Embryonic morphogenesis in Caenorhabditis elegans integrates the activity of LET-502 Rho-binding kinase, MEL-11 myosin phosphatase, DAF-2 insulin receptor and FEM-2 PP2c phosphatase

let-502 rho-binding kinase and mel-11 myosin phosphatase regulate Caenorhabditis elegans embryonic morphogenesis. Genetic analysis presented here establishes the following modes of let-502 action: (i) loss of only maternal let-502 results in abnormal early cleavages, (ii) loss of both zygotic and maternal let-502 causes elongation defects, and (iii) loss of only zygotic let-502 results in sterility. The morphogenetic function of let-502 and mel-11 is apparently redundant with another pathway since elimination of these two genes resulted in progeny that underwent near-normal elongation. Triple mutant analysis indicated that unc-73 (Rho/Rac guanine exchange factor) and mlc-4 (myosin light chain) act in parallel to or downstream of let-502/mel-11. In contrast mig-2 (Rho/Rac), daf-2 (insulin receptor), and age-1 (PI3 kinase) act within the let-502/mel-11 pathway. Mutations in the sex-determination gene fem-2, which encodes a PP2c phosphatase (unrelated to the MEL-11 phosphatase), enhanced mutations of let-502 and suppressed those of mel-11. fem-2's elongation function appears to be independent of its role in sexual identity since the sex-determination genes fem-1, fem-3, tra-1, and tra-3 had no effect on mel-11 or let-502. By itself, fem-2 affects morphogenesis with low penetrance. fem-2 blocked the near-normal elongation of let-502; mel-11 indicating that fem-2 acts in a parallel elongation pathway. The action of two redundant pathways likely ensures accurate elongation of the C. elegans embryo.     

The Caenorhabditis elegans mel-11 myosin phosphatase regulatory subunit affects tissue contraction in the somatic gonad and the embryonic epidermis and genetically interacts with the Rac signaling pathway

Caenorhabditis elegans embryonic elongation is driven by cell shape changes that cause a contraction of the epidermal cell layer enclosing the embryo. We have previously shown that this process requires a Rho-associated kinase (LET-502) and is opposed by the activity of a myosin phosphatase regulatory subunit (MEL-11). We now extend our characterization and show that mel-11 activity is required both in the epidermis during embryonic elongation and in the spermatheca of the adult somatic gonad. let-502 and mel-11 reporter gene constructs show reciprocal expression patterns in the embryonic epidermis and the spermatheca, and mutations of the two genes have opposite effects in these two tissues. These results are consistent with let-502 and mel-11 mediating tissue contraction and relaxation, respectively. We also find that mel-11 embryonic inviability is genetically enhanced by mutations in a Rac signaling pathway, suggesting that Rac potentiates or acts in parallel with the activity of the myosin phosphatase complex. Since Rho has been implicated in promoting cellular contraction, our results support a mechanism by which epithelial morphogenesis is regulated by the counteracting activities of Rho and Rac.     

The Caenorhabditis elegans nonmuscle myosin genes nmy-1 and nmy-2 function as redundant components of the let-502/Rho-binding kinase and mel-11/myosin phosphatase pathway during embryonic morphogenesis

Rho-binding kinase and the myosin phosphatase targeting subunit regulate nonmuscle contractile events in higher eukaryotes. Genetic evidence indicates that the C. elegans homologs regulate embryonic morphogenesis by controlling the actin-mediated epidermal cell shape changes that transform the spherical embryo into a long, thin worm. LET-502/Rho-binding kinase triggers elongation while MEL-11/myosin phosphatase targeting subunit inhibits this contractile event. We describe mutations in the nonmuscle myosin heavy chain gene nmy-1 that were isolated as suppressors of the mel-11 hypercontraction phenotype. However, a nmy-1 null allele displays elongation defects less severe than mutations in let-502 or in the single nonmuscle myosin light chain gene mlc-4. This results because nmy-1 is partially redundant with another nonmuscle myosin heavy chain, nmy-2, which was previously known only for its role in anterior/posterior polarity and cytokinesis in the early embryo. At the onset of elongation, NMY-1 forms filamentous-like structures similar to actin, and LET-502 is interspersed with these structures, where it may trigger contraction. MEL-11, which inhibits elongation, is initially cytoplasmic. In response to LET-502 activity, MEL-11 becomes sequestered away from the contractile apparatus, to the plasma membrane, when elongation commences. Upon completion of morphogenesis, MEL-11 again appears in the cytoplasm where it may halt actin/myosin contraction.     

