Biology subjects

Binti, S.

Publications and source records attributed to Binti, S..

7 recordsLinked to original sources

bioRxiv2026

Tracking cellular biomolecular condensate dynamics under proteostatic stress with middle-down phosphoproteomics

Coordination of biological function requires the partition of cellular components including into biomolecular condensates, but an overall landscape of how protein compartmentalize into higher-order assemblies under stress is still emerging. We apply proteome-wide solubility profiling to compare the compositions of NP-40-insoluble proteins and their phosphorylation status, using a new mass spectrometry-based hybrid bottom-up and chemical middle-down proteomics approach to analyze the solubility behavior of 8,740 proteins and 31,647 phosphopeptides under normal and ER stress conditions. Cell stress induces a pervasive differential partition of proteins in and out of detergent-insoluble cellular compartments. This differential partition is partially orthogonal to stress-induced abundance changes and comprises both phosphorylation-dependent and phosphorylation-independent mechanisms. Whereas phosphorylation-independent partition changes involve largely secretory pathway proteins and implicate higher-order assemblies of chaperones and clients, phosphorylation-based partitions suggest a dynamic rearrangement of biomolecular condensate compositions across cytoplasmic and nuclear ribonucleoprotein assemblies. The accumulation of serine/arginine rich (SR) proteins and other annotated nuclear speckle members in the condensate-rich proteome fractions emerges as a central feature of stress-induced remodeling. Our results establish global solubility dynamics as an integral component of proteome stress response and implicates broad involvements of splice factor spatial reorganization as a prominent facet of ER stress response.

biochemistry↗

bioRxiv2026

Proteogenomic analysis of the differential stability of cardiac protein isoforms

Alternative splicing is an important regulatory layer in gene expression, but knowledge on the isoform protein molecules continue to lag their canonical counterparts. An open question is whether alternative protein isoforms feature different half-life than the canonical counterpart, which could indicate differential usage and functional diversification. Here we combined a proteogenomics approach with heavy water-based protein turnover analysis to survey 24 pairs of canonical-alternative protein isoforms in the mouse heart. The results provide a reference on their numerical half-life and also reveal widespread differences in isoform stability.

biochemistry↗

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↗

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↗