Paper on Molecular Genetics

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Summarize the research article. Talk about the experiment- the methods they used (also talk about fig. 2) and compare the methods used to the method of cloning as described in the paper. Does the experiment answer the question? What is the impact of research to the scientific society and how is it beneficial to the human kind. Tell an additional experiment you would have done.

Put pictures (fig from the paper) for example; the intro (background info) should have a picture there describing C. elegans embryo development which is given in the paper and the pictures all together should just make up to 1 page, the rest 5 pages should be the written part. Use three additional research articles (sources).

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JCB: Article Cdc42 regulates junctional actin but not cell polarization in the Caenorhabditis elegans epidermis Yuliya Zilberman,1 Joshua Abrams,1 Dorian C. Anderson,1 and Jeremy Nance1,2 L. and Martin S. Kimmel Center for Biology and Medicine at the Skirball Institute of Biomolecular Medicine and 2Department of Cell Biology, New York University School of Medicine, New York, NY THE JOURNAL OF CELL BIOLOGY 1Helen During morphogenesis, adherens junctions (AJs) remodel to allow changes in cell shape and position while preserving adhesion. Here, we examine the function of Rho guanosine triphosphatase CDC-42 in AJ formation and regulation during Caenorhabditis elegans embryo elongation, a process driven by asymmetric epidermal cell shape changes. cdc42 mutant embryos arrest during elongation with epidermal ruptures. Unexpectedly, we find using time-lapse fluorescence imaging that cdc-42 is not required for epidermal cell polarization or junction assembly, but rather is needed for proper junctional actin regulation during elongation. We show that the RhoGAP PAC-1/ARH​GAP21 inhibits CDC-42 activity at AJs, and loss of PAC-1 or the interacting linker protein PICC-1/CCDC85A-C blocks elongation in embryos with compromised AJ function. pac-1 embryos exhibit dynamic accumulations of junctional F-actin and an increase in AJ protein levels. Our findings identify a previously unrecognized molecular mechanism for inhibiting junctional CDC42 to control actin organization and AJ protein levels during epithelial morphogenesis. Introduction Polarized cell shape changes provide forces that alter the morphology of tissues, organs, and embryos. For example, changes in the shapes of Caenorhabditis elegans epidermal cells transform the embryo from an ellipse into an elongated wormshaped cylinder in the absence of cell division. Epidermal cells are born on the dorsal surface of the embryo, then migrate ventrally and form new junctions with contralateral epidermal cells to wrap the embryo in skin (“ventral enclosure”; Chisholm and Hardin, 2005; Vuong-Brender et al., 2016). After completing ventral enclosure, epidermal cells begin to lengthen along their anterior-posterior axis and simultaneously shrink along their dorsal-ventral axis (“elongation”; Fig. 1 A). Actomyosin contractions in lateral epidermal cells provide the forces that alter epidermal cell shape during the early stage of elongation (Armenti and Nance, 2012; Cram, 2014; Vuong-Brender et al., 2016). Subsequently, the contraction of underlying muscles attached to epidermal cells provides forces that allow elongation to continue up to the fourfold stage (Armenti and Nance, 2012; Cram, 2014; Vuong-Brender et al., 2016). It is unclear how epidermal cells regulate adherens junctions (AJs) and their associated microfilaments during elongation to allow the remodeling needed for these asymmetric cell shape changes while still preserving cell adhesion. This problem is common to all types of epithelial cells that alter their shapes or change positions relative to neighbors during morphogenesis (Collinet and Lecuit, 2013; Röper, 2015). Correspondence to Jeremy Nance: Abbreviations used: AJ, adherens junction; CFB, circumferential bundle; CRIB, CDC-42/Rac interactive binding; DIC, differential