Institut für Biochemie I   Printer friendly
Actin Binding Proteins


Filamin, a dimeric organizer of cytoskeleton


Filamins are elongated homodimeric proteins that crosslink F-actin. Each monomer chain of filamin comprises an actin-binding domain, and a rod segment consisting of six (Dictyostelium filamin) up to 24 (human filamin) highly homologous repeats of approximately 96 amino acid residues, which adopt an immunoglobulin-like fold. Two hinges in the rod segment, together with the reversible unfolding of single repeats, might be the structural basis for the intrinsic flexibility of the actin networks generated by filamins. There are numerous filamin-binding proteins that associate, in most cases, along the repeats of the rod repeats. This rather promiscuous behaviour renders filamin a versatile scaffold between the actin network and finely tuned molecular cascades from the membrane to the cytoskeleton [1].

When subjected to a stretching force, filamin domain 4 unfolds at a lower force than all the other domains in the chain. Moreover, this domain shows a stable intermediate along its mechanical unfolding pathway. We investigate the folding kinetics and pathway of this domain. An obligatory and productive intermediate occurs on the folding pathway of the domain. Identical mechanical properties suggest that the unfolding and refolding intermediates are closely related. The folding process can be divided into two consecutive steps: in the first step 60 C-terminal amino acids form an intermediate at the rate of 55 s(-1); and in the second step the remaining 40 amino acids are packed on this core at the rate of 179 s(-1). This division increases the overall folding rate of this domain by a factor of ten compared with all other homologous domains of ddFLN that lack the folding intermediate [2].

Recently, a similar function of titin was published [3]. Myofibril elasticity, critical to muscle function, is dictated by the intrasarcomeric filament titin, which acts as a molecular spring. Titins conserved Ig-Ig motifs generate high-order in the structure of the filament, where conformationally stiff segments interspersed with pliant hinges form a regular pattern of dynamic super-motifs leading to segmental flexibility in the chain. Pliant hinges support molecular shape rearrangements that dominate chain behavior at moderate stretch, whereas stiffer segments predictably oppose high stretch forces upon full chain extension.

Dictyostelium strains lacking the F-actin cross-linking protein filamin (ddFLN) have a severe phototaxis defect at the multicellular slug stage. Filamins are rod-shaped homodimers that cross-link the actin cytoskeleton into highly viscous, orthogonal networks. Each monomer chain of filamin is comprised of an F-actin-binding domain and a rod domain. In rescue experiments only intact filamin re-established correct phototaxis in filamin minus mutants, whereas C-terminally truncated filamin proteins that had lost the dimerization domain and molecules lacking internal repeats but retaining the dimerization domain did not rescue the phototaxis defect. Deletion of individual rod repeats also changed their subcellular localization, and mutant filamins in general were less enriched at the cell cortex as compared with the full-length protein and were increasingly present in the cytoplasm. For correct phototaxis ddFLN is only required at the tip of the slug because expression under control of the cell type-specific extracellular-matrix protein A (ecmA) promoter and mixing experiments with wild type cells supported phototactic orientation. Likewise, in chimeric slugs wild type cells were primarily found at the tip of the slug, which acts as an organizer in Dictyostelium morphogenesis.

References:
  1. Popowicz GM, Schleicher M, Noegel AA, Holak TA, Filamins: promiscuous organizers of the cytoskeleton, Trends Biochem Sci. 31, 411-9 (2006)
  2. Schwaiger I, Schleicher M, Noegel AA, Rief M, The folding pathway of a fast-folding immunoglobulin domain revealed by single-molecule mechanical experiments, EMBO Rep. 6, 46-51 (2005)
  3. von Castelmur E, Marino M, Svergun DI, Kreplak L, Ucurum-Fotiadis Z, Konarev PV, Urzhumtsev A, Labeit D, Labeit S, Mayans O, A regular pattern of Ig super-motifs defines segmental flexibility as the elastic mechanism of the titin chain, Proc Natl Acad Sci U S A. 105, 1186-91 (2008)
  4. Khaire N, Muller R, Blau-Wasser R, Eichinger L, Schleicher M, Rief M, Holak TA, Noegel AA, Filamin-regulated F-actin assembly is essential for morphogenesis and controls phototaxis in dictyostelium, J Biol Chem. 282, 1948-55 (2007)


March 14, 2012
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