Zaidel-Bar Cell Adhesion Lab

It is well established that integrin-mediated adhesions (such as focal adhesions) are mechano-responsive: they grow when force is applied to them and they disassemble when tension is relieved. Stretching of cas and talin, exposing vinculin binding sites, recruitment of LIM domain proteins and tyrosine dephosphorylation of paxillin have all been implicated in integrin-mediated mechanosensing.

In recent years it is becoming apparent that adherens junctions are also sensitive to the level of tension at the junction. Inhibition of tension leads to disassembly of adherens junctions (albeit at a slower rate compared to focal adhesions), and increasing contractility within cells (by activating RhoA, for example) leads to reinforement of the junctions. The one mechanism proposed to date for junctional mechanosensing is the stretching of alpha-catenin by force, exposing a binding site for vinculin. We are working towards identifying novel components responsible for force-induced junction assembly by two methods: 1. An siRNA screen of candidate genes applied to a robust assay for force-induced assembly. 2. Differential mass spectroscopy of isolated cadherin adhesions under high and low tension conditions.

Previous work in Alexander Bershadsky’s lab and in other labs demonstrated a role for actin polymerization mediated by the formin Dia1 in adherens junction assembly and maintenance. However, several outstanding questions remain. First, since in all these experiments Dia1 is activated all over the cell it is not clear whether the effect on junctions is direct or not. Second, in these experiments Dia was activated either by expressing constitutively active RhoA, which activates Dia1, or by expressing a constitutively active mutant of Dia1 that sequesters RhoA and doesn’t localize normally. In both cases the level of active RhoA in the cell is affected, thus making it impossible to tease apart the effect of Dia-mediated actin polymerization from changes in myosin contractility. Finally, the mechanism of recruitment of Dia1 to junctions is unclear. Previous studies of localization of deletion constructs of Dia1 were performed in cells expressing endogenous Dia1, which may have compounded their results.

We are working to address the questions of localization and function of Dia1 at adherens junctions by creating new cleaner tools for activation of Dia1 and by working in cells knocked down for endogenous Dia1.

We are using powerful genetic tools, such as genome wide RNAi, tissue specific knockdown and forward genetics, to uncover novel components and regulators of cell-cell adhesion in C. elegans.

As a model system the “worm” has several significant strengths: it is simple (only ~1000 cells) and yet has the complexities of a living organism with different cell types and tissues. It is transparent, facilitating observation by Nomarski and fluorescent microscopy of adults and embryos. Its genome is fully sequenced (was the first multicellular organism to be sequenced!) and ~60% of its genes have homologs in humans. It is small and easy to maintain in the lab (eats bacteria!) and can be frozen and thawed.

The stage during development that is most sensitive to mutations in the cell-cell adhesion apparatus is epidermal morphogenesis, when the “skin” of the worm wraps around the embryo and the Lima bean shaped embryo morphs into a worm shape:

During enclosure (A) hypodermal cells migrate from the dorsal side to encompass the embryo and seal at the ventral midline (B). Early elongation (C) entails rapid remodeling of cell junctions. During elongation (D) an array of circumferential actin bundles distributes the squeezing force originating from actomysin contractions in the lateral cells. A’-D’ show AJM-GFP a marker of cell junctions; A’’-B’’ show F-actin with a live GFP reporter. (A-D and A’-D’ adapted from Hardin JD and Chisolm AD in WormBook Chapter “Epidermal Morphogenesis”).

Previously, we successfully constructed the integrin adhesome, which is a database of all known components of integrin mediated adhesion and the interactions between them. We have gone on to analyze the adhesome network using systems biology approaches. We performed a similar literature based construction of a network of adherens junction components, we named the Cadhesome, and are in the process of analyzing it and comparing it to the integrin adhesome.