Biology subjects

Grant, B. D.

Publications and source records attributed to Grant, B. D..

9 recordsLinked to original sources

bioRxiv2026

SCM-1/SCAMP Maintains Microdomain Boundaries and Cargo Sorting within the Endosomal System

After endocytosis, transmembrane cargo reaches sorting endosomes where it is partitioned into physically distinct recycling or degradative microdomains. While the J-domain protein RME-8/DNAJC13 is known to maintain these boundaries by actively removing degradative machinery from the recycling microdomain, other factors that contribute to this spatial organization remain poorly defined. Here, we identify the conserved tetraspan protein SCM-1/SCAMP as a key microdomain organizer, discovered through RME-8 proximity-dependent biotinylation screens in C. elegans and human cells. Leveraging the large endosomes of C. elegans coelomocytes, we show that SCM-1 is selectively enriched within the recycling microdomain. In scm-1 mutants, recycling and degradative microdomains still assemble but fail to remain spatially distinct, resulting in inappropriate microdomain overlap. This loss of boundary integrity occurs without increasing the recruitment of sorting machineries, indicating a mechanism distinct from the RME-8-mediated uncoating pathway. scm-1 mutants exhibit significant sorting defects, including misrouting of recycling cargo MIG-14/Wls and v-SNARE SNB-2/VAMP3 to late endosomes and lysosomes. We find that snb-2 mutants themselves missort MIG-14 to late endosomes and lysosomes, suggesting that SNB-2 sorting is key for recycling function. Our data suggest that both microdomains lose efficiency in scm-1 mutants, as cargo missorted into late endosomes and lysosomes is not depleted overall, and degradation of an independent ESCRT-dependent cargo is delayed. We conclude that SCM-1 ensures endosomal sorting fidelity by stabilizing microdomain boundary integrity, a process required for efficient recycling and degradation of transmembrane cargo.

cell biology↗

bioRxiv2025

PIKI-1, a class II phosphatidylinositol 3-kinase, functions in endocytic trafficking

Membrane trafficking, including endocytosis and exocytosis, is a complex process that is coordinated by trafficking-associated proteins, cargo molecules, the cytoskeleton, and membrane lipid composition. The NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking in Caenorhabditis elegans and are required for successful molting. Through a genetic approach, we isolated reduction-of-function mutations in piki-1 that suppress nekl-associated molting defects. piki-1 encodes the sole predicted C. elegans Class II phosphatidylinositol 3-kinase (PI3Ks), an understudied class of lipid modifiers that contribute to the production of phosphatidylinositol 3-phosphate (PI(3)P) and phosphatidylinositol 3,4-bisphosphate (PI(3,4)P2). Using a set of genetically encoded lipid sensors, we found that PIKI-1 was responsible for the production of PI(3,4)P2 in the C. elegans epidermis but played only a minor role in the control of PI(3)P levels. Consistent with this, both PI(3,4)P2 and PIKI-1 colocalized to early endosomes, and reduction of PIKI-1 function strongly affected early endosomal morphology and protein composition. Additionally, reduced PIKI-1 function led to excess tubulation of endosomal compartments associated with recycling or the degradation of cellular debris. In contrast to previous studies using mammalian cell culture, PIKI-1 was largely dispensable for clathrin-mediated endocytosis in the context of the worm epidermis, which is a polarized epithelium. Notably, reduction of PIKI-1 function strongly mitigated defects in early endosomes associated with the depletion of NEKL-2. We propose that reduction of PIKI-1 function may suppress nekl molting defects by partially restoring endocytic trafficking within specific compartments, including the early endosome. We also show that inhibition of the PI(3,4)P2-binding protein HIPR-1 (HIP1/HIPR1) suppresses nekl molting defects, suggesting that reduced PI(3,4)P2 levels alter endosomal protein recruitment in a manner that antagonizes NEKL-2 function. Author summaryThe uptake of materials from outside the cell and their subsequent delivery to specific intracellular locations are essential for cell function and survival. Two of the mechanisms that control this complex intracellular pathway involve the modification of proteins and of lipids, processes that are highly conserved across species. In this study, we used the model organism Caenorhabditis elegans, which is highly amenable to cell biological and genetic approaches, to establish a novel connection between these two regulatory mechanisms and demonstrate the importance of lipid modifications in maintaining the normal functioning of intracellular transport. Our results also provide insights into the fundamental cellular functions of proteins associated with human disease including cancer and metabolic disease.

