RESUMEN
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.
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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 Caenorhabditis elegans 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 eff-1 fusion-deficient mutants. Our RNAseq findings showed widespread transcriptomic changes that were enriched for epidermal genes and key molecular pathways involved in epidermal function during larval development. Subsequent single-molecule fluorescence in situ hybridization validated the altered expression of mRNA transcripts, confirming quantifiable changes in gene expression in the absence of embryonic epidermal fusion. These results underscore the significance of cell-cell fusion in shaping transcriptional programs during development and raise questions regarding the precise identities and specialized functions of different subclasses of nuclei within developing syncytial cells and tissues.
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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.
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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.
RESUMEN
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. Proteomics experiments also suggested potential roles for SYM-3/FAM102A and SYM-4/WDR44 family proteins in intracellular trafficking. Nonetheless, we found no evidence to support a critical function for SYM-3 or SYM-4 in the apical deposition of two aECM components, NOAH-1 and FBN-1. Moreover, loss of a key splicing regulator of fbn-1, MEC-8/RBPMS2, had surprisingly little effect on the abundance or deposition of FBN-1. Using a focused screening approach, we identified 32 additional proteins that likely contribute to the structure and function of the embryonic aECM. We also characterized morphogenesis defects in embryos lacking mir-51 microRNA family members, which display a similar phenotype to mec-8; sym double mutants. Collectively, these findings add to our knowledge of factors controlling embryonic morphogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Morfogénesis/genética , Matriz Extracelular/metabolismo , Desarrollo Embrionario/genéticaRESUMEN
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 STATEMENT: We 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.
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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.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Humanos , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endocitosis/genética , Endosomas/genética , Endosomas/metabolismo , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Clatrina/genética , Clatrina/metabolismo , Receptores de Proteínas Morfogenéticas Óseas/metabolismo , Transporte de Proteínas/genéticaRESUMEN
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 role for the sole C. elegans ADAM-meltrin metalloprotease family member, ADM-2, as a mediator of molting. Loss of adm-2, including mutations that disrupt the metalloprotease domain, led to the strong suppression of molting defects in partial loss-of-function nekl mutants. ADM-2 is expressed in the epidermis, and its trafficking through the endo-lysosomal network was disrupted after NEKL depletion. We 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 apical epidermal LRP-1, ADM-2 overexpression caused a reduction in LRP-1 levels. Consistent with this, several mammalian ADAMs, including the meltrin ADAM12, have been shown to regulate mammalian LRP1 via proteolysis. In contrast to mammalian homologs, however, the regulation of LRP-1 by ADM-2 does not appear to involve the metalloprotease function of ADM-2, nor is proteolytic processing of LRP-1 strongly affected in adm-2 mutants. Our findings suggest a noncanonical role for an ADAM family member in the regulation of a lipoprotein-like receptor and lead us to propose that endocytic trafficking may be important for both the internalization of factors that promote molting as well as the removal of proteins that can inhibit the process.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Endocitosis/genética , Mamíferos/metabolismo , Metaloproteasas/genética , Metaloproteasas/metabolismo , Muda/genéticaRESUMEN
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 (L2âL3 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.
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Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Regulación del Desarrollo de la Expresión Génica , Estadios del Ciclo de Vida/genética , Muda/genética , Quinasas Relacionadas con NIMA/genética , Quinasas Relacionadas con NIMA/metabolismo , Alelos , Animales , Animales Modificados Genéticamente , Sistemas CRISPR-Cas , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Endocitosis/genética , Epidermis/metabolismo , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Larva/genética , Larva/metabolismo , Mutación con Pérdida de Función , Transducción de Señal/genética , Inanición , Esteroles/metabolismo , Regulación hacia Arriba/genéticaRESUMEN
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. Here we report CRISPRcruncher, a computational tool that analyzes an input coding sequence and produces a complete list of all possible changes that could be made that will create new RE sites while preserving the original peptide sequence. Notably, for sequences tested, CRISPRcruncher identified approximately one new RE site per input nucleotide when mining for 4-bp or longer RE motifs and 0.5 new RE sites per input nucleotide when mining for 6-bp or longer motifs. Therefore, CRISPRcruncher represents a powerful new computational tool in the CRISPR arsenal.
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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. elegans. Loss of NEKL-2 or NEKL-3 activities leads to penetrant larval molting defects and to the abnormal localization of trafficking markers in arrested larvae. Using an auxin-based degron system, we also find that depletion of NEKLs in adult-stage C. elegans leads to gross clathrin mislocalization and to a dramatic reduction in clathrin mobility at the apical membrane. Using a non-biased genetic screen to identify suppressors of nekl molting defects, we identified several components and regulators of AP2, the major clathrin adapter complex acting at the plasma membrane. Strikingly, reduced AP2 activity rescues both nekl mutant molting defects as well as associated trafficking phenotypes, whereas increased levels of active AP2 exacerbate nekl defects. Moreover, in a unique example of mutual suppression, 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 required for molting. Notably, we find that human NEKs can rescue molting and trafficking defects in nekl mutant worms, suggesting that the control of intracellular trafficking is an evolutionarily conserved function of NEK family kinases.
