Biology subjects

Fay, D. S.

Publications and source records attributed to Fay, D. S..

10 recordsLinked to original sources

bioRxiv2025

Proximity Labeling of NIMA Kinase Complex Components in C. elegans

Proximity labeling has emerged as a powerful approach for identifying protein-protein interactions within living systems, particularly those involving weak or transient associations. Here, we present a comprehensive proximity labeling study of five conserved Caenorhabditis elegans proteins--NEKL-2, NEKL-3, MLT-2, MLT-3, and MLT-4--that form two NEKL-MLT kinase-scaffold subcomplexes involved in membrane trafficking and actin regulation. Using endogenously expressed TurboID fusions and a data-independent acquisition (DIA) mass spectrometry (MS) pipeline, we profiled NEKL-MLT interactomes across 23 experiments, including several methodological variations, applying stringent controls and filtering strategies. By analyzing and comparing experimental outcomes, we develop a set of intuitive quantitative metrics to assess experimental outcomes and quality. We demonstrate that DIA-based workflows produce sensitive physiologically relevant findings, even in the presence of experimental noise and variability across biological replicates. Our approach is validated through the identification of known NEKL-MLT binding partners and conserved genetic suppressors of nekl-mlt mutant phenotypes. Gene ontology enrichment further supports the involvement of newly identified NEKL-MLT interactors in processes including membrane trafficking, cytoskeletal regulation, and cell adhesion. Additionally, several novel proximate interactors were functionally validated using genetic assays. Our findings underscore the utility of DIA-MS in proximity labeling applications and highlight the value of incorporating internal controls, quantitative metrics, and biological validation to enhance confidence in candidate interactors. Overall, this study provides a scalable, organismal-level strategy for probing endogenous protein networks and offers practical guidelines for proximity labeling in multicellular systems.

molecular 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↗

bioRxiv2024

TAT-1, a phosphatidylserine flippase, affects molting and regulates membrane trafficking in the epidermis of C. elegans

Membrane trafficking is a conserved process required for the movement and distribution of proteins and other macromolecules within cells. The Caenorhabditis elegans NIMA-related kinases NEKL-2 (human NEK8/9) and NEKL-3 (human NEK6/7) are conserved regulators of membrane trafficking and are required for the completion of molting. We used a genetic approach to identify reduction-of-function mutations in tat-1 that suppress nekl-associated molting defects. tat-1 encodes the C. elegans ortholog of mammalian ATP8A1/2, a phosphatidylserine (PS) flippase that promotes the asymmetric distribution of PS to the cytosolic leaflet of lipid membrane bilayers. CHAT-1 (human CDC50), a conserved chaperone, was required for the correct localization of TAT-1, and chat-1 inhibition strongly suppressed nekl defects. Using a PS sensor, we found that TAT-1 was required for the normal localization of PS at apical endosomes and that loss of TAT-1 led to aberrant endosomal morphologies. Consistent with this, TAT-1 localized to early endosomes and to recycling endosomes marked with RME-1, the C. elegans ortholog of the human EPS15 homology (EH) domain-containing protein, EHD1. TAT-1, PS biosynthesis, and the PS-binding protein RFIP-2 (human RAB11-FIP2) were all required for the normal localization of RME-1 to apical endosomes. Consistent with these proteins functioning together, inhibition of RFIP-2 or RME-1 led to the partial suppression of nekl molting defects, as did the inhibition of PS biosynthesis. Using the auxin-inducible degron system, we found that depletion of NEKL-2 or NEKL-3 led to defects in RME-1 localization and that a reduction in TAT-1 function partially restored RME-1 localization in NEKL-3-depleted cells. ARTICLE SUMMARYEndocytosis is an essential process required for the movement of proteins and lipids within cells. NEKL-2 and NEKL-3, two evolutionarily conserved proteins in the nematode Caenorhabditis elegans, are important regulators of endocytosis. In the current study, the authors describe a new functional link between the NEKLs and several proteins with known roles in endocytosis including TAT-1, a conserved enzyme that moves lipids between the bilayers of cellular membranes. As previous work implicated NEKLs in developmental defects and cancer, the present study can provide new insights into how the misregulation of endocytosis affects human health and disease.

cell biology↗

bioRxiv2024

Loss of the Na+/K+ cation pump CATP-1 suppresses nekl-associated molting defects

The conserved C. elegans protein kinases NEKL-2 and NEKL-3 regulate multiple steps of membrane trafficking and are required for larval molting. Through a forward genetic screen we identified a loss-of-function mutation in catp-1 as a suppressor of molting defects in synthetically lethal nekl-2; nekl-3 double mutants. catp-1 is predicted to encode a membrane- associated P4-type ATPase involved in Na+-K+ exchange. Moreover, a mutation predicted to abolish CATP-1 ion-pump activity also suppressed nekl-2; nekl-3 mutants. Endogenously tagged CATP-1 was primarily expressed in epidermal (hypodermal) cells within punctate structures located at or near the apical plasma membrane. Through whole genome sequencing, we identified two additional nekl-2; nekl-3 suppressor strains containing coding-altering mutations in catp-1 but found that neither mutation, when introduced into nekl-2; nekl-3 mutants using CRISPR methods, was sufficient to elicit robust suppression of molting defects. Our data also suggested that the two catp-1 isoforms, catp-1a and catp-1b, may in some contexts be functionally redundant. On the basis of previously published studies, we tested the hypothesis that loss of catp-1 may suppress nekl-associated defects by inducing partial entry into the dauer pathway. Contrary to expectations, however, we failed to obtain evidence that loss of catp-1 suppresses nekl-2; nekl-3 defects through a dauer-associated mechanism or that loss of catp-1 leads to entry into the pre-dauer L2d stage. As such, loss of catp-1 may suppress nekl- associated molting and membrane trafficking defects by altering electrochemical gradients within membrane-bound compartments.