interference contrast; MET, mesenchymal-to-epithelial transition; sgRNA, single guide RNA; TIRF, total internal reflection fluorescence; ZF1, zinc finger 1. The Rockefeller University Press J. Cell Biol. Vol. 216 No. 11 3729–3744 C. elegans AJs contain highly conserved components, including the transmembrane homophilic adhesion protein HMR-1/E-cadherin and the cytoplasmic catenins HMP-1/αcatenin and HMP-2/β-catenin, which interact with the HMR1/E-cadherin cytoplasmic tail and link it to actin microfilaments (Costa et al., 1998; Korswagen et al., 2000; Kwiatkowski et al., 2010). Null mutations in hmr-1/E-cadherin, hmp-1/α-catenin, or hmp-2/β-catenin cause microfilaments to detach from AJs as epidermal cells elongate, leading to developmental arrest and epidermal rupture (Costa et al., 1998). In addition to α-catenin and β-catenin, the p120 catenin JAC-1 also binds to the cytoplasmic tail of HMR-1/E-cadherin (Pettitt et al., 2003). Although JAC-1 is not essential in C. elegans (Klompstra et al., 2015), its depletion enhances the phenotype of weak mutations in hmp-1 (Pettitt et al., 2003), indicating that JAC-1 is an important regulator of AJ function. AJs form through a two-step process of polarization and junction maturation. These events occur during the middle of embryogenesis, when epithelial precursor cells undergo a mesenchymal-to-epithelial transition (MET). During the polarization step of MET, clusters of AJ proteins found along the lateral membrane concentrate at the apicolateral region of the cell (Leung et al., 1999; McMahon et al., 2001; Achilleos et al., 2010). Concomitantly, polarity regulators begin to occupy distinct subdomains at the cell surface: the adaptor protein PAR-6 localizes apically, the scaffolding protein PAR-3 concentrates © 2017 Zilberman et al. This article is distributed under the terms of an Attribution– Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http​://www​.rupress​.org​/terms​/). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 4.0 International license, as described at https​://creativecommons​.org​/licenses​/by​-nc​-sa​/4​.0​/). JCB 3729 Figure 1. cdc-42 embryos have defects in ventral enclosure and elongation. (A) Stages of embryo elongation: bean stage (pre-elongation), comma stage (1.4-fold), and pretzel stage (>3-fold). Junctions between epidermal cells are indicated with black lines. Lateral epidermal cells (“seam” cells) are yellow. Double-headed arrows indicate the extension in anterior-posterior length of a cell as the embryo elongates. (B and C) Stills from DIC time-lapse movies of control and cdc-42(MZ) embryos shown at 30-min intervals. Genotypes were confirmed by single-embryo PCR after imaging. Arrows in C point to extruding cells. See Video 1. (D) Phenotypic classes of arrested embryos from DIC time-lapse imaging experiments (n = 39). (E) Rates of elongation in control (n = 13) and Class III (n = 9) embryos. Fold elongation was measured as schematized. t = 0 represents the comma stage. Values are the mean ± SD. Data for D and E were pooled from eight independent imaging experiments. P-values were calculated using a Mann-Whitney U test. ***, P ≤ 0.001. Bars, 5 µm. at AJs, the Discs large homologue DLG-1 accumulates at the basal side of AJs, and the Scribble protein LET-413 localizes to basolateral surfaces (Legouis et al., 2000; Bossinger et al., 2001; Firestein and Rongo, 2001; Köppen et al., 2001; McMahon et al., 2001; Aono et al., 2004; Achilleos et al., 2010). Whereas PAR-3 mediates polarization of other epithelial cell types in C. elegans (Köppen et al., 2001; Aono et al., 2004; Achilleos et al., 2010), epidermal cells polarize through an unknown PAR-3–independent mechanism. Junction maturation requires PAR-6, DLG-1, and LET-413; embryos lacking any of these polarity regulators arrest during elongation (Legouis et al., 2000; Bossinger et al., 2001; Firestein and Rongo, 2001; Köppen et al., 2001; McMahon et al., 2001; Totong et al., 2007). One important regulator of AJs that has not been examined during epidermal cell MET or elongation is the Rho GTPase CDC-42. Active CDC-42 can