Asunto(s)
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiología , Clatrina/metabolismo , Quinasas Relacionadas con NIMA/genética , Proteínas Quinasas/genética , Complejo 2 de Proteína Adaptadora/metabolismo , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/metabolismo , Membrana Celular/metabolismo , Endocitosis , Microscopía Intravital , Larva/crecimiento & desarrollo , Muda/genética , Mutación , Quinasas Relacionadas con NIMA/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Imagen de Lapso de TiempoRESUMEN
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. Hermaphrodites that lacked or had strongly reduced ARI1 activity had low fecundity and were partially defective in initiation of oocyte differentiation. We provide evidence that the C. elegans ARI1s likely function downstream or in parallel to FBF-1 and FBF-2, two closely related RNA-binding proteins that are required for the switch from spermatogenesis to oogenesis during late larval development. Previous studies have shown that the E2 enzymes UBC-18/UBCH7 and UBC-3/CDC34 can functionally collaborate with ARI1 family members. Our data indicated that UBC-18, but not UBC-3, specifically cooperates with the ARI1s in germline development. These findings provide new insights into the functions of RING-between-RING proteins and Ariadne E3s during development.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Células Germinativas/crecimiento & desarrollo , Células Germinativas/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina/metabolismo , Animales , Diferenciación Celular/fisiología , Oocitos/metabolismo , Oogénesis/fisiología , Filogenia , Proteínas de Unión al ARN/metabolismo , Espermatogénesis/fisiología , Enzimas Ubiquitina-Conjugadoras/metabolismoRESUMEN
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. To better understand the molecular and cellular functions of NEKLs during molting, we isolated genetic suppressors of nekl molting-defective mutants. Using two independent approaches, we identified CDC-42, a conserved Rho-family GTPase, and its effector protein kinase, SID-3/ACK1. Notably, CDC42 and ACK1 regulate actin dynamics in mammals, and actin reorganization within the worm epidermis has been proposed to be important for the molting process. Inhibition of NEKL-MLT activities led to strong defects in the distribution of actin and failure to form molting-specific apical actin bundles. Importantly, this phenotype was reverted following cdc-42 or sid-3 inhibition. In addition, repression of CDC-42 or SID-3 also suppressed nekl-associated defects in trafficking, a process that requires actin assembly and disassembly. Expression analyses indicated that components of the NEKL-MLT network colocalize with both actin and CDC-42 in specific regions of the epidermis. Moreover, NEKL-MLT components were required for the normal subcellular localization of CDC-42 in the epidermis as well as wild-type levels of CDC-42 activation. Taken together, our findings indicate that the NEKL-MLT network regulates actin through CDC-42 and its effector SID-3. Interestingly, we also observed that downregulation of CDC-42 in a wild-type background leads to molting defects, suggesting that there is a fine balance between NEKL-MLT and CDC-42-SID-3 activities in the epidermis.
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Actinas/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al GTP/metabolismo , Quinasas Relacionadas con NIMA/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Animales , Animales Modificados Genéticamente , Caenorhabditis elegans/genética , Caenorhabditis elegans/crecimiento & desarrollo , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Ciclo Celular/genética , Endocitosis , Epidermis/metabolismo , Proteínas de Unión al GTP/genética , Microscopía Confocal , Muda/genética , Mutación , Quinasas Relacionadas con NIMA/genética , Transporte de Proteínas , Proteínas Tirosina Quinasas/genética , Interferencia de ARN , Transducción de Señal/genéticaRESUMEN
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, Caenorhabditis elegans 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. This approach, however, is not well suited for uncovering mutations in complex genetic backgrounds, and HA polymorphisms can alter phenotypes. Other approaches make use of DNA variants present in the initial background or introduced by mutagenesis. This information is used to implicate genomic regions with high densities of DNA lesions that persist after backcrossing, but these methods can provide lower resolution than HA mapping. To identify suppressor mutations using WGS, we developed an approach termed the sibling subtraction method (SSM). This method works by eliminating variants present in both mutants and their nonmutant siblings, thus greatly reducing the number of candidates. We used this method 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 â¼50 suppressors of nekl-2; nekl-3 synthetic lethality using F1 clonal screening methods and a peel-1-based counterselection strategy. When applied to five of the 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.
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Caenorhabditis elegans/genética , Análisis Mutacional de ADN/métodos , Mutación , Secuenciación Completa del Genoma/métodos , Animales , Animales Modificados Genéticamente , Proteínas de Caenorhabditis elegans/genética , Mapeo Cromosómico , Regulación de la Expresión Génica , Genes de Helminto/genética , MutagénesisRESUMEN
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. In this review, we discuss what is known about molting, with a focus on studies in Caenorhabditis elegans. We also describe the key structural elements of the cuticle that must be released, newly synthesized, or remodeled for proper molting to occur.