cell biology↗

bioRxiv2023

Mechanical force of uterine occupation enables large vesicle extrusion from proteostressed maternal neurons

Large vesicle extrusion from neurons may contribute to spreading pathogenic protein aggregates and promoting inflammatory responses, two mechanisms leading to neurodegenerative disease. Factors that regulate extrusion of large vesicles, such as exophers produced by proteostressed C. elegans touch neurons, are poorly understood. Here we document that mechanical force can significantly potentiate exopher extrusion from proteostressed neurons. Exopher production from the C. elegans ALMR neuron peaks at adult day 2 or 3, coinciding with the C. elegans reproductive peak. Genetic disruption of C. elegans germline, sperm, oocytes, or egg/early embryo production can strongly suppress exopher extrusion from the ALMR neurons during the peak period. Conversely, restoring egg production at the late reproductive phase through mating with males or inducing egg retention via genetic interventions that block egg-laying can strongly increase ALMR exopher production. Overall, genetic interventions that promote ALMR exopher production are associated with expanded uterus lengths and genetic interventions that suppress ALMR exopher production are associated with shorter uterus lengths. In addition to the impact of fertilized eggs, ALMR exopher production can be enhanced by filling the uterus with oocytes, dead eggs, or even fluid, supporting that distention consequences, rather than the presence of fertilized eggs, constitute the exopher-inducing stimulus. We conclude that the mechanical force of uterine occupation potentiates exopher extrusion from proximal proteostressed maternal neurons. Our observations draw attention to the potential importance of mechanical signaling in extracellular vesicle production and in aggregate spreading mechanisms, making a case for enhanced attention to mechanobiology in neurodegenerative disease.

neuroscience↗

bioRxiv2023

A Conserved Requirement for RME-8/DNAJC13 in Neuronal Autolysosome Reformation

Autophagosomes fuse with lysosomes, forming autolysosomes that degrade engulfed cargo. To maintain lysosomal capacity, autolysosome reformation (ALR) must regenerate lysosomes from autolysosomes using a membrane tubule-based process. Maintaining lysosomal capacity is required to maintain proteostasis and cellular health, especially in neurons where lysosomal dysfunction has been repeatedly implicated in neurodegenerative disease. Cell biological studies have linked the DNA-J domain Hsc70 co-chaperone RME-8/DNAJC13 to endosomal coat protein regulation, while human genetics studies have linked RME-8/DNAJC13 to neurological disease, including Parkinsonism and Essential Tremor. We report new analysis of the requirements for the RME-8/DNAJC13 protein in neurons, focusing on C. elegans mechanosensory neurons in the intact animal, and in primary mouse cortical neurons in culture. We find that loss of RME-8/DNAJC13 in both systems results in accumulation of grossly elongated autolysosomal tubules. Further C. elegans analysis revealed a similar autolysosome tubule accumulation defect in mutants known to be required for ALR in mammals, including bec-1/beclin and vps-15/PIK3R4/p150 that regulate type-III PI3-kinase VPS-34, and dyn-1/dynamin that severs ALR tubules. Clathrin is also an important ALR regulator implicated in autolysosome tubule formation and release. In C. elegans we found that loss of RME-8 causes severe depletion of clathrin from neuronal autolysosomes, a phenotype shared with bec-1 and vps-15 mutants. We conclude that RME-8/DNAJC13 plays a conserved but previously unrecognized role in autolysosome reformation, likely affecting ALR tubule initiation and/or severing. Additionally, in both systems, we found that loss of RME-8/DNAJC13 appeared to reduce autophagic flux, suggesting feedback regulation from ALR to autophagy. Our results connecting RME-8/DNAJC13 to ALR and autophagy provide a potential mechanism by which RME-8/DNAJC13 could influence neuronal health and the progression of neurodegenerative disease.

cell biology↗

bioRxiv2023

Active zone protein SYD-2/Liprin-α acts downstream of LRK-1/LRRK2 to regulate polarized trafficking of synaptic vesicle precursors through clathrin adaptor protein complexes