interface with PAR proteins by binding directly to the PAR-6 CRIB (CDC-42/Rac interactive binding) domain (Gotta et al., 2001; Aceto et al., 2006), and interfering with CDC-42 function in many epithelial cell types disrupts polarity or the turnover of AJ components, leading to epithelial defects (Tepass, 2012; Duquette and Lamarche-Vane, 2014; Mack and Georgiou, 2014). For example, Cdc42 is required for apical membrane differentiation in the blastoderm 3730 JCB • Volume 216 • Number 11 • 2017 of Drosophila melanogaster embryos (Hutterer et al., 2004) and in epithelial MDCK cells grown in 3D culture (MartinBelmonte et al., 2007). Removing Cdc42 in fully polarized Drosophila epithelia causes defects in AJ organization by altering endocytosis or exocytosis, depending on the tissue (Georgiou et al., 2008; Harris and Tepass, 2008; Leibfried et al., 2008). CDC-42 can also regulate junction stability by controlling actin polymerization and turnover. For instance, multiple actinnucleating factors, such as the Arp2/3 complex and formins, function downstream of CDC-42 to influence F-actin organization at junctions (Otani et al., 2006; Verma et al., 2012; Phng et al., 2015; Rao and Zaidel-Bar, 2016). In turn, junctional F-actin can modulate AJs by affecting endocytosis, or by physically controlling E-cadherin clustering (Collinet and Lecuit, 2013; Truong Quang et al., 2013; Wu et al., 2015). Because CDC-42 is widely distributed in cells and regulates many different events (Erickson and Cerione, 2001), its activity at specific subcellular locations is often locally controlled. Like other Rho GTPases, CDC-42 is active when bound to GTP and inactive when bound to GDP. RhoGEFs activate CDC-42 and other Rho GTPases by promoting GTP binding, whereas RhoGAPs function as inhibitors by promoting GTP hydrolysis (Bos et al., 2007). Studies using constitutively active and dominant negative versions of CDC-42 have shown that its activity must be regulated to ensure normal junction formation and maintenance (Kroschewski et al., 1999; Rojas et al., 2001; Bruewer et al., 2004; Elbediwy et al., 2012), indicating that RhoGEFs and RhoGAPs are likely to influence CDC-42 activity at junctions. In cultured mammalian epithelial cells, CDC-42 activity is tuned at different stages of junction biogenesis by a small number of GEFs and GAPs, including the RhoGEFs Tuba and Dbl3/ARH​GEF21 (Otani et al., 2006; Zihni et al., 2014) and the RhoGAPs Rich1 and SH3BP1 (Wells et al., 2006; Elbediwy et al., 2012). However, it remains unclear which RhoGEFs or RhoGAPs function in vivo to regulate CDC-42 activity as junctions remodel during morphogenesis. Here, we show that, unexpectedly, CDC-42 is dispensable for polarization and junction maturation during MET in the C. elegans epidermis, but is essential during elongation to control the polarized cell shape changes and junctional actin dynamics of epidermal cells. We identify the conserved RhoGAP PAC-1/ARH​ GAP21, which localizes specifically to AJs and likely functions with its binding partner PICC-1/ CCDC85A-C, as a negative regulator of CDC-42 activity at these sites. pac-1 mutant embryos form ectopic, dynamic, actin extensions at AJs, exhibit increased AJ protein levels, and fail to elongate in a sensitized genetic background. Our findings reveal that RhoGAP-mediated CDC-42 regulation enables tight control over actin organization and AJ protein levels in vivo, allowing epithelial cells to remodel their junctions efficiently as cells change shape. Results CDC-42 is required for proper ventral enclosure and embryo elongation C. elegans has a single cdc-42 homologue, which is expressed ubiquitously in embryos, larvae, and adults (Anderson et al., 2008; Armenti et al., 2014b; Neukomm et al., 2014). Within the embryo, CDC-42 protein arises from both maternal and zygotic sources. Reducing levels of maternal CDC-42 results in a loss of polarity and defects in spindle orientation in early embryos, culminating in embryonic lethality (Gotta et al., 2001; Kay and Hunter, 2001; Anderson et al., 2008). We examined cdc-42 function in later embryos by using