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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 Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Proteínas Cullin/metabolismo , Proteínas Ligasas SKP Cullina F-box/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas Cullin/genética , Proteínas Ligasas SKP Cullina F-box/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
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. Genetic analyses demonstrated that MLT proteins functionally cooperate with the conserved NIMA kinase family members NEKL-2/NEK8 and NEKL-3/NEK6/NEK7 to promote cuticle shedding. MLT and NEKL proteins were specifically required within the hyp7 epidermal syncytium, and fluorescently tagged mlt and nekl alleles were expressed in puncta within this tissue. Expression studies further showed that NEKL-2-MLT-2-MLT-4 and NEKL-3-MLT-3 colocalize within largely distinct assemblies of apical foci. MLT-2 and MLT-4 were required for the normal accumulation of NEKL-2 at the hyp7-seam cell boundary, and loss of mlt-2 caused abnormal nuclear accumulation of NEKL-2 Correspondingly, MLT-3, which bound directly to NEKL-3, prevented NEKL-3 nuclear localization, supporting the model that MLT proteins may serve as molecular scaffolds for NEKL kinases. Our studies additionally showed that the NEKL-MLT network regulates early steps in clathrin-mediated endocytosis at the apical surface of hyp7, which may in part account for molting defects observed in nekl and mlt mutants. This study has thus identified a conserved NEKL-MLT protein network that regulates remodeling of the apical extracellular matrix and intracellular trafficking, functions that may be conserved across species.
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Repetición de Anquirina , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/fisiología , Matriz Extracelular/metabolismo , Quinasas Relacionadas con NIMA/metabolismo , Alelos , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Citoplasma/metabolismo , Endocitosis , Matriz Extracelular/genética , Muda/genética , Mutación , Quinasas Relacionadas con NIMA/genética , Proteínas Quinasas/genética , Transporte de ProteínasRESUMEN
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. Resistance of the epidermis to this force and to actomyosin-based circumferential constricting forces is mediated by FBN-1, a ZP domain protein related to vertebrate fibrillins. fbn-1 was required specifically within the epidermis and FBN-1 was expressed in epidermal cells and secreted to the apical surface as a putative component of the embryonic sheath. Tiling array studies indicated that fbn-1 mRNA processing requires the conserved alternative splicing factor MEC-8/RBPMS. The conserved SYM-3/FAM102A and SYM-4/WDR44 proteins, which are linked to protein trafficking, function as additional components of this network. Our studies demonstrate the importance of the apical extracellular matrix in preventing mechanical deformation of the epidermis during development.
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Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/fisiología , Desarrollo Embrionario , Epidermis/patología , Proteínas de Microfilamentos/metabolismo , Estrés Mecánico , Animales , Fenómenos Biomecánicos , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/química , Epidermis/embriología , Epidermis/metabolismo , Exones/genética , Fibrilinas , Transferencia Resonante de Energía de Fluorescencia , Genes de Helminto , Morfogénesis , Mutación/genética , Faringe/fisiología , Fenotipo , Estructura Terciaria de Proteína , Empalme del ARN/genética , Vertebrados/metabolismoRESUMEN
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. Rather, our evidence suggests that PHA-1 acts in the arcade or anterior epithelial cells of the pharynx to promote attachment of the pharynx to the future buccal capsule. In addition, PHA-1 appears to be required in the epidermis for embryonic morphogenesis, in the excretory system for osmoregulation, and in the somatic gonad for normal ovulation and fertility. PHA-1 activity is also required within at least a subset of intestinal cells for viability. To better understand the role of PHA-1 in the epidermis, we analyzed several apical junction markers in pha-1(tm3671) homozygous embryos. PHA-1 regulates the expression of several components of two apical junction complexes including AJM-1-DLG-1/discs large complex and the classical cadherin-catenin complex, which may account for the role of PHA-1 in embryonic morphogenesis.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Faringe/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Caenorhabditis elegans/embriología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Cateninas/genética , Cateninas/metabolismo , Diferenciación Celular , Células Epiteliales/citología , Células Epiteliales/metabolismo , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Especificidad de Órganos , Faringe/citología , Faringe/embriología , Uniones Estrechas/metabolismoRESUMEN
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. Depletion of PBAF subunits SWSN-7/BAF200 and PBRM-1/BAF180 results in decreased expression of ESRE genes and increased sensitivity to thermal stress. When overexpressed, SWSN-7/BAF200 and PBRM-1/BAF180 led to increased ESRE transcription, enhanced thermotolerance, and induction of a nuclear ESRE-binding activity. Our data support a model in which PBAF is recruited by an ESRE-binding protein to genomic ESRE sites. We also show that the closely related SWI/SNF complex, BAF, which regulates stress induction through DAF-16/FOXO, does not contribute to ESRE gene expression or bind directly to ESRE sites. To our knowledge, this is the first report demonstrating direct and specific regulation of a stress response network by the PBAF nucleosome-remodeling complex in vivo in metazoa. In addition, we show that PBAF cooperates with the histone demethylase, JMJC-1/NO66, to promote expression of ESRE genes following stress.