Endocytic transport pathways

Overview

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The Grant lab has long focused on uncovering the fundamental players and mechanisms that produce directed transport between membrane systems in vivo. Our work has focused most intensely on endocytic trafficking, and the intricate functions that allow endosomes to sort and transport cargo to specific destinations within the cell. Endocytic trafficking, the transport of membrane-associated proteins and lipids back and forth between the plasma membrane and endosomes, is a vital mechanism by which cells sense and interact with their environment and is essential to all eukaryotic life. Endocytic transport controls the composition of the plasma membrane, mediates nutrient uptake, and regulates key processes such as cell division, cell migration, cell polarity, antigen processing by the immune system, nervous system activity, and growth factor receptor signaling during development and disease (Grant and Donaldson, 2009).

Our work is unique in that we pioneered the microscopic nematode C. elegans for the analysis of the fundamental biology of endocytic transport, allowing our research group to make repeated discoveries that would prove generalizable to other systems, including human cells. By combining the great technical advantages of advanced C. elegans molecular genetics with cell biological and biochemical methods, our research has provided many new insights into the basic trafficking processes shared by all multicellular animals. Our high throughput genetic screens identified hundreds of genes required for endocytic traffic and the secretory pathway (Balklava et al., 2007). Many of the endocytic regulators that we discovered first are named RME proteins, including RME-1 and RME-8, the analysis of which proved particularly influential. Our most recent work has delved into multiple facets of under-explored biology, with our studies contributing understanding of the formation and maintenance of endosomal microdomains (Norris et al, 2017; Norris and Grant 2020; Norris et al., 2022), endosomal tubule formation and fission (Rodriguez-Polanco et al., 2023), autophagic lysosome reformation (Swords et al., 2024), giant vesicle ejection by stressed neurons (Wang et al., 2023; Cooper et al., 2021; Arnold et al., 2023), and the phagocytic interactions of neighboring cells with emerging neuronal vesicles (Wang et al., 2023), all analyses within the physiological context of the living animal.

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Figure 5

Proteostasis, Aging Neurons, and Giant Extracellular Vesicles (Exophers)

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Healthy aging of the brain is highly dependent upon a range of protein quality control systems, and such quality control capacity is often disrupted in neurodegenerative disease. Recently it has come to light that diseased neurons can transfer toxic products such as aggregated proteins to neighboring cells, likely leading to spreading pathology within the brain. How neurons generate and send out extracellular material in vivo is a question that must be addressed as we consider therapeutic intervention. We have been collaborating closely with Monica Driscoll’s lab to understand the mechanisms behind formation of a new class of neuron-derived giant vesicles called “exophers” that carry toxic aggregates and organelles out of the cell and are induced by several cell stressors (Fig. 5) (Cooper et al., 2021; Arnold et al., 2023). Similar mechanisms of giant vesicle budding and transfer of aggregates, lipids, and damaged organelles have been recently identified in C. elegans muscle, mouse cardiomyocytes, and mouse and human brain, strongly implying that discoveries we make about how this process operates in C. elegans will be widely relevant across species and tissues. Our contributions to this work have discovered specific mechanisms for triggering exopher production, such as association of intermediate filaments with aggresomes, and specific pathways in the uptake and degradation of exophers by the neighboring hypodermal cell (Fig.6) (Cooper et al., 2021; Wang et al., 2023; Arnold et al., 2023). Importantly our studies identified a surprisingly strict and non-autonomous requirement for phagocytic recognition of nascent exophers by the hypodermal cell for the neuron to complete formation of the exopher, a mechanism that we liken to synaptic pruning by glia (Wang et al., 2023).

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Autophagic Lysosome Reformation

Autophagic Lysosome Reformation

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Two phenomena have driven a recent interest in deciphering trafficking mechanisms relevant to neuronal heath and function. One of these was inspired by recent human genetics studies linking RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We extended our RME-8/DNAJC13 protein studies into neurons to find that that loss of RME-8/DNAJC13 in C. elegans and mouse neurons (in collaboration with Qian Cai) result in accumulation of grossly elongated autolysosomal tubules (Swords et al., 2024). In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with PI(3)P regulators bec-1/beclin and vps-15 (Swords et al., 2024). We concluded that RME-8/DNAC13 plays a conserved but previously unrecognized role in autophagic lysosome reformation affecting ALR tubule severing. While we continue to define how this mechanism works, our studies already provide new insight into how lysosomal recycling tubules are released, suggest that ALR feeds back to autophagic flux control, and provides a likely explanation for how RME-8 contributes to neurodegenerative disease by contributing to lysosome function/dysfunction.

