Abstract. Overexpression in insect cells of the full coding sequence of the human membrane cytoskeletal linker ezrin (1-586) was compared with that of a NHEterminal domain (ezrin 1-233) and that of a COOHterminal domain (ezrin 310-586). Ezrin (1-586), as well as ezrin (1-233) enhanced cell adhesion of infected Si x ) cells without inducing gross morphological changes in the cell structure. Ezrin (310-586) enhanced cell adhesion and elicited membrane spreading followed by microspike and lamellipodia extensions by mobilization of Sf9 cell actin. Moreover some microspikes elongated into thin processes, up to 200 #m in length, resembling neurite outgrowths by a mechanism requiring microtubule assembly. Kinetics of videomicroscopic and drug-interference studies demonstrated that mobilization of actin was required for tubulin assembly to proceed. A similar phenotype was observed in CHO cells when a comparable ezrin domain was transiently overexpressed. The shortest domain promoting cell extension was localized between residues 373-586. Removal of residues 566-586, involved in in vitro actin binding (Turunen, O., T. Wahlstr6m, and A. Vaheri. 1994. J. Cell Biol. 126:1445-1453, suppressed the extension activity. Coexpression of ezrin (1-233) with ezrin (310-586) in the same insect cells blocked the constitutive activity of ezrin COOH-terminal domain. The inhibitory activity was mapped within ezrin 115 first NH2-terminal residues. We conclude that ezrin has properties to promote cell adhesion, and that ezrin NHe-terminal domain negatively regulates membrane spreading and elongation properties of ezrin COOH-terminal domain.

Zebrafish (Danio rerio) embryos are increasingly used as an infection model to study the function of the vertebrate innate immune system in host-pathogen interactions. The ease of obtaining large numbers of embryos, their accessibility due to external development, their optical transparency as well as the availability of a wide panoply of genetic/immunological tools and transgenic reporter line collections, contribute to the versatility of this model. In this respect, the present manuscript describes the use of zebrafish as an in vivo model system to investigate the chronology of Mycobacterium abscessus infection. This human pathogen can exist either as smooth (S) or rough (R) variants, depending on cell wall composition, and their respective virulence can be imaged and compared in zebrafish embryos and larvae. Micro-injection of either S or R fluorescent variants directly in the blood circulation via the caudal vein, leads to chronic or acute/lethal infections, respectively. This biological system allows high resolution visualization and analysis of the role of mycobacterial cording in promoting abscess formation. In addition, the use of fluorescent bacteria along with transgenic zebrafish lines harbouring fluorescent macrophages produces a unique opportunity for multi-color imaging of the host-pathogen interactions. This article describes detailed protocols for the preparation of homogenous M. abscessus inoculum and for intravenous injection of zebrafish embryos for subsequent fluorescence imaging of the interaction with macrophages. These techniques open the avenue to future investigations involving mutants defective in cord formation and are dedicated to understand how this impacts on M. abscessus pathogenicity in a whole vertebrate. Video LinkThe video component of this article can be found at

The ERM proteins-ezrin, radixin, and moesin-are key players in membrane-cytoskeleton interactions. In insect cells infected with recombinant baculoviruses, amino acids 1-115 of ezrin were shown to inhibit an actin-and tubulin-dependent cell-extension activity located in ezrin C-terminal domain (ezrin310_586), whereas full-length ezrin1l586 did not induce any morphological change. To refine the mapping of functional domains of ezrin, 30 additional constructs were overexpressed in Sf9 cells, and the resulting effect of each was qualitatively and semiquantitatively compared. The removal of amino acids 13-30 was sufficient to release a cell-extension phenotype. This effect was abrogated if the 21 distal-most C-terminal amino acids were subsequently deleted (ezrin31l565), confirming the existence of a head-to-tail regulation in the whole molecule. Surprisingly, the deletion in full-length ezrin of the same 21 amino acids provided strong cell-extension competence to ezrin1j565, and this property was recovered in N-terminal constructs as short as ezrin1l310. Within ezrinl310_, amino acid sequences 13-30 and 281-310 were important determinants and acted in cooperation to induce cytoskeleton mobilization. In addition, these same residues are part of a new actin-binding site characterized in vitro in ezrin N-terminal domain.