a degron-based strategy to deplete maternal CDC-42 protein from the early embryo just after its function in the one-cell embryo is complete, while simultaneously eliminating zygotic cdc-42 expression with a cdc42 null allele, gk388 (Anderson et al., 2008). Proteins tagged with the PIE-1 zinc finger 1 (ZF1) domain are recognized by the E3 ligase adaptor ZIF-1, which causes the tagged proteins to degrade rapidly from early embryonic somatic cells after the one-cell stage (Reese et al., 2000; DeRenzo et al., 2003; Nance et al., 2003). However, because ZIF-1 appears to be present only in early embryos (Armenti et al., 2014b), ZF1-tagged proteins that are expressed zygotically at later embryonic stages do not degrade. CDC-42 fused at its N-terminus with HA tags and the ZF1 domain (Anderson et al., 2008) largely rescued the lethality of cdc-42 mutants (84% [389/463] of cdc-42[gk388]; ha-zf1-cdc-42 embryos were viable) and degraded rapidly from early embryonic somatic cells before its zygotic expression began during the middle of embryogenesis (Fig. S1, A–B; Anderson et al., 2008). To obtain embryos lacking cdc-42 activity, we allowed cdc-42; ha-zf1-cdc-42/+ hermaphrodites to self-fertilize (Fig. S1 C, Strategy I). One quarter of the resulting progeny should lack the ha-zf1-cdc-42 transgene, and because the maternal HA-ZF1-CDC-42 protein is degraded, these embryos will be left with no maternal or zygotic source of CDC-42 (Fig. S1 C, right). Hereafter, we refer to such embryos as cdc42(MZ). Of the self-progeny of cdc-42; ha-zf1-cdc-42/+ heterozygotes, 24% (116/479) died before hatching. PCR genotyping showed that 84% (15/18) of randomly selected dead embryos were cdc-42(MZ), whereas the remaining 16% (3/18) contained the ha-zf1-cdc-42 transgene but failed to rescue. Thus, although ha-zf1-cdc-42 transgene largely rescues the cdc-42(gk388) mutant phenotype, it does not do so completely. All healthy genotyped L1 larvae contained the ha-zf1-cdc-42 transgene (19/19), whereas a small number of cdc-42(MZ) embryos died soon after hatching into sickly larvae. We conclude that cdc-42 has an essential function during the second half of embryonic development, after zygotic cdc-42 expression begins. We used 3D time-lapse differential interference contrast (DIC) imaging to examine the dynamics of ventral enclosure and elongation in live cdc-42(MZ) embryos (Fig. 1, B and C; and Video 1). Of 135 embryos imaged from a cdc-42; ha-zf1cdc-42/+ mother, 39 (29%) did not hatch. Based on the results of our genotyping, 86% of unhatched embryos are predicted to be cdc-42(MZ) embryos, whereas the remaining small percentage of arrested embryos are predicted to contain the ha-zf1-cdc-42 transgene. Arrested embryos were assigned to three phenotypic classes according to the final stage of morphogenesis they completed (Fig. 1 D): Class I embryos (17/39, 44%) failed to complete ventral enclosure and therefore arrested before elongation; Class II embryos ruptured in the head ventral region early in elongation, typically at the 1.5-fold stage (8/39, 20%; Fig. 1 C); Class III embryos arrested later during elongation, at or after the twofold stage, with extruded cells (14/39, 36%). Embryos that arrested during elongation also elongated at a slower rate than embryos that developed to hatching (Fig. 1 E). Based on these observations and our genotyping data, we conclude that loss of cdc-42 activity causes significant morphogenetic defects that are apparent during ventral enclosure and elongation, and that cause embryonic arrest before elongation is complete. CDC-42 is dispensable for polarity establishment and junction maturation We next tested if there were defects in epidermal cell polarization or junction maturation in cdc-42(MZ) embryos that could explain their arrest phenotype. CDC-42 is thought to promote epithelial cell polarization by recruiting PAR-6 to the apical surface; this interaction occurs through the PAR-6 CRIB domain, which binds directly to active CDC-42 (Joberty et al., 2000; Johansson et al., 2000; Lin et al., 2000; Qiu et al., 2000; Gotta et al., 2001; Hutterer et al., 