Ribosomal protein RPS-14 modulates let-7 microRNA function in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates developmental timing at the larval-to-adult transition in Caenorhabditis elegans. Dysregulation of let-7 results in irregular hypodermal and vulval development. Disrupted let-7 function is also a feature of human lung cancer. However, little is known about the mechanism and co-factors of let-7. Here we demonstrate that ribosomal protein RPS-14 is able to modulate let-7 function in C. elegans. The RPS-14 protein co-immunoprecipitated with the nematode Argonaute homolog, ALG-1. Reduction of rps-14 gene expression by RNAi suppressed the aberrant vulva and hypodermis development phenotypes of let-7(n2853) mutant animals and the mis-regulation of a reporter bearing the lin-41 3′UTR, a well established let-7 target. Our results indicate an interactive relationship between let-7 miRNA function and ribosomal protein RPS-14 in regulation of terminal differentiation that may help in understanding the mechanism of translational control by miRNAs.     

Functional Genomic Analysis of the let-7 Regulatory Network in Caenorhabditis elegans

The let-7 microRNA (miRNA) regulates cellular differentiation across many animal species. Loss of let-7 activity causes abnormal development in Caenorhabditis elegans and unchecked cellular proliferation in human cells, which contributes to tumorigenesis. These defects are due to improper expression of protein-coding genes normally under let-7 regulation. While some direct targets of let-7 have been identified, the genome-wide effect of let-7 insufficiency in a developing animal has not been fully investigated. Here we report the results of molecular and genetic assays aimed at determining the global network of genes regulated by let-7 in C. elegans. By screening for mis-regulated genes that also contribute to let-7 mutant phenotypes, we derived a list of physiologically relevant potential targets of let-7 regulation. Twenty new suppressors of the rupturing vulva or extra seam cell division phenotypes characteristic of let-7 mutants emerged. Three of these genes, opt-2, prmt-1, and T27D12.1, were found to associate with Argonaute in a let-7–dependent manner and are likely novel direct targets of this miRNA. Overall, a complex network of genes with various activities is subject to let-7 regulation to coordinate developmental timing across tissues during worm development.     

Asymmetric Wolbachia Segregation during Early Brugia malayi Embryogenesis Determines Its Distribution in Adult Host Tissues

Wolbachia are required for filarial nematode survival and fertility and contribute to the immune responses associated with human filarial diseases. Here we developed whole-mount immunofluorescence techniques to characterize Wolbachia somatic and germline transmission patterns and tissue distribution in Brugia malayi, a nematode responsible for lymphatic filariasis. In the initial embryonic divisions, Wolbachia segregate asymmetrically such that they occupy only a small subset of cells in the developing embryo, facilitating their concentration in the adult hypodermal chords and female germline. Wolbachia are not found in male reproductive tissues and the absence of Wolbachia from embryonic germline precursors in half of the embryos indicates Wolbachia loss from the male germline may occur in early embryogenesis. Wolbachia rely on fusion of hypodermal cells to populate adult chords. Finally, we detect Wolbachia in the secretory canal lumen suggesting living worms may release bacteria and/or their products into their host.     

A Putative Catenin–Cadherin System Mediates Morphogenesis of the Caenorhabditis elegans Embryo

