Actin: its cumbersome pilgrimage through cellular compartments

In this article, we follow the history of one of the most abundant, most intensely studied proteins of the eukaryotic cells: actin. We report on hallmarks of its discovery, its structural and functional characterization and localization over time, and point to present days’ knowledge on its position as a member of a large family. We focus on the rather puzzling number of diverse functions as proposed for actin as a dual compartment protein. Finally, we venture on some speculations as to its origin

A six-module human nebulin fragment bundles actin filaments and induces actin polymerization

Gonsior SM, Gautel M, Hinssen H. A six-module human nebulin fragment bundles actin filaments and induces actin polymerization. JOURNAL OF MUSCLE RESEARCH AND CELL MOTILITY. 1998;19(3):225-235.We have investigated the interaction of a 6-repeat recombinant human nebulin fragment (S6R2R7) with F-actin, with Mg2+-induced actin paracrystals, and G-actin, respectively. This fragment corresponds to super-repeat 6, repeat 2 to 7 of human nebulin, and is located in the N-terminal part of the super-repeat region of the nebulin molecule. The S6R2R7 fragment included an immuno-tag of three amino-acid residues (EEF) at one end which was detectable by a monoclonal anti-tubulin YL1/2. By a cosedimentation assay, interaction between F-actin and S6R2R7 was observed. Electron microscopy revealed the formation of large bundle-like aggregates containing highly parallelized actin filaments, apparently caused by actin bundling of the nebulin fragment. Compared with Mg2+-induced actin paracrystals where the helices of the actin filaments are arranged in register, the filaments in the actin-nebulin bundles seem to be packed in a different way and show no obvious periodicity. The bundles were also visible in the light microscope, and immunofluorescence microscopy revealed binding of the nebulin fragment S6R2R7 to both preformed Mg2+ paracrystals and to F-actin. We also analyzed the effect of S6R2R7 on actin under non-polymerizing conditions by cosedimentation assays and pyrene actin fluorimetry, as well as fluorescence microscopy and electron microscopy. Nebulin-induced actin polymerization was observed with an enhancement of the nucleation step indicating a stabilization of actin nuclei by S6R2R7. Light and electron microscopy revealed bundle-like actin-nebulin aggregates similar to those formed by pre-assembled F-actin and S6R2R7. Thus, even in the absence of salt, S6R2R7 promotes actin polymerization and induces formation of tightly packed actin filament bundles. We assume that the actin filaments are crosslinked by the nebulin fragments, indicating a rather low cooperativity of binding to a single filament. (C) Chapman & Hall Ltd

Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody

Gonsior SM, Platz S, Buchmeier S, Scheer U, Jockusch BM, Hinssen H. Conformational difference between nuclear and cytoplasmic actin as detected by a monoclonal antibody. JOURNAL OF CELL SCIENCE. 1999;112(6):797-809.Using a reconstituted complex: of profilin and skeletal muscle actin as an antigen, we generated a monoclonal mouse antibody against actin, termed 2G2, As revealed by immunoblots of proteolytic actin fragments and by pepscan analysis, the antibody recognises a nonsequential epitope on actin which is located within three different regions of the sequence, consisting of aa131-139, aa155-169, and aa176-187, In the actin model derived from X-ray diffraction, these sequences lie spatially close together in the region of the nucleotide-binding cleft, but do not form a coherent patch. In immunoblots, 2G2 reacts with all SDS-denatured actin isoforms and with actins of many vertebrates. In contrast, its immunofluorescence reactivity is highly selective and fixation-dependent, In fibroblasts and myogenic cells, fixed and extracted by formaldehyde/detergent, stress fibres or myofibrils, respectively, remained unstained. Likewise, after microinjection into living cells, 2G2 did not bind to such microfilament bundles. Extraction of myosin and tropomyosin did not alter this pattern indicating that the lack in reactivity is probably not due to epitope-masking by actin-binding proteins, More likely, the reason for the lack of reactivity with filamentous actin is that its epitope is not accessible in F-actin, However, the antibody revealed a distinct pattern of nuclear dots in differentiated myogenic cells but not in myoblasts, and of fibrillar structures in nuclei of Xenopus oocytes, In contrast, after methanol treatment, a 2G2-specific staining of stress fibres and myofibrils was observed, but no nuclear dot staining. We conclude that 2G2, in addition to binding to SDS- and methanol-denatured actin, recognises a specific conformation of native actin which is present in the nucleus and specified by compaction of the antibody-reactive region into a coherent patch. This conformation is apparently present in differentiated myogenic cells and oocytes, but not in cytoplasmic actin filament bundles