Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia.

The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

Dynamics and ultrastructure of developmental cell fusions in the Caenorhabditis elegans hypodermis.

Cell fusions produce multinucleate syncytia that are crucial to the structure of essential tissues in many organisms [1-5]. In humans the entire musculature, much of the placenta, and key cells in bones and blood are derived from cell fusion. Yet the developmental fusion of cell membranes has never been directly observed and is poorly understood. Similarity between viral fusion proteins and recently discovered cellular proteins implies that both cell-cell and virus-cell fusion may occur by a similar mechanism [6-8]. Paradoxically, however, fusion of enveloped viruses with cells involves an opening originating as a single pore [9-11], whereas electron microscopy studies of cell-cell fusion describe simultaneous breakdown of large areas of membrane [12, 13]. Here, we have shown that developmental cell fusion is indeed consistent with initiation by a virus-like, pore-forming mechanism. We examined live cell fusions in the epithelia of Caenorhabditis elegans embryos by a new method that integrates multiphoton, confocal, and electron microscopy. The fusion aperture always originated at a single point restricted to the apical adherens junction and widened slowly as a radial wavefront. The fusing membranes dispersed by vesiculation, rather than simple unfolding of the conjoined double bilayer. Thus, in these cells fusion appears to require two specialized sequential processes: formation of a unique primary pore and expansion of the opening by radial internalization of the interacting cell membranes.

Pip92: a short-lived, growth factor-inducible protein in BALB/c 3T3 and PC12 cells.

pip92 is a cellular immediate-early gene inducible by serum growth factors in fibroblasts. It is also induced in the rat pheochromocytoma cell line PC12 by agents that cause proliferation, neuronal differentiation, and membrane depolarization. We show that the pip92-encoded polypeptide is a proline-rich protein of 221 amino acids, has an extremely short half-life, and is localized in the cytoplasm. We hypothesize that Pip92 plays a role in mediating the cellular responses to a variety of extracellular signals.

Genomic structure, cDNA sequence, and expression of gly96, a growth factor-inducible immediate-early gene encoding a short-lived glycosylated protein.

We report the cDNA sequence and genomic structure of gly96, an immediate early gene inducible by serum growth factors in mouse fibroblasts. It encodes a 153-amino acid protein that does not share significant sequence similarity with any known protein. In the adult mouse, gly96 is expressed predominantly in the lung, testes and the uterus. We have identified the Gly96 protein in Balb/c 3T3 cells using affinity-purified antibodies recognizing the Gly96 polypeptide. We show that Gly96 is glycosylated and has a short half-life in serum stimulated fibroblasts.

Getting into shape: epidermal morphogenesis in Caenorhabditis elegans embryos.

The change in shape of the C. elegans embryo from an ovoid ball of cells into a worm-shaped larva is driven by three events within the cells of the hypodermis (epidermis): (1) intercalation of two rows of dorsal cells, (2) enclosure of the ventral surface by hypodermis, and (3) elongation of the embryo. While the behavior of the hypodermal cells involved in each of these processes differs dramatically, it is clear that F-actin and microtubules have essential functions in each of these processes, whereas contraction of actomyosin structures appears to be involved specifically in elongation. Molecular analysis of these processes is revealing components specific to C. elegans as well as components found in other systems. Since C. elegans hypodermal cells demonstrate dramatically different behaviors during intercalation, enclosure and elongation, the study of cytoskeletal dynamics in these processes may reveal both unique and conserved activities during distinct epithelial morphogenetic movements. BioEssays 23:12-23, 2001.

Sequential signalling during Caenorhabditis elegans vulval induction.

During the induction of the Caenorhabditis elegans vulva, cell signalling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p and P8.p) express the tertiary cell fate (Fig. 1a). We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor-cell signal-transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor-cell signal transduction is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.

Pulsed-field gel electrophoresis of circular DNA.

Mobility of supercoiled (form I) and nicked circular (form II) plasmid DNAs was determined on two major forms of pulsed-field electrophoresis, CHEF and OFAGE. Plasmids with molecular lengths ranging from 2.30 to 17.8 kilobase pairs (kb) were used with Saccharomyces cerevisiae chromosomes as standards. Agarose gel concentrations were varied from 0.3 to 2.0 percent, with higher percentage gels resolving forms I and II of smaller plasmids. The pulsing range of 3.7 to 240 seconds resulted in quite variable Saccharomyces chromosomal mobilities on both 0.5 and 1.0 percent gels, while both form I and II of all plasmid DNAs showed relatively constant mobilities with some increase at the shortest pulse times. Using a 30 second pulse time and gel concentrations of at least 1.0 percent, the usual order of migration of plasmid forms for a 17.8 kb plasmid could be changed. We interpret this result as an increase in the relative mobility of form II in our pulsed-field gel conditions.

LET-23 receptor localization by the cell junction protein LIN-7 during C. elegans vulval induction.

In C. elegans, the anchor cell signal induces Pn.p cells to form the vulva by activating a conserved receptor tyrosine kinase pathway. lin-2 and lin-7 mutants exhibit a vulvaless phenotype similar to the phenotype observed when this signaling pathway is defective. We have found that LIN-7 is a cell junction-associated protein that binds to the LET-23 receptor tyrosine kinase. LET-23 is also localized to the cell junctions, and both LIN-2 and LIN-7 are required for this localization. LET-23 overexpression rescues the lin-2 or lin-7 vulvaless phenotype, suggesting that increased receptor density can compensate for mislocalization. These results suggest that proper localization of LET-23 receptor to the Pn.p cell junctions is required for signaling activity.

The beta-catenin homolog BAR-1 and LET-60 Ras coordinately regulate the Hox gene lin-39 during Caenorhabditis elegans vulval development.

In C. elegans, the epithelial Pn.p cells adopt either a vulval precursor cell fate or fuse with the surrounding hypodermis (the F fate). Our results suggest that a Wnt signal transduced through a pathway involving the beta-catenin homolog BAR-1 controls whether P3.p through P8.p adopt the vulval precursor cell fate. In bar-1 mutants, P3.p through P8.p can adopt F fates instead of vulval precursor cell fates. The Wnt/bar-1 signaling pathway acts by regulating the expression of the Hox gene lin-39, since bar-1 is required for LIN-39 expression and forced lin-39 expression rescues the bar-1 mutant phenotype. LIN-39 activity is also regulated by the anchor cell signal/let-23 receptor tyrosine kinase/let-60 Ras signaling pathway. Our genetic and molecular experiments show that the vulval precursor cells can integrate the input from the BAR-1 and LET-60 Ras signaling pathways by coordinately regulating activity of the common target LIN-39 Hox.