During morphogenesis of the Caenorhabditis elegans embryo, hypodermal (or epidermal) cells migrate to enclose the embryo in an epithelium and, subsequently, change shape coordinately to elongate the body (Priess, J.R., and D.I. Hirsh. 1986. Dev. Biol. 117:156– 173; Williams-Masson, E.M., A.N. Malik, and J. Hardin. 1997. Development [Camb.]. 124:2889–2901). We have isolated mutants defective in morphogenesis that identify three genes required for both cell migration during body enclosure and cell shape change during body elongation. Analyses of hmp-1, hmp-2, and hmr-1 mutants suggest that products of these genes anchor contractile actin filament bundles at the adherens junctions between hypodermal cells and, thereby, transmit the force of bundle contraction into cell shape change. The protein products of all three genes localize to hypodermal adherens junctions in embryos. The sequences of the predicted HMP-1, HMP-2, and HMR-1 proteins are related to the cell adhesion proteins α-catenin, β-catenin/Armadillo, and classical cadherin, respectively. This putative catenin–cadherin system is not essential for general cell adhesion in the C. elegans embryo, but rather mediates specific aspects of morphogenetic cell shape change and cytoskeletal organization.The morphology of the animal body and its tissues arise as embryonic cells change their shapes and/or positions (Mittenthal and Jacobson, 1990). Many of these changes are mediated by dynamic rearrangements of cytoskeletal components (Wessells et al., 1971). Cells can organize diverse patterns of microtubules and actin filaments, and movement of actin filaments by myosin proteins is thought to generate the force that drives many morphogenetic processes. An important step toward understanding the mechanical basis of morphogenesis is the identification and characterization of molecules that pattern the cytoskeleton and translate force into concerted cell movements. For cells to change shape coordinately or move relative to each other, forces generated within an individual cell must be transmitted to adhesive junctions at the plasma membrane and exerted on neighboring cells or the extracellular matrix (Gumbiner, 1996). The best characterized cell–cell junction is the adherens junction. This type of junction usually forms a subapical, beltlike structure that mechanically links the lateral surfaces of adjacent epithelial cells. Adherens junctions contain transmembrane proteins of the cadherin family that mediate homotypic adhesion. Cadherins are thought to connect to the actin cytoskeleton indirectly through the proteins α-catenin and β-catenin. Catenin–cadherin complexes also are associated with sites of contact between blastomeres in vertebrate and invertebrate embryos. In Drosophila, mice, and Xenopus, gene inactivation of catenins or cadherins disrupts general cell adhesion and apicobasal polarity of blastomeres and epithelial cells (Heasman et al., 1994; Larue et al., 1994; Haegel et al., 1995; Cox et al., 1996; Müller and Wieschaus, 1996; Kafron et al., 1997; Torres et al., 1997). Thus, it has been difficult to define direct requirements for these proteins in cytoskeletal organization and morphogenesis, although there is evidence for specific roles in tracheal cell migration (Tanaka-Matakatsu et al., 1996) and axon outgrowth (Iwai et al., 1997) in Drosophila.The Caenorhabditis elegans embryo provides a model system for studying how cells move and change shape to generate body and tissue morphologies. At hatching, the outermost cellular layer of the body consists of a monolayer of 85 epithelial cells called hypodermal cells that are linked together by adherens junctions (White, 1988). During embryogenesis, hypodermal cells are involved in two distinct processes that transform the initially ellipsoidal mass of embryonic cells into a long, thin worm; these processes are called body enclosure and body elongation (Sulston et al., 1983; Priess and Hirsh, 1986; Williams-Masson et al., 1997). The hypodermal cells are born on the dorsal surface of the embryo. As the hypodermal cells develop adherens junction connections, they begin to spread as a sheet across the embryo until the contralateral edges of the sheet meet at the ventral midline. In the anterior of the embryo, ventral hypodermal cells on the periphery of the spreading sheet develop filopodial extensions that may function to draw the contralateral edges of the sheet together (Williams-Masson et al., 1997). In the posterior of the embryo, the contralateral edges appear to be drawn together by a purse-string–like contraction that completes the enclosure process (Williams-Masson et al., 1997). In several respects, these processes are similar to epithelial cell movements described in a variety of systems, such as wound healing in vertebrates (Martin and Lewis, 1992) and dorsal closure in Drosophila (Young et al., 1993). At the completion of body enclosure in C. elegans, the apical surfaces of the hypodermal cells resemble rectangles that are elongated along the circumferential contour of the embryo''s body. These apical