Direct interaction of actin filaments with F-BAR protein pacsin2.

Two mechanisms have emerged as major regulators of membrane shape: BAR domain-containing proteins, which induce invaginations and protrusions, and nuclear promoting factors, which cause generation of branched actin filaments that exert mechanical forces on membranes. While a large body of information exists on interactions of BAR proteins with membranes and regulatory proteins of the cytoskeleton, little is known about connections between these two processes. Here, we show that the F-BAR domain protein pacsin2 is able to associate with actin filaments using the same concave surface employed to bind to membranes, while some other tested N-BAR and F-BAR proteins (endophilin, CIP4 and FCHO2) do not associate with actin. This finding reveals a new level of complexity in membrane remodeling processes.

Whole-mount immunoelectron tomography of chromosomes and cells.

Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.

EILATox-Oregon Workshop: blind study evaluation of Vitotox test with genotoxic and cytotoxic sample library.

In order to assess the robustness, sensitivity and specificity of a recently developed Vitotox test, 17 blind coded chemicals and three environmental water samples were tested at the EILATox-Oregon Workshop using the Thermo Electron Vitotox kit. The Vitotox test is a rapid geno- and cytotoxicity test using standard 96- or 384-well microtitre plates. The genotoxicity test is based on two genetically modified Salmonella typhimurium strains containing bacterial luciferase operon from Vibrio fisheri under the SOS inducible promoter. The SOS system is an inducible network in Escherichia coli that responds to DNA damage and activates DNA repair. The Vitotox genotoxicity test bacteria strain carries bacterial luciferase genes under the control of SOS inducible promoter and therefore any DNA damage inside the cells induces the production of bacterial luciferase. The luciferase expression is then followed with a microtitre plate luminometer for 3 h after mixing different dilutions of sample with the test bacteria. The genotoxicity index is calculated for each dilution and the genotoxicity of the sample is interpreted based on kinetic time curves and genotoxicity vs concentration/dilution curves. Cytotoxicity of the sample is determined simultaneously with another test strain containing the same luciferase operon controlled by the constitutive promoter. This bacterium produces constant bioluminescence and any decrease of the bioluminescence production is used as a marker for cytotoxicity. As a miniaturized microtitre plate assay the Vitotox test requires a very small quantity of the sample material. The samples used in the workshop were diluted 1 : 10 or 1 : 100 before testing. Genotoxicity and cytotoxicity data were collected at dilutions of 1 : 10-1 : 2000. When the samples of the EILATox-Oregon Workshop were tested using the Vitotox test, four coded chemicals out of 17 were determined to be genotoxic. Seven chemicals and one environmental sample were found to be cytotoxic. Three chemical samples were found to be both geno- and cytotoxic.

FAP52 regulates actin organization via binding to filamin.

FAP52, a focal adhesion-associated phosphoprotein, is a member of a FAP52/PACSIN/syndapin family of proteins. They share a multidomain structure and are implicated in actin-based and endocytotic functions. We show, by using both native and recombinant proteins, that FAP52 selectively binds to the actin cross-linking protein filamin (ABP-280). This was based on an affinity purification followed by a sequence determination by mass spectrometry, co-immunoprecipitation, overlay binding, and surface plasmon resonance analysis. Binding studies with deletion mutants showed that the sites of the interaction map to the highly alpha-helical N-terminal part of FAP52 and to the C-terminal region of filamin, which also contains binding sites to some transmembrane signaling proteins. In immunofluorescence and immunoelectron microscopy of cultured fibroblasts, a different overall subcellular distribution was seen for filamin and FAP52 except for a stress fiber-focal adhesion junction where they showed a notable overlap. Overexpression of the full-length and mutant forms of FAP52 led to an extensive reorganization of actin and filamin in cultured fibroblasts. Thus, the results show that FAP52 interacts with filamin, and we propose that this interaction is important in linking and coordinating the events between focal adhesions and the actin cytoskeleton.

Focal adhesion protein FAP52 self-associates through a sequence conserved among the members of the PCH family proteins.

FAP52 is a recently described focal adhesion-associated protein. It is a member of an emerging PCH (pombe Cdc15 homology) family of proteins characterized by a common domain organization and involvement in actin cytoskeleton organization, cytokinesis, and vesicular trafficking. Using gel filtration, surface plasmon resonance, and native polyacrylamide gel electrophoresis analysis, combined with chemical cross-linking of both native and recombinant protein, we show that FAP52 self-associates in vitro and suggest that it occurs predominantly as a trimer also in vivo. Analysis of the various domains of FAP52 by surface plasmon resonance showed that the highly alpha-helical region in the N-terminal half of the protein provides the self-association interface. Overexpression of the oligomerization domain in cultured cells was accompanied by major alterations in cellular morphology, actin organization, and the structure of focal adhesions, suggesting that an orderly coming together of FAP52 molecules is crucial for a proper actin filament organization and cytoskeletal structure. Comparison of the primary structures shows that all of the members of the PCH family have, in their N-terminal halves, a similar, highly alpha-helical region, suggesting that they all have a capacity to self-associate.