Biology subjects

Binti, S.

Publications and source records attributed to Binti, S..

3 recordsLinked to original sources

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↗

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↗