Synaptic vesicle proteins (SVps) are thought to travel in heterogeneous carriers dependent on the motor UNC-104/KIF1A. In C. elegans neurons, we found that some SVps are transported along with lysosomal proteins by the motor UNC-104/KIF1A. LRK-1/LRRK2 and the clathrin adaptor protein complex AP-3 are critical for the separation of lysosomal proteins from SVp transport carriers. In lrk-1 mutants, both SVp carriers and SVp carriers containing lysosomal proteins are independent of UNC-104, suggesting that LRK-1 plays a key role in ensuring UNC-104-dependent transport of SVps. Additionally, LRK-1 likely acts upstream of the AP-3 complex and regulates the membrane localization of AP-3. The action of AP-3 is necessary for the active zone protein SYD-2/Liprin- to facilitate the transport of SVp carriers. In the absence of the AP-3 complex, SYD-2/Liprin- acts with UNC-104 to instead facilitate the transport of SVp carriers containing lysosomal proteins. We further show that the mistrafficking of SVps into the dendrite in lrk-1 and apb-3 mutants depends on SYD-2, likely by regulating the recruitment of the AP-1/UNC-101. We propose that SYD-2 acts in concert with both the AP-1 and AP-3 complexes to ensure polarized trafficking of SVps.

cell biology↗

bioRxiv2022

Conserved NIMA kinases regulate multiple steps of endocytic trafficking

Human NIMA-related kinases have primarily been studied for their roles in cell cycle progression (NEK1/2/6/7/9), checkpoint-DNA-damage control (NEK1/2/4/5/10/11), and ciliogenesis (NEK1/4/8). We previously showed that Caenorhabditis elegans NEKL-2 (NEK8/9 homolog) and NEKL-3 (NEK6/7 homolog) regulate apical clathrin-mediated endocytosis (CME) in the worm epidermis and are essential for molting. Here we show that NEKL-2 and NEKL-3 also have distinct roles in controlling endosome function and morphology. Specifically, loss of NEKL-2 led to enlarged early endosomes with long tubular extensions but showed minimal effects on other compartments. In contrast, NEKL-3 depletion caused pronounced defects in early, late, and recycling endosomes. Consistently, NEKL-2 was strongly localized to early endosomes, whereas NEKL-3 was localized to multiple endosomal compartments. Loss of NEKLs also led to variable defects in the recycling of two resident cargoes of the trans-Golgi network (TGN), MIG-14/Wntless and TGN-38/TGN38, which were missorted to lysosomes after NEKL depletion. In addition, defects were observed in the uptake of clathrin-dependent (SMA-6/Type I BMP receptor) and independent cargoes (DAF-4/Type II BMP receptor) from the basolateral surface of epidermal cells after NEKL-2 or NEKL-3 depletion. Complementary studies in human cell lines further showed that siRNA knockdown of the NEKL-3 orthologs NEK6 and NEK7 led to missorting of the mannose 6-phosphate receptor from endosomes. Moreover, in multiple human cell types, depletion of NEK6 or NEK7 disrupted both early and recycling endosomal compartments, including the presence of excess tubulation within recycling endosomes, a defect also observed after NEKL-3 depletion in worms. Thus, NIMA family kinases carry out multiple functions during endocytosis in both worms and humans, consistent with our previous observation that human NEKL-3 orthologs can rescue molting and trafficking defects in C. elegans nekl-3 mutants. Our findings suggest that trafficking defects could underlie some of the proposed roles for NEK kinases in human disease. Author SummaryIntracellular trafficking is an evolutionary conserved process whereby cargoes, which include proteins, lipids, and other macromolecules, are internalized by cells, packaged into vesicles, and distributed to their proper places within the cell. This study demonstrated that two conserved NIMA-related kinases, NEKL-2 and NEKL-3, are required for the transport of multiple cargoes in the epidermis of C. elegans. NEKL-2 and NEKL-3 function at organelles, called endosomes, to regulate their morphology and control the sorting of cargoes between different intracellular compartments. In the absence of NEKL activities, various cargoes, including components of the BMP and Wnt signaling pathways, were misregulated. Our studies are further supported by results showing that the human counterparts of NEKL-3, NEK6 and NEK7, were also required for maintaining endosome morphologies and for the proper sorting of cargo in human cells. Notably, NIMA-kinases are well studied for their roles in cell cycle regulation, and overexpression of these kinases is linked to cancer formation and poor prognosis. Our study suggests their role in cancer progression could be partly due to the abnormal intracellular trafficking of conserved signaling components with known roles in cancer formation.

cell biology↗

bioRxiv2022

Intermediate Filaments Associate with Aggresome-like Structures and Influence Rate of Cellular Expulsion of Neuronal Disease Aggregates

