No abstract

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.

We tested the model that neurons and epithelial cells use a shared mechanism for polarized protein sorting by comparing the pathways for localizing basolateral and postsynaptic proteins in C. elegans. GLR-1 glutamate receptors are localized to postsynaptic elements of central synapses and, when ectopically expressed, to basolateral membranes of epithelial cells. Proper localization of GLR-1 in both neurons and epithelia requires the PDZ protein LIN-10, defining LIN-10 as a shared component of the basolateral and postsynaptic localization pathways. Changing the GLR-1 carboxy-terminal sequence from a group I PDZ-binding consensus (-TAV) to a group II consensus (-FYV) restores GLR-1 synaptic localization in lin-10 mutants. Thus, these interneurons utilize at least two separate postsynaptic localization pathways.

Caenorhabditis elegans RAB-10 functions in endocytic recycling in polarized cells, regulating basolateral cargo transport in the intestinal epithelia and postsynaptic cargo transport in interneurons. Here we show binding of RAB-10 to EHBP-1, a CH-domain protein, and demonstrate a requirement for EHBP-1 in RAB-10–regulated transport in both of these tissues.

Dendritic morphology determines many aspects of neuronal function, including action potential propagation and information processing. However, the question remains as to how distinct neuronal dendrite branching patterns are established. Here, we report that postsynaptic density-95 (PSD-95), a protein involved in dendritic spine maturation and clustering of synaptic signaling proteins, plays a novel role in regulating dendrite outgrowth and branching, independent of its synaptic functions. In immature neurons, overexpression of PSD-95 decreases the proportion of primary dendrites that undergo additional branching, resulting in a marked reduction of secondary dendrite number. Conversely, knocking down PSD-95 protein in immature neurons increases secondary dendrite number. The effect of PSD-95 is activity-independent and is antagonized by cypin, a nonsynaptic protein that regulates PSD-95 localization. Binding of cypin to PSD-95 correlates with formation of stable dendrite branches. Finally, overexpression of PSD-95 in COS-7 cells disrupts microtubule organization, indicating that PSD-95 may modulate microtubules to regulate dendritic branching. Whereas many factors have been identified which regulate dendrite number, our findings provide direct evidence that proteins primarily involved in synaptic functions can also play developmental roles in shaping how a neuron patterns its dendrite branches.

Synaptic connections undergo a dynamic process of stabilization or elimination during development, and this process is thought to be critical in memory and learning and in establishing the specificity of synaptic connections. The type II calcium- and calmodulin-dependent protein kinase (CaMKII) has been proposed to be pivotal in regulating synaptic strength and in maturation of synapses during development. Here we describe how CaMKII regulates the formation of central glutamatergic synapses in Caenorhabditis elegans. During larval development, the density of ventral nerve cord synapses containing the GLR-1 glutamate receptor is held constant despite marked changes in neurite length. The coupling of synapse number to neurite length requires both CaMKII and voltage-gated calcium channels. CaMKII regulates GLR-1 by at least two distinct mechanisms: regulating transport of GLR-1 from cell bodies to neurites; and regulating the addition or maintenance of GLR-1 to postsynaptic elements.

Epidermal growth factor (EGF) signalling regulates growth and differentiation. Here, we examine the function of EGF signalling in Caenorhabditis elegans lifespan. We find that EGF signalling regulates lifespan via the Ras-MAPK pathway and the PLZF transcription factors EOR-1 and EOR-2. As animals enter adulthood, EGF signalling upregulates the expression of genes involved in the ubiquitin proteasome system (UPS), including the Skp1-like protein SKR-5, while downregulating the expression of HSP16-type chaperones. Using reporters for global UPS activity, protein aggregation, and oxidative stress, we find that EGF signalling alters protein homoeostasis in adults by increasing UPS activity and polyubiquitination, while decreasing protein aggregation. We show that SKR-5 and the E3/E4 ligases that comprise the ubiquitin fusion degradation (UFD) complex are required for the increase in UPS activity observed in adults, and that animals that lack SKR-5 or the UFD have reduced lifespans and indications of oxidative stress. We propose that as animals enter fertile adulthood, EGF signalling switches the mechanism for maintaining protein homoeostasis from a chaperonebased approach to an approach involving protein elimination via augmented UPS activity.

Cellular junctions are critical for intercellular communication and for the assembly of cells into tissues. Cell junctions often consist of tight junctions, which form a permeability barrier and prevent the diffusion of lipids and proteins between cell compartments, and adherens junctions, which control the adhesion of cells and link cortical actin filaments to attachment sites on the plasma membrane. Proper tight junction formation and cell polarity require the function of membrane-associated guanylate kinases (MAGUKs) that contain the PDZ protein-protein interaction domain. In contrast, less is known about how adherens junctions are assembled. Here we describe how the PDZ-containing protein DLG-1 is required for the proper formation and function of adherens junctions in Caenorhabditis elegans. DLG-1 is a MAGUK protein that is most similar in sequence to mammalian SAP97, which is found at both synapses of the CNS, as well as at cell junctions of epithelia. DLG-1 is localized to adherens junctions, and DLG-1 localization is mediated by an amino-terminal domain shared with SAP97 but not found in other MAGUK family members. DLG-1 recruits other proteins and signaling molecules to adherens junctions, while embryos that lack DLG-1 fail to recruit the proteins AJM-1 and CPI-1 to adherens junctions. DLG-1 is required for the proper organization of the actin cytoskeleton and for the morphological elongation of embryos. In contrast to other proteins that have been observed to affect adherens junction assembly and function, DLG-1 is not required to maintain cell polarity. Our results suggest a new function for MAGUK proteins distinct from their role in cell polarity. INTRODUCTIONCell adhesion facilitates the assembly of individual cells into organized tissues, and several different cell adhesion mechanisms fulfill this role (Gumbiner, 1996). One of these adhesion mechanisms is the formation of the tight junction or zonula occludens, which functions to regulate the permeability of the cell layer and to polarize the cell surface into apical and basolateral compartments (Mitic and Anderson, 1998). Tight junctions (in vertebrates) and septate junctions (in invertebrates) maintain the separation between the apical and basolateral surfaces by hindering the diffusion of lipids and proteins (Powell, 1981;Gumbiner, 1987). In contrast to tight junctions, a second mechanism of cell adhesion is the adherens junction, which is responsible for maintaining adhesion between neighboring cells and is important for intercellular communication (Muller, 2000;Vasioukhin and Fuchs, 2001).While many proteins have important roles in assembling these junctions, mutations in these proteins result in large defects in cell polarity as well. For example, mutations in the Drosophila genes bazooka (baz), crumbs (crb), discs lost (dlt), scribble (scrib), lethal (1) discs large (dlg), and lethal giant larvae (lgl), and the Caenorhabditis elegans gene let-413 disrupt epithelial cell polarity and prevent the formation of cell junctions (Legouis et al., 20...