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Fig. 3. Espin isoforms, domains and activities. (A-C) Isoform and domain dependence for forming the centrosomal actin bundle (CAB) by wild-type espins and the nuclear actin bundle (NAB) by jerker (je) espins. (A) Representatives of the four major espin-isoform size classes. ABM, actin-bundling module; AR, ankyrin-like repeat; je, jerker peptide (red and white diagonal stripe; sequence shown in F), the frameshifted peptide that replaces the C-terminal part of the espin actin-bundling module at the site of the jerker deafness mutation (black arrow, illustrated for espin 4); PR, proline-rich peptide; xABS, 23-aa additional F-actin-binding site; purple colour indicates peptides unique to espin 4. (B) Effect of truncation or deletion mutations indicating a requirement for the actin-bundling module, WH2 domain and, in the case of jerker espin 2B, the 23-aa additional F-actin-binding site. (C) Restoration of CAB or NAB forming activity by adding an upstream WH2 domain to the espin 2B WH2-domain-deletion construct. *3LA, WH2 domain with its 3 leucine residues (asterisks in D) mutated to alanine. (D) Peptide encoded by espin WH2-domain exon with its 17-aa core underlined. *, leucines mutated to alanine in *3LA construct (activities shown in C). (E) Pull-down assay showing WH2-domain-dependent binding of ATP-actin monomer to espin 2B. <, actin band. (F) Sequence of jerker peptide. (G,H) Pyrene-actin polymerization assay for the designated construct (0.4 µM) in the presence (G) or absence (H) of 10 nM Arp2/3 complex showing absence of nucleation-promoting and nucleation activity of espins in vitro. Espin "WA", espin C-terminal peptide that begins 12 aa upstream of the 17-aa core of the WH2 domain. s, second.

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