surfaces begin to change shape, constricting along the circumferential contour of the body and elongating along the anterior–posterior (longitudinal) axis. The coordinate changes in the shapes of the hypodermal cells appear to cause the body to decrease in circumference and to elongate about fourfold along its longitudinal axis (Sulston et al., 1983; Priess and Hirsh, 1986). Before body elongation, the apical cytoskeleton of each hypodermal cell reorganizes to form an array of parallel actin filament bundles oriented along the circumferential contour of the body (Priess and Hirsh, 1986; Costa et al., 1997). The parallel filament bundles bridge two opposing sides of each hypodermal cell, apparently connecting to the subapical adherens junction. Contraction of the filament bundles has been proposed as the force that elongates the embryo; the bundles become shorter and thicker during elongation, and drugs that disrupt actin filament organization prevent elongation. Apical constriction of cells has been shown in other systems to drive the invagination of epithelial sheets; because of the closed, cylindrical geometry of the hypodermal sheet in C. elegans, an analogous apical constriction might instead drive body elongation (Priess and Hirsh, 1986). Although the morphology and properties of the hypodermal cells strongly suggest that they mediate body elongation, almost all of the elongation-defective mutants described thus far have mutations in genes encoding muscle or basement membrane components. Body-wall muscles underlie the hypodermis, separated by a basement membrane (Hresko et al., 1994; diagram in Fig. ​Fig.88 a). Mutations in any of several genes that eliminate embryonic muscle contraction prevent elongation beyond a twofold increase in body length; this phenotype is called Pat¹ (paralyzed, arrested elongation at twofold; Williams and Waterston, 1994). Some of the genes of the Pat class have been shown to encode muscle-specific proteins. Because the muscles and myofilaments are oriented longitudinally, muscle contraction would be expected to oppose body elongation; thus, it is not yet understood why muscle function is required for complete elongation. The genes let-2 and emb-9 encode basement membrane collagens, and mutations in these genes produce elongation defects similar to those of Pat mutants (Guo et al., 1991; Sibley et al., 1993; Williams and Waterston, 1994). The only gene identified that is both required for proper body elongation and apparently expressed in hypodermal cells is let-502 (Wissmann et al., 1997). The predicted LET-502 protein is related to Rho-binding kinases, which can activate myosin light chain kinase, suggesting that LET-502 could have a role in hypodermal cells for the contraction of the array of actin filament bundles. Open in a separate windowFigure 8Models of morphogenetic forces and molecular organization at hypodermal cell junctions. (A) Oblique view of a schematic cross-section of an embryo after fusion of the dorsal hypodermal cells. CFBs are shown as thin lines, and adherens junctions are shown as thick lines. Bands of longitudinally oriented body wall muscles (m) are shown underlying the hypodermal cells. (B) Mechanical model of forces between the dorsal hypodermis and a lateral hypodermal cell. The connection between each CFB and the adherens junction (AJ) is represented as an open circle. In the lateral hypodermal cell, the connections are pulled downward by contraction of the CFBs within the lateral cell itself and pulled upward as CFBs in the dorsal cell contract. Note that the adherens junction at the two ends of the lateral cell (shown as two springs) are oriented such that they could dissipate some of the force exerted by contractions in the dorsal cell. (C) Two molecular models for the linkage between a filament (CF) in a CFB to a filament (AJF) in the adherens junction. HMR-1 is shown at the membrane contacts between two cells and associated with HMP-2. In the top cell, HMP-1 links a CF and an AJF directly; in the bottom cell, HMP-1 links different AJFs together while another factor (X) provides the link between the AJFs and CFs. To expand our understanding of the molecular basis for morphogenesis, we have isolated and characterized a group of mutants that display similar defects in embryo morphogenesis. In this paper, we present evidence that a C. elegans catenin–cadherin system mediates morphogenetic cell shape changes and specific aspects of cytoskeletal organization. We show that the genes hmp-1, hmp-2, and hmr-1 are required for the proper migration of hypodermal cells during body enclosure and for body elongation. We demonstrate that hmp-1, hmp-2, and hmr-1 can encode proteins related to α-catenin, β-catenin, and cadherin, respectively. We show that the protein products of these genes are localized to adherens junctions in the hypodermis. Our results indicate that these proteins anchor the parallel actin filament bundles to the adherens junctions in hypodermal cells and that this coupling translates the force of bundle contraction into cell shape change.     