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Figure 4

Endosomal Microdomains: Retromer, ESCRT, and Their Cross-Regulation

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The most fundamental function of an endosome is to sort cargo. The endosome field has expended tremendous effort to define and understand the peripheral membrane complexes that sort cargo, with one major advance anchored in the recognition that endosomal sorting complexes can define functional microdomains on the endosomal limiting membrane (Norris and Grant, 2020). Our studies were the first to develop an in vivo light microscopy-based system capable of reliably visualizing, measuring, and manipulating endosomal microdomains, taking advantage of the naturally large (1-5 micron diameter) endosomes of C. elegans coelomocyte cells (Fig. 4) (Norris et al., 2017). Our studies discovered for the first time that the recycling Retromer containing microdomains and degradative ESCRT containing microdomains on endosomes are distinct and cross-regulate—the RME-8 Hsc70-co-chaperone associates with SNX-1 in the recycling microdomain, preventing the assembly of degradative ESCRT-0 and clathrin within the recycling microdomain. Without RME-8, the microdomains mix and recycling cargo inappropriately degrades (Norris and Grant, 2017; Norris et al., 2020).

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FIg 2

Recycling Transport Through a Tubular Endocytic Network

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We were the first to show that C. elegans RME-1 and mammalian mRME-1/EHD1 (with Fred Maxfield) are required for recycling endosome to plasma membrane transport. With our collaborator Lois Greene we also showed that proteins of the RME-1 family are ATPases and require ATP binding for homo-oligomerization and association with endosomes (Lee et al., 2005). We later identified several RME-1 interacting proteins that collaborate with RME-1 in this function, including the BAR-domain protein AMPH-1/Amphiphysin/BIN1 (Shi et al., 2007; Pant et al., 2009; Liu and Grant, 2015). Furthermore, with our collaborator Steve Caplan, we showed that mammalian Amph2/BIN1 is also required for recycling endosome function (Pant et al., 2009). Importantly, in collaboration with Chavela Carr we advanced this work by establishing a synthetic liposome and pure recombinant protein reconstitution system for these proteins. In vitro we found that pure recombinant RME-1 forms a distinctive spiral coat that tubulates liposomes, consistent with our proposal that RME-1/EHD family proteins function as part of the tubulation and fission machinery on endosomal tubules (Pant et al., 2009).

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YP170::GFP

Identifying New Endocytosis Components

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One highly successful focus has been our group’s comprehensive identification of novel proteins that control endocytic transport, and our subsequent dissection of their mechanisms of action. Using the tagged ligand assay system that we invented following yolk protein YP170, our laboratory systematically tested nearly every gene in the worm genome by RNAi for effects on the basolateral secretion of yolk proteins by the intestinal epithelium and its subsequent endocytosis by the oocytes (Balklava et al., 2007). After secondary assays our studies identified hundreds of endocytosis and secretion regulators, many of which are highly conserved among metazoans (including humans) but are not found in simpler systems like yeast (Sato et al., 2005; Sato et al., 2008). One broad conclusion we drew from these screens was the critical importance of endocytic recycling, which must occur to support endocytic uptake. Deep analysis of our mutant collection showed that many of the novel trafficking regulators we discovered controlled receptor recycling steps rather than directly affecting uptake. Our data were pivotal in defining endocytic recycling in general as an active process, rather than a passive default pathway, and also served as motivation for our group to move on to deciphering recycling mechanisms, which were extremely poorly defined across all systems.

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in Vivo Tissue Models for Trafficking Studies fi

Developing New in Vivo Tissue Models for Trafficking Studies

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Our laboratory is widely known for field-leading work pioneering unique and extensive systems of tools and reagents for in vivo analysis of endocytic transport in the C. elegans germline (oocytes), intestine (a simple epithelial tube), coelomocytes (scavenger cells), hypodermis (skin), and most recently neurons, especially mechanosensory touch neurons. Our C. elegans germline studies were the key to identifying many novel factors essential for metazoan membrane trafficking (Sato et al., 2005; Sato et al., 2006; Balklava et al., 2007; Sato et al., 2008; Sato et al., 2009). Our C. elegans intestinal studies forged new understanding of tubular endocytic networks mediating cargo recycling pathways (Chen et al., 2006; Shi et al., 2007; Pant et al., 2009; Shi et al., 2010; Shi et al., 2012; Liu and Grant, 2015; Gleason et al., 2016; Wang et al., 2016; Rodriguez-Polanco et al., 2023), while our studies in coelomocytes have been key for direct analysis of cross-regulation among endosomal microdomains (Norris et al, 2017; Norris and Grant 2020; Norris et al., 2022). Our work in neurons has defined new mechanisms controlling lysosome reformation after autophagy (Swords et al., 2024), and a novel type of giant extracellular vesicle released by neurons (Wang et al., 2023; Cooper et al., 2021; Arnold et al., 2023; Wang et al., 2024). A distinctive theme across these studies is our development of molecular reagents that we manipulate and visualize within the living animal to address biological mechanisms in native context.

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