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 import, export, 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. Using a genetic approach we identified reduction-of-function mutations in tat-1 that suppress nekl-associated molting defects.

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

The conserved Caenorhabditis elegans protein kinases NEKL-2 and NEKL-3 regulate membrane trafficking and are required for larval molting. Through a forward genetic screen we identified a mutation in catp-1 as a suppressor of molting defects in synthetically lethal nekl-2; nekl-3 double mutants. catp-1 encodes a membrane-associated P4-type ATPase involved in Na+-K+ exchange.

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

Unlabelled: Membrane trafficking is a conserved process required for the movement and distribution of proteins and other macromolecules within cells. The 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 that suppress -associated molting defects.

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.

Epidermal cell fusion promotes the transition from an embryonic to a larval transcriptome in .

Cell fusion is a fundamental process in the development of multicellular organisms, yet its impact on gene regulation, particularly during crucial developmental stages, remains poorly understood. The epidermis comprises 8-10 syncytial cells, with the largest integrating 139 individual nuclei through cell-cell fusion governed by the fusogenic protein EFF-1. To explore the effects of cell fusion on developmental progression and associated gene expression changes, we conducted transcriptomic analyses of fusion-deficient mutants.

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

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 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.

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

The conserved 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 as a suppressor of molting defects in synthetically lethal double mutants. is predicted to encode a membrane- associated P4-type ATPase involved in Na -K exchange.

Effectors of 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. At present, the factors that contribute to aECM function are mostly unknown, including the aECM components themselves, their posttranslational regulators, and the pathways required for their secretion. Here we showed that two proteins previously linked to aECM function, SYM-3/FAM102A and SYM-4/WDR44, colocalize to intracellular and membrane-associated puncta and likely function in a complex.

Pathways that affect anterior morphogenesis in embryos.

Unlabelled: During embryogenesis the nascent 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.

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.

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.

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.

CRISPRcruncher: A tool for engineering restriction sites into coding regions.

CRISPR/Cas9 genome editing strategies often rely on the placement of an introduced restriction endonuclease (RE) site adjacent to the genomic edit of interest. This allows for rapid initial PCR-based detection of cells and organisms containing the edit of interest and may also be used for subsequent genotyping. Nevertheless, engineering RE sites at optimal locations within coding regions can be difficult due to the many hundreds of potential endonuclease options and the strict requirement to maintain the correct amino acid sequence.

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 in vivo. 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.

Regulation of germ cell development by ARI1 family ubiquitin ligases in C. elegans.

RING-between-RING (RBR) E3 ubiquitin ligases are implicated in various developmental processes, and mutations in genes encoding RBR proteins HHARI/ARIH1 and Parkin are associated with human diseases. Here we show by phylogenetic analysis that the ARI1 family has undergone a dramatic expansion within the Caenorhabditis clade in recent history, a characteristic shared by some genes involved in germline development. We then examined the effects of deleting all ARI1 family members in the nematode Caenorhabditis elegans, which to our knowledge represents the first complete knockout of ARI1 function in a metazoan.

Actin organization and endocytic trafficking are controlled by a network linking NIMA-related kinases to the CDC-42-SID-3/ACK1 pathway.

Molting is an essential process in the nematode Caenorhabditis elegans during which the epidermal apical extracellular matrix, termed the cuticle, is detached and replaced at each larval stage. The conserved NIMA-related kinases NEKL-2/NEK8/NEK9 and NEKL-3/NEK6/NEK7, together with their ankyrin repeat partners, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, are essential for normal molting. In nekl and mlt mutants, the old larval cuticle fails to be completely shed, leading to entrapment and growth arrest.

Use of a Sibling Subtraction Method for Identifying Causal Mutations in by Whole-Genome Sequencing.

Whole-genome sequencing (WGS) is an indispensable tool for identifying causal mutations obtained from genetic screens. To reduce the number of causal mutation candidates typically uncovered by WGS, researchers have developed several strategies. One involves crossing N2-background mutants to the polymorphic Hawaiian (HA) strain, which can be used to simultaneously identify mutant strain variants and obtain high-density mapping information.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in development.

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans).

Molting in .

Molting is an essential developmental process for the majority of animal species on Earth. During the molting process, which is a specialized form of extracellular matrix (ECM) remodeling, the old apical ECM, or cuticle, is replaced with a new one. Many of the genes and pathways identified as important for molting in nematodes are highly conserved in vertebrates and include regulators and components of vesicular trafficking, steroid-hormone signaling, developmental timers, and hedgehog-like signaling.

Conserved Ankyrin Repeat Proteins and Their NIMA Kinase Partners Regulate Extracellular Matrix Remodeling and Intracellular Trafficking in Caenorhabditis elegans.

Molting is an essential developmental process in nematodes during which the epidermal apical extracellular matrix, the cuticle, is remodeled to accommodate further growth. Using genetic approaches, we identified a requirement for three conserved ankyrin repeat-rich proteins, MLT-2/ANKS6, MLT-3/ANKS3, and MLT-4/INVS, in Caenorhabditis elegans molting. Loss of mlt function resulted in severe defects in the ability of larvae to shed old cuticle and led to developmental arrest.

FBN-1, a fibrillin-related protein, is required for resistance of the epidermis to mechanical deformation during C. elegans embryogenesis.

During development, biomechanical forces contour the body and provide shape to internal organs. Using genetic and molecular approaches in combination with a FRET-based tension sensor, we characterized a pulling force exerted by the elongating pharynx (foregut) on the anterior epidermis during C. elegans embryogenesis.

Analysis of PHA-1 reveals a limited role in pharyngeal development and novel functions in other tissues.

PHA-1 encodes a cytoplasmic protein that is required for embryonic morphogenesis and attachment of the foregut (pharynx) to the mouth (buccal capsule). Previous reports have in some cases suggested that PHA-1 is essential for the differentiation of most or all pharyngeal cell types. By performing mosaic analysis with a recently acquired pha-1 null mutation (tm3671), we found that PHA-1 is not required within most or all pharyngeal cells for their proper specification, differentiation, or function.

The conserved PBAF nucleosome-remodeling complex mediates the response to stress in Caenorhabditis elegans.

To adapt to stress, cells must undergo major changes in their gene expression profiles. We have previously described a largely uncharacterized stress response pathway in Caenorhabditis elegans that acts through an evolutionarily conserved motif, termed ESRE, for ethanol and stress response element. We characterize here the requirements for ESRE gene expression and show that the ESRE network is regulated by a conserved SWI/SNF family nucleosome remodeling complex termed PBAF.

Implicating SCF complexes in organogenesis in Caenorhabditis elegans.

Development of the Caenorhabditis elegans foregut (pharynx) is regulated by a network of proteins that includes the Retinoblastoma protein (pRb) ortholog LIN-35; the ubiquitin pathway components UBC-18 and ARI-1; and PHA-1, a cytoplasmic protein. Loss of pha-1 activity impairs pharyngeal development and body morphogenesis, leading to embryonic arrest. We have used a genetic suppressor approach to dissect this complex pathway.