Morphogenesis beyond Cytokinetic Arrest in Saccharomyces cerevisiae

The budding yeast lyt1 mutation causes cell lysis. We report here that lyt1 is an allele of cdc15, a gene which encodes a protein kinase that functions late in the cell cycle. Neither cdc15-1 nor cdc15-lyt1 strains are able to septate at 37°C, even though they may manage to rebud. Cells lyse after a shmoo-like projection appears at the distal pole of the daughter cell. Actin polarizes towards the distal pole but the septins remain at the mother–daughter neck. This morphogenetic response reflects entry into a new round of the cell cycle: the preference for polarization from the distal pole was lost in bud1 cdc15 double mutants; double cdc15-lyt1 cdc28-4 mutants, defective for START, did not develop apical projections and apical polarization was accompanied by DNA replication. The same phenomena were caused by mutations in the genes CDC14, DBF2, and TEM1, which are functionally related to CDC15. Apical polarization was delayed in cdc15 mutants as compared with budding in control cells and this delay was abolished in a septin mutant. Our results suggest that the delayed M/G1 transition in cdc15 mutants is due to a septin-dependent checkpoint that couples initiation of the cell cycle to the completion of cytokinesis.     

Alternative Splicing in Class V Myosins Determines Association with Rab10

Rab proteins influence vesicle trafficking pathways through the assembly of regulatory protein complexes. Previous investigations have documented that Rab11a and Rab8a can interact with the tail region of myosin Vb and regulate distinct trafficking pathways. We have now determined that a related Rab protein, Rab10, can interact with myosin Va, myosin Vb, and myosin Vc. Rab10 localized to a system of tubules and vesicles that have partially overlapping localization with Rab8a. Both Rab8a and Rab10 were mislocalized by the expression of dominant-negative myosin V tails. Interaction with Rab10 was dependent on the presence of the alternatively spliced exon D in myosin Va and myosin Vb and the homologous region in myosin Vc. Yeast two-hybrid assays and fluorescence resonance energy transfer studies confirmed that Rab10 binding to myosin V tails in vivo required the alternatively spliced exon D. In contrast to our previous work, we found that Rab11a can interact with both myosin Va and myosin Vb tails independent of their splice isoform. These results indicate that Rab GTPases regulate diverse endocytic trafficking pathways through recruitment of multiple myosin V isoforms.Eukaryotic cells are comprised of networks of highly organized membranous structures that require the efficient and timely movement of diverse intracellular proteins for proper function. Molecular motors provide the physical force needed to move these materials along microtubules and actin microfilaments. Unconventional myosin motors, such as those belonging to classes V, VI, and VII, have roles in the trafficking and recycling of membrane-bound structures in eukaryotic cells (1) and are recruited to discrete vesicle populations. Myosin VI is involved in clathrin-mediated endocytosis (2), whereas myosin VIIa participates in the proper development of stereocilia of inner ear hair cells and the transport of pigment granules in retinal pigmented epithelial cells (3, 4). Similarly, the three members of vertebrate class V myosins, myosin Va, myosin Vb, and myosin Vc, are required for the proper transport of a wide array of membrane cargoes, such as the melanosomes of pigment cells, synaptic vesicles in neurons, apical recycling endosomes in polarized epithelial cells, and bulk recycling vesicles in non-polarized cells (5).Members of the Rab family of small GTPases regulate many cellular systems, including membrane trafficking (6, 7). Certain Rab proteins associate with and regulate the function of class V myosins. Rab27a, in a complex with the adaptor protein melanophilin/Slac2-a, is required to localize myosin Va to the surface of melanin-filled pigment granules in vertebrates (8-10), whereas Rab27a and Slac2-c/MyRIP associate with both Myosin Va and myosin VIIa (3, 11). Rab11a, in a complex with its adaptor protein Rab11-FIP2, associates with myosin Vb on recycling endosomes (12-14) where the tripartite complex regulates the recycling of a variety of cargoes (15-19). In addition, Rab8a associates with both myosin Vb (20) and myosin Vc (21) as part of the non-clathrin-mediated tubular recycling system (20). Recently, Rab11a has also been shown to associate with myosin Va in the transport of AMPA receptors in dendritic spines (22), contributing to the model of myosin V regulation by multiple Rab proteins.Previous investigations have documented alternative splicing of myosin Va in a tissue-specific manner (23-28). Alternate splicing occurs in a region lying between the coiled-coil region of the neck of the motor and the globular tail region. Three exons in particular are subject to alternative splicing: exons B, D, and F (23-25). Exon F is critical for association with melanophilin/Slac2 and Rab27a (8, 9, 29, 30). Additionally, exon B is required for the interaction of myosin Va with dynein light chain 2 (DLC2) (27, 28). Currently no function for the alternatively spliced exon D has been reported. Similar to myosin Va, myosin Vb contains exons A, B, C, D, and E, whereas no exon F has yet been identified in myosin Vb (Fig. 1A). In addition, exon B in myosin Vb does not resemble the dynein light chain 2 (DLC2) binding region in myosin Va (27, 28), and therefore, it likely does not interact with DLC2. On the other hand, exon D is highly conserved among Myosin Va, myosin Vb, and myosin Vc, suggesting a common function in these molecular motors.Open in a separate windowFIGURE 1.Tissue distribution of human myosin Va and myosin Vb splice isoforms. A, schematic of the alternative exon organization in the tails of myosin Va and myosin Vb. It is known that exons B, D, and F are subject to alternative splicing in myosin Va, whereas there is only evidence that exon D is alternatively spliced in myosin Vb, which does not contain exon F. B, alignment of exon D sequences from mouse and human myosin V''s. myosin Va and myosin Vb both contain exon D (amino acids 1320-1346 of myosin Va and 1315-1340 of myosin Vb), whereas myosin Vc contains an exon D-like region (amino acids 1124-1147 of human myosin Vc) that is not known to be alternatively spliced. Alignment of the exon D regions from all three motors reveals a high degree of homology, especially in the center of the exon. Asterisks indicate amino acid identities. C, PCR-based analysis of human tissue panels reveals the alternative splicing pattern of exon D in myosin Va and myosin Vb. Primers flanking the region encoding exon D for both motors were used to amplify cDNA from human MTC™ panels (Clontech). cDNA amplified from HeLa cell RNA as well as myosin Va and myosin Vb tail constructs were used as positive controls. Variants expressing exon D (upper bands) and lacking exon D (lower bands) were visible. Per., peripheral; Pos., positive.Here we report that Rab10, a protein related to Rab8a and thought to have similar function (31-35), localizes to a system of tubules and vesicles overlapping in distribution with Rab8a in HeLa cells. Utilizing dominant-negative myosin V tail constructs, we show that Rab8a and Rab10 can interact with Myosin Va, myosin Vb, and myosin Vc in vivo. In addition, we have determined that the alternatively spliced exon D in both myosin Va and myosin Vb is required for interaction with Rab10. In contrast to our previous findings, we demonstrate that Rab11a is able to interact with both myosin Va and myosin Vb tails in an exon independent-manner. These results reveal that multiple Rab proteins potentially regulate all three class V myosin motors.     

