ABSTRACT
The ability to generate asymmetry at the cell cortex underlies cell polarization and asymmetric cell division. Here we demonstrate a novel role for the tumor suppressor Merlin and closely related ERM proteins (Ezrin, Radixin, and Moesin) in generating cortical asymmetry in the absence of external cues. Our data reveal that Merlin functions to restrict the cortical distribution of the actin regulator Ezrin, which in turn positions the interphase centrosome in single epithelial cells and three-dimensional organotypic cultures. In the absence of Merlin, ectopic cortical Ezrin yields mispositioned centrosomes, misoriented spindles, and aberrant epithelial architecture. Furthermore, in tumor cells with centrosome amplification, the failure to restrict cortical Ezrin abolishes centrosome clustering, yielding multipolar mitoses. These data uncover fundamental roles for Merlin/ERM proteins in spatiotemporally organizing the cell cortex and suggest that Merlin's role in restricting cortical Ezrin may contribute to tumorigenesis by disrupting cell polarity, spindle orientation, and, potentially, genome stability.
Subject(s)
Cytoskeletal Proteins/metabolism , Neurofibromin 2/metabolism , Animals , Caco-2 Cells , Cell Cycle/physiology , Cell Line, Tumor , Cell Polarity , Centrosome/metabolism , Cytoskeletal Proteins/genetics , Epithelial Cells/cytology , Epithelial Cells/metabolism , Humans , Mice , Neurofibromin 2/genetics , Spindle Apparatus/metabolismABSTRACT
The neurofibromatosis type 2 (NF2) tumor suppressor, Merlin, is a FERM (Four point one, Ezrin, Radixin, Moesin) domain-containing protein whose loss results in defective morphogenesis and tumorigenesis in multiple tissues. Like the closely related ERM proteins (Ezrin, Radixin, and Moesin), Merlin may organize the plasma membrane by assembling membrane protein complexes and linking them to the cortical actin cytoskeleton. We previously found that Merlin is a critical mediator of contact-dependent inhibition of proliferation and is required for the establishment of stable adherens junctions (AJs) in cultured cells. Here, we delineate the molecular function of Merlin in AJ establishment in epidermal keratinocytes in vitro and confirm that a role in AJ establishment is an essential function of Merlin in vivo. Our studies reveal that Merlin can associate directly with α-catenin and link it to Par3, thereby providing an essential link between the AJ and the Par3 polarity complex during junctional maturation.
Subject(s)
Adherens Junctions/metabolism , Cell Polarity , Epidermis/embryology , Epidermis/growth & development , Neurofibromatosis 2/metabolism , Neurofibromin 2/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Adhesion Molecules/genetics , Cell Adhesion Molecules/metabolism , Cell Cycle Proteins , Epidermal Cells , Epidermis/physiology , Keratinocytes/cytology , Keratinocytes/physiology , Mice , Mice, Transgenic , Neurofibromatosis 2/genetics , Neurofibromin 2/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , alpha Catenin/genetics , alpha Catenin/metabolismABSTRACT
A new small molecule inhibitor of bacterial cell division has been discovered using a high-throughput screen in Escherichia coli. Although the lead screening hit (534F6) exhibited modest inhibition of the GTPase activity of FtsZ (20+/-5% at 100microM of compound), a primary target for bacterial cell division inhibitors, several analogs caused potent bacterial growth inhibition with negligible antagonism of FtsZ GTPase activity. A library of analogs has been prepared and several alkyne-tagged photoaffinity probes have been synthesized for use in experiments to elucidate the primary target of this compound.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , Cell Division/drug effects , Escherichia coli/drug effects , Growth Inhibitors/chemical synthesis , Pyrrolidines/chemical synthesis , Sulfonamides/chemical synthesis , Anti-Bacterial Agents/pharmacology , Cell Division/physiology , Escherichia coli/cytology , Escherichia coli/physiology , Escherichia coli Proteins/antagonists & inhibitors , Escherichia coli Proteins/physiology , Growth Inhibitors/pharmacology , Microbial Sensitivity Tests , Pyrrolidines/pharmacology , Sulfonamides/pharmacologyABSTRACT
FtsZ, the ancestral homolog of eukaryotic tubulins, is a GTPase that assembles into a cytokinetic ring structure essential for cell division in prokaryotic cells. Similar to tubulin, purified FtsZ polymerizes into dynamic protofilaments in the presence of GTP; polymer assembly is accompanied by GTP hydrolysis. We used a high-throughput protein-based chemical screen to identify small molecules that target assembly-dependent GTPase activity of FtsZ. Here, we report the identification of five structurally diverse compounds, named Zantrins, which inhibit FtsZ GTPase either by destabilizing the FtsZ protofilaments or by inducing filament hyperstability through increased lateral association. These two classes of FtsZ inhibitors are reminiscent of the antitubulin drugs colchicine and Taxol, respectively. We also show that Zantrins perturb FtsZ ring assembly in Escherichia coli cells and cause lethality to a variety of bacteria in broth cultures, indicating that FtsZ antagonists may serve as chemical leads for the development of new broad-spectrum antibacterial agents. Our results illustrate the utility of small-molecule chemical probes to study FtsZ polymerization dynamics and the feasibility of FtsZ as a novel therapeutic target.