Under conditions of proteostasis disequilibrium, neurons can enhance intracellular and extracellular protective mechanisms to guard against neurotoxicity. In mammals, an intracellular response to severe proteostasis imbalance that results from proteosome inhibition is the formation of juxtanuclear intermediate filament-surrounded, aggregate-filled aggresomes, which sequester threatening aggregates for later disposal via lysosomal degradation. Highly proteo-stressed neurons can also engage the assistance of neighboring cells in aggregate removal by loading threatening materials into large exopher vesicles that are transferred to neighboring cells for remote degradation of contents, a process that has been suggested to be analogous to the process that enables aggregate spreading in the human brain in neurodegenerative disease. In C. elegans these large extruded vesicles are called exophers. Here we document that players involved in aggresome biology are required for the elimination of potentially deleterious materials in neuronal exophers. We show that in proteostressed C. elegans touch receptor neurons, intermediate filament proteins IFD-1 and IFD-2 can assemble into juxtanuclear structures with multiple molecular and cellular characteristics of mammalian aggresomes. IFD-concentrating structures depend upon orthologs of mammalian adapter proteins, dynein motors, and microtubule integrity for aggregate collection into juxtanuclear compartments where they associate with ubiquitinated and neurotoxic polyglutamine expansion proteins. Strikingly, disruption of aggresome-decoration genes encoding IFDs or disruption of the BAG/14-3-3/Hsc70 adapter that promote aggregate loading of aggresome-like organelles, lowers exopher production via a cell autonomous mechanism. Although aggresome-like structures are not mandatory exopher cargo, IFD compartments can be extruded from neurons in exophers, revealing a previously unreported strategy to eliminate neuronal aggresome-like organelles via transfer to neighboring cells. Human IF neurofilament light chain hNFL can partially substitute for C. elegans IFD-2 proteins in promoting exopher production, indicating conservation of the capacity of intermediate filaments to influence neuronal aggregate extrusions across phyla. In sum, we identify a requirement for specific intermediate filaments, counterparts of human biomarkers of neuronal injury and disease and major components of Parkinsons disease Lewy bodies, in C. elegans neuronal aggresome-like organelle formation and large vesicle exopher extrusion from stressed neurons.

cell biology↗

bioRxiv2022

Mutagenesis and structural modeling implicate RME-8 IWN domains as conformational control points

After endocytosis, transmembrane cargo is differentially sorted into degradative or recycling pathways. This process is facilitated by recruitment into physically distinct degradative or recycling microdomains on the limiting membrane of individual endosomes. Endosomal sorting complexes required for transport (ESCRT) mark the degradative microdomain, while the recycling domain is marked by the retromer complex and associated proteins RME-8 and SNX-1. The separation of endosomal microdomains is also controlled by RME-8 and SNX-1, at least in part via removal of degradative component HRS/HGRS-1 from the recycling microdomain. This activity is likely due to recruitment and activation of chaperone Hsc70 to the recycling microdomain by the RME-8 DNAJ domain. To better understand the mechanism of RME-8 function we performed a new phylogenetic analysis of RME-8 and identified new conserved sequence features. In a complementary approach, we performed structure-function analysis that identified the C-terminus as important for microdomain localization and likely substrate binding, while N-terminal sequences beyond the known single N-terminal PH-like domain are important for endosome recruitment. Random mutagenesis identified IWN4, and by analogy IWN3, to be important for the inhibitory DNAJ domain binding. Combining AlphaFold structural predictions with in vivo truncation and point mutation analysis of RME-8, we propose a model whereby SNX-1 and the IWN domains control the conformation of RME-8 and hence the productive exposure of the DNAJ domain. Furthermore, we propose that the activation of RME-8 is cyclical, with SNX-1 acting as an activator and a target of RME-8 uncoating activity.

cell biology↗

bioRxiv2021

SPE-51, a sperm secreted protein with an Immunoglobulin-like domain, is required for sperm-egg fusion in C. elegans

Despite the importance of fertilization, the molecular basis of sperm-egg interaction is not well understood. In a forward genetics screen for fertility mutants in Caenorhabditis elegans we identified spe-51. Mutant worms make sperm that are unable to fertilize the oocyte but otherwise normal by all available measurements. The spe-51 gene encodes a secreted protein that includes an immunoglobulin (Ig)-like domain and a hydrophobic sequence of amino acids. The SPE-51 protein acts cell-autonomously and localizes to the surface of the spermatozoa. This is the first example of a secreted protein required for the interactions between the sperm and egg with genetic validation for a specific function in fertilization. Our analyses of these genes begin to build a paradigm for sperm-secreted or reproductive tract-secreted proteins that coat the sperm surface and influence their survival, motility, and/or the ability to fertilize the egg.

developmental biology↗