CDC28 phosphorylates Cac1p and regulates the association of chromatin assembly factor i with chromatin

Chromatin Assembly Factor I (CAF-I) plays a key role in the replication-coupled assembly of nucleosomes. It is expected that its function is linked to the regulation of the cell cycle, but little detail is available. Current models suggest that CAF-I is recruited to replication forks and to chromatin via an interaction between its Cac1p subunit and the replication sliding clamp, PCNA, and that this interaction is stimulated by the kinase CDC7. Here we show that another kinase, CDC28, phosphorylates Cac1p on serines 94 and 515 in early S phase and regulates its association with chromatin, but not its association with PCNA. Mutations in the Cac1p-phosphorylation sites of CDC28 but not of CDC7 substantially reduce the in vivo phosphorylation of Cac1p. However, mutations in the putative CDC7 target sites on Cac1p reduce its stability. The association of CAF-I with chromatin is impaired in a cdc28–1 mutant and to a lesser extent in a cdc7–1 mutant. In addition, mutations in the Cac1p-phosphorylation sites by both CDC28 and CDC7 reduce gene silencing at the telomeres. We propose that this phosphorylation represents a regulatory step in the recruitment of CAF-I to chromatin in early S phase that is distinct from the association of CAF-I with PCNA. Hence, we implicate CDC28 in the regulation of chromatin reassembly during DNA replication. These findings provide novel mechanistic insights on the links between cell-cycle regulation, DNA replication and chromatin reassembly.     

Two Cdc2 Kinase Genes with Distinct Functions in Vegetative and Infectious Hyphae in Fusarium graminearum

Eukaryotic cell cycle involves a number of protein kinases important for the onset and progression through mitosis, most of which are well characterized in the budding and fission yeasts and conserved in other fungi. However, unlike the model yeast and filamentous fungi that have a single Cdc2 essential for cell cycle progression, the wheat scab fungus Fusarium graminearum contains two CDC2 orthologs. The cdc2A and cdc2B mutants had no obvious defects in growth rate and conidiation but deletion of both of them is lethal, indicating that these two CDC2 orthologs have redundant functions during vegetative growth and asexual reproduction. However, whereas the cdc2B mutant was normal, the cdc2A mutant was significantly reduced in virulence and rarely produced ascospores. Although deletion of CDC2A had no obvious effect on the formation of penetration branches or hyphopodia, the cdc2A mutant was limited in the differentiation and growth of infectious growth in wheat tissues. Therefore, CDC2A plays stage-specific roles in cell cycle regulation during infectious growth and sexual reproduction. Both CDC2A and CDC2B are constitutively expressed but only CDC2A was up-regulated during plant infection and ascosporogenesis. Localization of Cdc2A- GFP to the nucleus but not Cdc2B-GFP was observed in vegetative hyphae, ascospores, and infectious hyphae. Complementation assays with chimeric fusion constructs showed that both the N- and C-terminal regions of Cdc2A are important for its functions in pathogenesis and ascosporogenesis but only the N-terminal region is important for its subcellular localization. Among the Sordariomycetes, only three Fusarium species closely related to F. graminearum have two CDC2 genes. Furthermore, F. graminearum uniquely has two Aurora kinase genes and one additional putative cyclin gene, and its orthologs of CAK1 and other four essential mitotic kinases in the budding yeast are dispensable for viability. Overall, our data indicate that cell cycle regulation is different between vegetative and infectious hyphae in F. graminearum and Cdc2A, possibly by interacting with a stage-specific cyclin, plays a more important role than Cdc2B during ascosporogenesis and plant infection.     

TP53 and Let-7a micro-RNA Regulate K-Ras Activity in HCT116 Colorectal Cancer Cells

Recent reports have indicated that KRAS and TP53 mutations predict response to therapy in colorectal cancer. However, little is known about the relationship between these two common genetic alterations. Micro-RNAs (miRNAs), a class of noncoding RNA implicated in cellular processes, have been increasingly linked to KRAS and TP53. We hypothesized that lethal-7a (let-7a) miRNA regulates KRAS through TP53. To investigate the relationship between KRAS, TP53, and let-7a, we used HCT116 KRAS^mut human colorectal cancer cells with four different genotypic modifications in TP53 (TP53^−/−, TP53^+/−, TP53^mut/+, and TP53^mut/−). Using these cells we observed that K-Ras activity was higher in cells with mutant or knocked out TP53 alleles, suggesting that wild-type TP53 may suppress K-Ras activity. Let-7a was present in HCT116 KRAS^mut cells, though there was no correlation between let-7a level and TP53 genotype status. To explore how let-7a may regulate K-Ras in the different TP53 genotype cells we used let-7a inhibitor and demonstrated increased K-Ras activity across all TP53, thus corroborating prior reports that let-7a regulates K-Ras. To assess potential clinical implications of this regulatory network, we examined the influence of TP53 genotype and let-7a inhibition on colon cancer cell survival following chemoradiation therapy (CRT). We observed that cells with complete loss of wild-type TP53 alleles (^−/− or ^−/mut) were resistant to CRT following treatment with 5-fluorouracil and radiation. Further increase in K-Ras activity with let-7a inhibition did not impact survival in these cells. In contrast, cells with single or double wild-type TP53 alleles were moderately responsive to CRT and exhibited resistance when let-7a was inhibited. In summary, our results show a complex regulatory system involving TP53, KRAS, and let-7a. Our results may provide clues to understand and target these interactions in colorectal cancer.     

