma, and lung cancer. During the course of our studies, we demonstrated that two oncogenic fusion proteins, TFG-NTRK1 and TFG-ALK, created by distinct chromosomal translocation events, localize to subdomains of the endoplasmic reticulum (sites of COPII vesicle formation) and dramatically upregulate ERK activity. Using phosphoproteomic approaches, we are mapping downstream effectors of TFG-NTRK1 and TFG-ALK that simultaneously drive cell transformation and regulate vesicle secretion. By altering membrane transport in the early secretory pathway, TFG fusion proteins may modulate cargo export, potentially enhancing the secretion of growth factors that help to sustain a rich tumor microenvironment.
Importantly, the Ras-Raf-MEK-ERK signaling cascade is a known effector of several ligand-activated growth factor receptors, including EGFR and Her2. Upon stimulation, these receptors hyperactivate Ras, leading to upregulated ERK signaling. To terminate signaling, the receptor is typically downregulated and sequestered within the lumen of the endosome, a process dependent on the ESCRT machinery. Failure to properly route activated receptors for turnover can lead to constitutive mitogenic signaling and potentially oncogenic transformation. Consistent with this idea, numerous mutations that perturb transport through the endocytic system are also associated with cancer. Thus, our studies of endocytosis and ESCRT function also contribute to a better understanding of the mechanisms that govern the termination of receptor signaling, which is essential for normal development.
Neurodegenerative disease
Synaptic transmission depends on the constant microtubule-based transport of organelles and vesicles to and from the distal portions of axons and dendrites. Defects in these processes can lead to a variety of neurodegenerative disorders. In particular, hereditary spastic paraplegias (HSPs) arise from a length-dependent axonopathy of corticospinal motor neurons. This diverse group of disorders is characterized by progressive lower-limb spasticity and weakness and has been linked to more than 100 unique genetic loci, many of which encode proteins that function in cytoskeleton and organelle homeostasis. However, mechanistic insights into the direct effect of mutations that cause HSPs are lacking.
In collaboration with a number of groups from around the world, we have identified and characterized the impact of point mutations observed in adolescent patients who exhibit complicated forms of HSP, which alters highly conserved residues within Trk-fused gene (TFG). In addition to pronounced leg spasticity, vision problems due to optic atrophy are noted in some cases. Wasting of hand and leg muscles, as well as electromyography findings, indicate additional neuropathy, whereas there is limited evidence for sensory involvement in these cases. Importantly, our previously published findings indicated that TFG functions on subdomains of the endoplasmic reticulum (ER) to regulate anterograde vesicle transport, which is mediated by COPII-coated carriers. Although COPII function has been implicated directly in dendritic growth, its potential role in axonal development and maintenance remains less clear. A single amino acid change in TFG (p.R106C) that causes HSP impairs the ability of TFG to oligomerize normally, which adversely affects its function in vivo. In non-neuronal cells, our studies demonstrate that depletion of TFG results in the collapse of the ER network onto the underlying microtubule cytoskeleton, dramatically altering its morphology and dynamics. Furthermore, the distribution of mitochondria within cells lacking TFG function is highly abnormal, suggesting that TFG may function to coordinate interactions between multiple organelles and microtubules. Our goal is to explore this idea and define new mechanisms that sustain and enhance neuron viability and activity during development and aging.