genetics↗

bioRxiv2024

A conserved protein tyrosine phosphatase, PTPN-22, functions in diverse developmental processes in C. elegans

Protein tyrosine phosphatases non-receptor type (PTPNs) have been studied extensively in the context of the adaptive immune system; however, their roles beyond immunoregulation are less well explored. Here we identify novel functions for the conserved C. elegans phosphatase PTPN-22, establishing its role in nematode molting, cell adhesion, and cytoskeletal regulation. Through a non-biased genetic screen, we found that loss of PTPN-22 phosphatase activity suppressed molting defects caused by loss-of-function mutations in the conserved NIMA-related kinases NEKL-2 (human NEK8/NEK9) and NEKL-3 (human NEK6/NEK7), which act at the interface of membrane trafficking and actin regulation. To better understand the functions of PTPN-22, we carried out proximity labeling studies to identify candidate interactors of PTPN-22 during development. Through this approach we identified the CDC42 guanine-nucleotide exchange factor DNBP-1 (human DNMBP) as an in vivo partner of PTPN-22. Consistent with this interaction, loss of DNBP-1 also suppressed nekl-associated molting defects. Genetic analysis, co-localization studies, and proximity labeling revealed roles for PTPN-22 in several epidermal adhesion complexes, including C. elegans hemidesmosomes, suggesting that PTPN-22 plays a broad role in maintaining the structural integrity of tissues. Localization and proximity labeling also implicated PTPN-22 in functions connected to nucleocytoplasmic transport and mRNA regulation, particularly within the germline, as nearly one-third of proteins identified by PTPN-22 proximity labeling are known P granule components. Collectively, these studies highlight the utility of combined genetic and proteomic approaches for identifying novel gene functions.

genetics↗

bioRxiv2023

Pathways that affect anterior morphogenesis in C. elegans embryos

During embryogenesis the nascent Caenorhabditis elegans epidermis secretes an apical extracellular matrix (aECM) that serves as an external stabilizer, preventing deformation of the epidermis by mechanical forces exerted during morphogenesis. We showed that two conserved proteins linked to this process, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function together in a complex. Proteomics data also suggested potential roles for FAM102A and WDR44 family proteins in intracellular trafficking, consistent with their localization patterns. Nonetheless, we found no evidence to support a clear function for SYM-3 or SYM-4 in the apical deposition of two aECM components, FBN-1 and NOAH. Surprisingly, loss of MEC-8/RBPMS2, a conserved splicing factor and regulator of fbn-1, had little effect on the abundance or deposition of FBN-1 to the aECM. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. Lastly, we examined morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a related embryonic phenotype to mec-8; sym double mutants. Collectively, our findings add to our knowledge of pathways controlling embryonic morphogenesis. SUMMARY STATEMENTWe identify new proteins in apical ECM biology in C. elegans and provide evidence that SYM-3/FAM102A and SYM-4/WDR44 function together in trafficking but do not regulate apical ECM protein deposition.

developmental biology↗

bioRxiv2021

An unexpected role for the conserved ADAM-family metalloprotease ADM-2 in Caenorhabditis elegans molting

Molting is a widespread developmental process in which the external extracellular matrix (ECM), the cuticle, is remodeled to allow for organismal growth and environmental adaptation. Studies in the nematode Caenorhabditis elegans have identified a diverse set of molting-associated factors including signaling molecules, intracellular trafficking regulators, ECM components, and ECM-modifying enzymes such as matrix metalloproteases. C. elegans NEKL-2 and NEKL-3, two conserved members of the NEK family of protein kinases, are essential for molting and promote the endocytosis of environmental steroid-hormone precursors by the epidermis. Steroids in turn drive the cyclic induction of many genes required for molting. Here we report a novel role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a negative regulator of molting. Whereas loss of adm-2 led to strong suppression of molting defects in partial loss-of-function nekl mutants, overexpression of ADM-2 induced molting defects in wild-type animals. CRISPR genome editing implicated the Zn-binding motif within the metalloprotease domain as critical for mediating the effects of ADM-2 on molting. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We also identified the epidermally expressed low-density lipoprotein receptor-related protein, LRP-1, as a candidate target of ADM-2 regulation. Whereas loss of ADM-2 activity led to the upregulation of LRP-1, ADM-2 overexpression caused a reduction in LRP-1 abundance, suggesting that ADM-2 may function as a sheddase for LRP-1. We propose that loss of adm-2 suppresses molting defects in nekl mutants by eliminating a negative regulator of LRP-1, thereby compensating for defects in the efficiency of LRP-1 and sterol uptake. Our findings emphasize the importance of endocytic trafficking for both the internalization of factors that promote molting and the removal of proteins that would otherwise be deleterious to the molting process. Author SummaryThe molecular and cellular features of molting in nematodes share many similarities with cellular and developmental processes that occur in mammals. This includes the degradation and reorganization of extracellular matrix materials that surround cells, as well as the intracellular machineries that allow cells to communicate and sample their environments. In the current study, we found an unexpected function for a conserved protein that degrades proteins on the external surface of cells. Rather than promoting molting through extracellular matrix reorganization, the ADM-2 protease can inhibit the molting process. This observation can be explained by data showing that ADM-2 negatively regulates LRP-1, a membrane protein that promotes molting by facilitating the uptake of molecular building blocks at the cell surface that are needed for molting-related signaling. Our data provide insights into the mechanisms controlling molting and link several conserved proteins to show how they function together during development.