Characterization and expression patterns of <Emphasis Type="Italic">let-7</Emphasis> microRNA in the silkworm (<Emphasis Type="Italic">Bombyx mori</Emphasis>)

Background  

lin-4 and let-7, the two founding members of heterochronic microRNA genes, are firstly confirmed in Caenorhabditis elegans to control the proper timing of developmental programs in a heterochronic pathway. let-7 has been thought to trigger the onset of adulthood across animal phyla. Ecdysone and Broad-Complex are required for the temporal expression of let-7 in Drosophila melanogaster. For a better understanding of the conservation and functions of let-7, we seek to explore how it is expressed in the silkworm (Bombyx mori).     

Genes implicated in Caenorhabditis elegans and human health regulate stress resistance and physical abilities in aged Caenorhabditis elegans

Recently, nine Caenorhabditis elegans genes, grouped into two pathways/clusters, were found to be implicated in healthspan in C. elegans and their homologues in humans, based on literature curation, WormBase data mining and bioinformatics analyses. Here, we further validated these genes experimentally in C. elegans. We downregulated the nine genes via RNA interference (RNAi), and their effects on physical function (locomotion in a swim assay) and on physiological function (survival after heat stress) were analysed in aged nematodes. Swim performance was negatively affected by the downregulation of acox-1.1, pept-1, pak-2, gsk-3 and C25G6.3 in worms with advanced age (twelfth day of adulthood) and heat stress resistance was decreased by RNAi targeting of acox-1.1, daf-22, cat-4, pig-1, pak-2, gsk-3 and C25G6.3 in moderately (seventh day of adulthood) or advanced aged nematodes. Only one gene, sad-1, could not be linked to a health-related function in C. elegans with the bioassays we selected. Thus, most of the healthspan genes could be re-confirmed by health measurements in old worms.     

MEL-47, a novel protein required for early cell divisions in the nematode <Emphasis Type="Italic">Caenorhabditis elegans</Emphasis>

In the early Caenorhabditis elegans embryo, a rapid succession of cell divisions, many of them asymmetric, form blastomeres that differ in size, cell cycle duration and developmental potential. These early cell cycles are highly regulated and controlled by maternally contributed products. We describe here a novel gene, mel-47, that is required maternally for the proper execution of the early cell cycles. mel-47(yt2) mutants arrest as completely disorganized embryos with 50–80 cells of variable size. The earliest defects we found are changes in the absolute and relative duration of the very early embryonic cell cycles. In particular, the posterior cell of the two-cell embryo divides late compared with its anterior sister. Frequently the daughter cells remain connected through chromatin bridges after the early cleavage divisions indicating that the chromosomes do not segregate properly. The cell cycle delay can be suppressed by knocking down a DNA replication check point. Therefore we propose that mel-47 is required for proper DNA replication in the early embryo. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Functions of the novel RhoGAP proteins RGA-3 and RGA-4 in the germ line and in the early embryo of C. elegans

We have identified two redundant GTPase activating proteins (GAPs) - RGA-3 and RGA-4 - that regulate Rho GTPase function at the plasma membrane in early Caenorhabditis elegans embryos. Knockdown of both RhoGAPs resulted in extensive membrane ruffling, furrowing and pronounced pseudo-cleavages. In addition, the non-muscle myosin NMY-2 and RHO-1 accumulated on the cortex at sites of ruffling. RGA-3 and RGA-4 are GAPs for RHO-1, but most probably not for CDC-42, because only RHO-1 was epistatic to the two GAPs, and the GAPs had no obvious influence on CDC-42 function. Furthermore, knockdown of either the RHO-1 effector, LET-502, or the exchange factor for RHO-1, ECT-2, alleviated the membrane-ruffling phenotype caused by simultaneous knockdown of both RGA-3 and RGA-4 [rga-3/4 (RNAi)]. GFP::PAR-6 and GFP::PAR-2 were localized at the anterior and posterior part of the early C. elegans embryo, respectively showing that rga-3/4 (RNAi) did not interfere with polarity establishment. Most importantly, upon simultaneous knockdown of RGA-3, RGA-4 and the third RhoGAP present in the early embryo, CYK-4, NMY-2 spread over the entire cortex and GFP::PAR-2 localization at the posterior cortex was greatly diminished. These results indicate that the functions of CYK-4 are temporally and spatially distinct from RGA-3 and RGA-4 (RGA-3/4). RGA-3/4 and CYK-4 also play different roles in controlling LET-502 activation in the germ line, because rga-3/4 (RNAi), but not cyk-4 (RNAi), aggravated the let-502(sb106) phenotype. We propose that RGA-3/4 and CYK-4 control with which effector molecules RHO-1 interacts at particular sites at the cortex in the zygote and in the germ line.     

