Formation of a centrosomal actin bundle (CAB) in neuronal cells transfected with GFP-espin (green). (A-C) Colcalization of GFP-espin (B) and F-actin labeled with Texas Red-phalloidin (C, red) in a large perinuclear actin bundle in a transfected PC12 cell (A, phase). (D,E) GFP-espin (E) in a large perinuclear actin bundle in a transfected primary hippocampal neuron (D, phase). (F-H) GFP-espin in bundle assembly intermediates observed in PC12 cells 4-8 h after transfection (in order of appearance) highlighting relationship to microtubule-organizing center labeled with γ-tubulin antibody (red dots). (I) Inhibition of CAB formation upon cotransfection of PC12 cell with GFP espin and myc-dynamitin constructs (dynamitin expression was confirmed with myc antibody; not shown) (J) Absence of CAB formation in PC12 cell transfected with GFP-espin WH2 domain-deletion construct. (K) Restoration of CAB formation in a PC12 cell transfected with the GFP-espin WH2 domain-deletion construct containing an upstream WH2 domain. Blue, nuclei labeled with DAPI. Bars, 5 μm.

Espin isoform, 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. AR, ankyrin-like repeat; PR, proline-rich peptide; xABS, 23-aa additional F-actin-binding site; ABM, actin-bundling module; 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); purple, 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 espin2B WH2 domain-deletion construct. *3L->A, 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 *3L->A 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 COOH-terminal peptide that begins 12 aa upstream of the 17-aa core of the WH2 domain.

Domain dependence of NAB formation. (A-D) Targeting of the GFP-jerker peptide construct (B) to nucleoli, as revealed by labeling with nucleolin antibody (C). (E,F) Colocalization of GFP-jerker espin (E) and F-actin labeled with Texas Red-phalloidin (F) in a NAB. (G,H) Targeting of the GFP-jerker peptide construct to the nucleolus (G) does not cause F-actin accumulation (H). (I,J) The GFP-jerker espin WH2 domain-deletion construct is targeted to nucleoli and to small foci in the nucleoplasm (I), but fails to accumulate F-actin in the nucleus (J). (K,L) Reintroduction of an upstream WH2 domain into GFP-jerker espin WH2 domain-deletion construct results in restoration of NAB forming activity, causing the colocalization of GFP construct (K) and F-actin (L) in a large NAB. (M,N) The GFP-villin-jerker peptide is targeted to nucleoli (M), but does not cause accumulation of nuclear F-actin (N). (O,P) Introduction of the espin WH2 domain upstream of the villin-jerker peptide construct confers NAB forming activity and causes colocalization of the GFP-WH2 domain-villin-jerker peptide construct (O) with F-actin (P) in fine NABs. Blue, nuclei labeled with DAPI. Bar, 10 μm.

EM analysis of CABs in transfected PC12 cells without (A) and with (B-D) S1 decoration showing that CABs are PABs. Nu in B, nucleus. (C,D) Left and right ends of bundle in B, respectively. Barbed ends face to the right. Arrowheads, microtubules. Bars, 0.2 μm (A,C,D) or 1 μm (B).

Formation of nuclear actin bundle (NAB) in LLC-PK1 cells transfected with GFP-jerker espin 2B (green). (A-C) Colocalization of GFP-jerker espin (B) and F-actin labeled with Texas Red-phalloidin (C, red) in a large actin bundle in a transfected cells (A, phase; blue in B, DAPI). (D) Confocal image showing that the GFP-jerker espin-labeled actin bundle is internal to the nuclear lamina revealed by lamin antibody (red). (E-T) Four rows of NAB assembly intermediates observed 4-8 h after transfection (in order of appearance) highlighting relationship to the nucleolus (phase dense) at early stages. Bars, 15 μm (B) or 5 μm (D,F).