2004; Aceto et al., 2006). Unexpectedly, PAR-6 still accumulated at the apical membrane of epidermal cells (Fig. 2 B, red arrow) in cdc-42(MZ) embryos, even though the apical to cytoplasmic ratio of PAR-6 immunostaining in the epidermis of cdc-42(MZ) mutants was decreased twofold compared with control embryos (Fig. 2, A, E, and F). To rule out the possibility that a trace amount of HA-ZF1-CDC-42 beyond our limit of detection remained in cdc-42(MZ) embryos and was sufficient to recruit PAR-6, we examined the localization of a previously characterized form of PAR-6 with mutations in the CRIB domain that make it unable to bind CDC-42 (Aceto et al., 2006). PAR-6ΔCRIBGFP, expressed from endogenous regulatory sequences and Cdc42 regulates junctions but not polarization • Zilberman et al. 3731 Figure 2. cdc-42 is not required for epidermal cell polarity or junction maturation. (A and B) PAR-6 staining of control embryos (cdc-42 mutants rescued with ha-zf1-cdc-42) and cdc42(MZ) embryos. Red arrows indicate PAR-6 enrichment at the apical domain in epidermis; white arrows indicate enrichment at the apical domain in intestine. Red lines show t where the intensity profile shown in E was taken. (C and D) PAR-6-GFP and PAR-6ΔCRIB-GFP imaged live in par-6(MZ) embryos. (E) Intensity profile of PAR-6 in control (representative of n = 24) and in cdc-42(MZ) (representative of n = 20) epidermal cells (taken from micrographs shown in A and B). (F) Quantification of apical membrane to cytoplasm intensity ratio of PAR-6 immunostaining in the epidermis of control (n = 24) and cdc-42(MZ) (n = 20) embryos. Individual data points from two independent experiments were pooled. (G) Quantification of apical membrane to cytoplasm intensity ratio in the epidermis of PAR-6-GFP (n = 13) and PAR-6ΔCRIB-GFP (n = 14) in live par-6(MZ) embryos. Individual data points from two independent experiments were pooled. (H–I) Frames from time-lapse movies of control and cdc-42(MZ) embryos expressing HMR-1-GFP, showing polarization and junction maturation of dorsal epidermal cells. Insets show a side view of lateral epidermal cells, in which apical enrichment of HMR-1-GFP to junctions is observed by 50 min. t = 0 is defined as the initiation of polarization, when HMR-1-GFP puncta are first detected in the intestine. See Videos 2 and 3. (J) Quantification of junctional enrichment of HMR-1-GFP in epidermal cells at t = 50 min (control, n = 9 embryos; cdc42(MZ), n = 12 embryos). (K and L) Immunostaining of junctional HMP-1 and basolateral LET-413 in control and cdc-42(MZ) embryos. Insets below are of boxed region. Line and error bars in graphs (F, G, and J) indicate mean ± SD. P-values were calculated using a Mann-Whitney U test. *, P ≤ 0.05; ***, P ≤ 0.001. NS, P > 0.05. Bars, 5 µm. examined in a par-6(MZ) mutant background, localized similarly to endogenous PAR-6 in cdc-42(MZ) mutants, with strong apical enrichment in epithelial cells that was less pronounced but still evident in epidermal cells (compared with PAR-6GFP in wild-type; Fig. 2, C, D, and G). However, par-6(MZ); par-6ΔCRIB-gfp embryos arrested before the 1.7-fold stage of elongation (27/27), in contrast with par-6(MZ); par-6-gfp embryos, which nearly all survived (95%, 232/243). Thus, whereas neither CDC-42 nor the PAR-6 CRIB domain is essential for PAR-6 apical enrichment, both contribute to enriching PAR-6 apically within epidermal cells, and the CRIB domain is needed for PAR-6 function. Because PAR-6 is required for junction maturation, we asked whether the decreased apical enrichment of PAR-6 in 3732 JCB • Volume 216 • Number 11 • 2017 cdc-42(MZ) epidermal cells blocked or slowed junction formation or maturation. We examined junctions when epidermal cells first polarized via MET using fluorescence time-lapse imaging of two junction proteins: the E-cadherin HMR-1-GFP expressed from a knockin allele, and the Discs large protein DLG-1-RFP expressed from an integrated transgene. To genotype live cdc-42(MZ) embryos, we created a functional zf1-yfpcdc-42 knockin allele, plac ...
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