Identification of specific let-7 microRNA binding complexes in Caenorhabditis elegans

Little is known about the protein complexes required for microRNA formation and function. Here we used native gel electrophoresis to identify miRNA ribonucleoprotein complexes (miRNPs) in Caenorhabditis elegans. Our data reveal multiple distinct miRNPs that assemble on the let-7 miRNA in vitro. The formation of these complexes is affected but not abolished by alg-1 or alg-2 null mutations. The largest complex (M*) with an estimated molecular mass of >669 kDa cofractionates with the known RISC factors ALG-1, VIG-1, and TSN-1. The M* complex and two complexes, M3 and M4, with similar molecular weights of ~500 kDa, also assemble on all other miRNAs used in our experiments. Two smaller complexes, M1 (~160 kDa) and M2 (~250 kDa), assemble on the members of the let-7 miRNAs family but not lin-4 or mir-234, and their formation is highly dependent on specific sequences in the 5′ seed region of let-7. Moreover, an unidentified protein, p40, which only appears in the M1 and M2 complexes, was detected by UV triggered cross-linking to let-7 but not to lin-4. The cross-linking of p40 to let-7 is also dependent on the let-7 sequence. Another unidentified protein, p13, is detected in all let-7 binding complexes and lin-4 cross-linked products. Our data suggest that besides being present in certain large miRNPs with sizes similar to reported RISC, the let-7 miRNA also assembles with specific binding proteins and forms distinct small complexes.     

GA signaling expands: The plant UBX domain-containing protein 1 is a binding partner for the GA receptor

The plant Ubiquitin Regulatory X (UBX) domain-containing protein 1 (PUX1) functions as a negative regulator of gibberellin (GA) signaling. GAs are plant hormones that stimulate seed germination, the transition to flowering, and cell elongation and division. Loss of Arabidopsis (Arabidopsis thaliana) PUX1 resulted in a “GA-overdose” phenotype including early flowering, increased stem and root elongation, and partial resistance to the GA-biosynthesis inhibitor paclobutrazol during seed germination and root elongation. Furthermore, GA application failed to stimulate further stem elongation or flowering onset suggesting that elongation and flowering response to GA had reached its maximum. GA hormone partially repressed PUX1 protein accumulation, and PUX1 showed a GA-independent interaction with the GA receptor GA-INSENSITIVE DWARF-1 (GID1). This suggests that PUX1 is GA regulated and/or regulates elements of the GA signaling pathway. Consistent with PUX1 function as a negative regulator of GA signaling, the pux1 mutant caused increased GID1 expression and decreased accumulation of the DELLA REPRESSOR OF GA1-3, RGA. PUX1 is a negative regulator of the hexameric AAA+ ATPase CDC48, a protein that functions in diverse cellular processes including unfolding proteins in preparation for proteasomal degradation, cell division, and expansion. PUX1 binding to GID1 required the UBX domain, a binding motif necessary for CDC48 interaction. Moreover, PUX1 overexpression in cell culture not only stimulated the disassembly of CDC48 hexamer but also resulted in co-fractionation of GID1, PUX1, and CDC48 subunits in velocity sedimentation assays. Based on our results, we propose that PUX1 and CDC48 are additional factors that need to be incorporated into our understanding of GA signaling.

The plant protein PUX1 interacts with the gibberellin hormone receptor and functions as a negative regulator of gibberellin hormone responses, including seed germination, plant growth, and flowering.     

Mlc1p Is a Light Chain for the Unconventional Myosin Myo2p in Saccharomyces cerevisiae

In Saccharomyces cerevisiae, the unconventional myosin Myo2p is of fundamental importance in polarized growth. We explore the role of the neck region and its associated light chains in regulating Myo2p function. Surprisingly, we find that precise deletion of the six IQ sites in the neck region results in a myosin, Myo2-Δ6IQp, that can support the growth of a yeast strain at 90% the rate of a wild-type isogenic strain. We exploit this mutant in a characterization of the light chains of Myo2p. First, we demonstrate that the localization of calmodulin to sites of polarized growth largely depends on the IQ sites in the neck of Myo2p. Second, we demonstrate that a previously uncharacterized protein, Mlc1p, is a myosin light chain of Myo2p. MLC1 (YGL106w) is an essential gene that exhibits haploinsufficiency. Reduced levels of MYO2 overcome the haploinsufficiency of MLC1. The mutant MYO2-Δ6IQ is able to suppress haploinsufficiency but not deletion of MLC1. We used a modified gel overlay assay to demonstrate a direct interaction between Mlc1p and the neck of Myo2p. Overexpression of MYO2 is toxic, causing a severe decrease in growth rate. When MYO2 is overexpressed, Myo2p is fourfold less stable than in a wild-type strain. High copies of MLC1 completely overcome the growth defects and increase the stability of Myo2p. Our results suggest that Mlc1p is responsible for stabilizing this myosin by binding to the neck region.