Formin-like 2 drives amoeboid invasive cell motility downstream of RhoC.

Invasive cell migration is a key step for cancer metastasis and involves Rho GTPase-controlled reorganization of the actin cytoskeleton. Altered Rho GTPase expression is found in various malignancies. Particularly, the closely related GTPases RhoA and RhoC are upregulated in many aggressive tumours, but specific effectors that distinguish between these two GTPases to explain mechanistic differences have not been identified. The formins are by far the largest family of Rho GTPase effectors and are characterized by the actin-nucleating formin homology 2 domain. Using siRNA-based screening against all 15 human formins, we systematically analysed their functions in 3D cell motility using three different cancer cell lines. These results reveal distinct requirements for specific formins in amoeboid versus mesenchymal invasive cell migration. Importantly, by knocking down all Rho proteins, we identified formin-like 2 (FMNL2) as a specific RhoC effector, showing selective interaction of FMNL2 with active RhoC, but not RhoA or RhoB. Functional analysis shows that RhoC regulates autoinhibition of FMNL2, whereas suppression of FMNL2 inhibits RhoC-, but not RhoA-dependent, rounded invasive cell migration. Thus, our data uncover a novel regulatory and functional interaction between RhoC and FMNL2 for modulating cell shape and invasiveness and provide mechanistic insight into RhoC-specific signalling events.

A switch in disulfide linkage during minicollagen assembly in Hydra nematocysts.

The smallest known collagens with only 14 Gly-X-Y repeats referred to as minicollagens are the main constituents of the capsule wall of nematocysts. These are explosive organelles found in Hydra, jellyfish, corals and other Cnidaria. Minicollagen-1 of Hydra recombinantly expressed in mammalian 293 cells contains disulfide bonds within its N- and C-terminal Cys-rich domains but no interchain cross-links. It is soluble and self-associates through non-covalent interactions to form 25-nm-long trimeric helical rod-like molecules. We have used a polyclonal antibody prepared against the recombinant protein to follow the maturation of minicollagens from soluble precursors present in the endoplasmic reticulum and post-Golgi vacuoles to the disulfide-linked insoluble assembly form of the wall. The switch from intra- to intermolecular disulfide bonds is associated with 'hardening' of the capsule wall and provides an explanation for its high tensile strength and elasticity. The process is comparable to disulfide reshuffling between the NC1 domains of collagen IV in mammalian basement membranes.

Homophilic adhesion by cadherins.

Cadherins mediate cell-cell adhesion through homophilic interactions. High-resolution structures have greatly enhanced our understanding of this phenomenon over the past few years. Nonetheless, some of the original concepts about cadherin interactions need revision, with the new structural and additional mutagenesis data currently available. Furthermore, in vivo studies on cadherins have provided supplementary information.

A new crystal structure, Ca2+ dependence and mutational analysis reveal molecular details of E-cadherin homoassociation.

Electron microscopy of ECADCOMP, a recombinant E-cadherin ectodomain pentamerized by the assembly domain of cartilage oligomeric matrix protein, has been used to analyze the role of cis-dimerization and trans-interaction in the homophilic association of this cell adhesion molecule. The Ca2+ dependency of both interactions was investigated. Low Ca2+ concentrations (50 microM) stabilized the rod-like structure of E-cadherin. At medium Ca2+ concentration (500 microM), two adjacent ectodomains in a pentamer formed cis-dimers. At high Ca2+ concentration (>1 mM), two cis-dimers from different pentamers formed a trans-interaction. The X-ray structure of an N-terminal domain pair of E-cadherin revealed two molecules per asymmetric unit in an intertwisted X-shaped arrangement with closest contacts in the Ca2+-binding region between domains 1 and 2. Contrary to previous data, Trp2 was docked in the hydrophobic cavity of its own molecule, and was therefore not involved in cis-dimerization of two molecules. This was supported further by W2A and A80I (a residue involved in the hydrophobic cavity surrounding Trp2) mutations in ECADCOMP which both led to abrogation of the trans- but not the cis-interaction. Structural and biochemical data suggest a link between Ca2+ binding in the millimolar range and Trp2 docking, both events being essential for the trans-association.