genetics↗

bioRxiv2021

A life cycle alteration can correct molting defects in Caenorhabditis elegans

Molting is a widespread feature in the development of many invertebrates, including nematodes and arthropods. In Caenorhabditis elegans, the highly conserved protein kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (NEKLs) promote molting through their involvement in the uptake and intracellular trafficking of epidermal cargos. We found that the relative requirements for NEKL-2 and NEKL-3 differed at different life-cycle stages and under different environmental conditions. Most notably, the transition from the second to the third larval stage (L2[->]L3 molt) required a higher level of NEKL function than during several other life stages or when animals had experienced starvation at the L1 stage. Specifically, larvae that entered the pre-dauer L2d stage could escape molting defects when transiting to the (non-dauer) L3 stage. Consistent with this, mutations that promote entry into L2d suppressed nekl-associated molting defects, whereas mutations that inhibit L2d entry reduced starvation-mediated suppression. We further showed that loss or reduction of NEKL functions led to defects in the transcription of cyclically expressed molting genes, many of which are under the control of systemic steroid hormone regulation. Moreover, the timing and severity of these transcriptional defects correlated closely with the strength of nekl alleles and with their stage of arrest. Interestingly, transit through L2d rescued nekl-associated expression defects in suppressed worms, providing an example of how life-cycle decisions can impact subsequent developmental events. Given that NEKLs are implicated in the uptake of sterols by the epidermis, we propose that loss of NEKLs leads to a physiological reduction in steroid-hormone signaling and consequent defects in the transcription of genes required for molting.

developmental biology↗

bioRxiv2019

Control of clathrin-mediated endocytosis by NIMA family kinases

Endocytosis, the process by which cells internalize plasma membrane and associated cargo, is regulated extensively by posttranslational modifications. Previous studies suggested the potential involvement of scores of protein kinases in endocytic control, of which only a few have been validated within their native context. Here we show that the conserved NIMA-related kinases NEKL-2/NEK8/9 and NEKL-3/NEK6/7 (the NEKLs) control clathrin-mediated endocytosis in C. elegans. Loss of NEKLs leads to clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Strikingly, reducing the levels of active AP2, the major clathrin adapter complex, rescues nekl mutant defects, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, NEKL inhibition alleviates defects associated with reduced AP2 activity, attesting to the tight link between NEKL and AP2 functions. We also show that NEKLs are required for the clustering and internalization of membrane cargo and that human NEKs rescue defects in nekl mutant worms.

cell biology↗

bioRxiv2017

Use of a Sibling Subtraction Method (SSM) for Identifying Causal Mutations in C. elegans by Whole-Genome Sequencing

Whole-genome sequencing (WGS) has become an indispensible tool for identifying causal mutations obtained from genetic screens. To reduce the number of causal mutation candidates typically uncovered by WGS, C. elegans researchers have developed several strategies. One involves crossing N2-background mutants to the polymorphic strain CB4856, which can be used to simultaneously identify mutant-strain variants and obtain high-density mapping information. This approach, however, is not well suited for uncovering mutations in complex genetic backgrounds, and CB4856 polymorphisms can alter phenotypes. Several other approaches make use of DNA variants introduced by mutagenesis. This information is used to implicate genomic regions with high densities of DNA lesions that persist after mutant backcrossing, but these methods provide lower mapping resolution than use of CB4856. To identify suppressor mutations using WGS, we developed a new approach termed the Sibling Subtraction Method (SSM). This method works by eliminating variants present in both mutants and their non-mutant siblings, thus greatly reducing the number of candidates. We used this method here with two members of the C. elegans NimA-related kinase family, nekl-2 and nekl-3. Combining weak aphenotypic alleles of nekl-2 and nekl-3 leads to penetrant molting defects and larval arrest. We isolated ~50 suppressors of nekl-2; nekl-3 synthetic lethality using F1-clonal screening methods and a toxin-based counter-selection strategy. When applied to five of the identified suppressors, SSM led to only one to four suppressor candidates per strain. Thus SSM is a powerful approach for identifying causal mutations in any genetic background and provides an alternative to current methods.

genetics↗