Espins Are Multifunctional Actin Cytoskeletal Regulatory Proteins in the Microvilli of Chemosensory and Mechanosensory Cells

Immunofluorescence localization of espins to the dendritic microvilli of rat VNO sensory neurons observed in tissue sections (A-E) and in isolated neurons (F, G). A, Espin antibody (red) labels the luminal aspect of the concave face, which contains the sensory neurons (sn), as revealed by olfactory marker protein (OMP) antibody (green). The concave and convex faces are closely apposed in this section. Arrows, Isolated espin-positive cells in epithelium of convex face. Scale bar, 50 μm. B, Higher magnification view showing espin antibody labeling (red) of microvilli emanating from the distal ends of the neuronal dendrites, which are labeled with TuJ1 tubulin antibody (green). Scale bar, 10 μm. C-E, Double-labeling with espin antibody (red in C, E) and olfactory marker protein (OMP) antibody (D, E, green), which highlights the dendritic knobs (D, E, arrowheads) of the VNO sensory neurons. Scale bar, 5 μm. F, G, Double labeling with espin antibody (red) and calretinin antibody (green) showing intense espin staining in the collection of microvilli located at the distal end of the dendrite of isolated rat VNO neurons. Scale bars, 10 μm.

Immunofluorescence localization of espins to the microvilli of SCCs and taste cells. A-F, Sections of mouse (A) or rat (B-F) VNO. Espin antibody labeling (red) is concentrated in the microvilli (B, D-F, arrows) of cells in the epithelium of the convex face with the properties of SCCs: the cells are labeled with α-gustducin antibody (A, C, green) and in contact with nerve fibers that are labeled with PGP 9.5 antibody (D, E, green). These cells are also labeled by IP₃R3 antibody (F, green). F, Arrowheads indicate espin antibody staining in the dendritic microvilli of VNO sensory neurons across the VNO lumen. Scale bars: A,20 μm; C-F,10 μm. G-J, Sections of mouse (G) or rat (H-J) tongue showing taste buds of circumvallate papillae. Espin antibody labeling (G, H, J, red) is concentrated in the microvilli of taste cells (H, J, arrows). The espinpositive cells are labeled with IP₃R3 antibody (I, J, green), whereas fewer are labeled with α-gustducin antibody (G, green). Scale bars: G, 50 μm; J, 10 μm.

Immunofluorescence localization of espins to the microvilli of Merkel cells and retinal Müller glial cells. A, B, Sections of mouse facial skin (A) and hard palate (B). Espin antibody labeling (green) is concentrated in the spiky microvilli of Merkel cells (asterisks), which are located in the basal layer of the stratified squamous epithelium. The section in B is double labeled with TuJ1 tubulin antibody (red) to highlight the nerve fibers that contact the Merkel cells. Scale bars, 5 μm. C-G, Sections of rat retina. C, D, Espin antibody labeling (green) is concentrated in the microvilli of Müller glial cells but is also present at lower levels in the thin cytoplasmic processes of Müller cells, which are better revealed by double labeling with vimentin antibody (D, blue). Scale bar, 20 μm. E-G, The espin antibody labeling of Müller cell microvilli (E, G, green) colocalizes with the F-actin in the PAB at their core, as revealed by double-labeling with Texas Red-phalloidin (F, G, red). Scale bar, 5 μm.

Western blot detection and molecular biological characterization of novel espin isoforms in sensory tissues. A, B, Western blots of SDS extracts prepared from the designated tissue. Note the presence of multiple bands that react with espin antibody (B) but not preimmune IgG (A), including a 30-35 kDa multiplet (B, bracket with asterisk) that does not correspond to any of the known espin isoforms. (This multiplet appears to run slightly slower in the retina sample, because it was close to the gel edge.) B, Numbers to the left, Molecular mass in kilodaltons. B, Abbreviations to the right, Positions of known espin isoforms (SC, Sertoli cell espin; PC1, Purkinje cell espin 1; PC2, Purkinje cell espin 2; Sm, small espin). C, Western blot showing that the newly identified espin 3A and espin 3B isoforms comigrate with the 30-35 kDa multiplet (asterisk) when expressed in untagged form in transiently transfected LLC-PK1-CL4 epithelial cells. D, Stick-figure diagrams of the structures deduced by PCR for espin 3A and espin 3B, highlighting their utilization of espin gene exons (l-z) and their relationships to the other major espin isoforms, which are designated using the new nomenclature. In parentheses are the abbreviations of the espin isoforms in the old nomenclature (see under B, above). The exons encoding the extended N terminus of espin 1, including its eight ankyrin-like repeats, are not shown. Note that the exon-lettering scheme has also been revised (Sekerková et al., 2003) to reflect the identification of a new exon, q, which is unique to espin 3B (asterisk). Exons t and v are unique to espin 4. PR1/xAB, Proline-rich peptide 1 plus additional F-actin-binding site; PR2, proline-rich peptide 2; WH2, WASP homology domain 2; ABM, actin-bundling module.

Espin isoform distribution among sensory cells assessed at the protein level using whole espin antibody (Whole) and the LIS espin antibody. A-E, Immunofluorescence of transfected cells expressing untagged espin 2B (A) or espin 3B (B-E) using whole (B,C) or LIS (A, D, E) antibody (red). C, E, Overlays of B and D, respectively, showing espin-induced coarse cytoplasmic F-actin bundles labeled with fluorescein-phalloidin (green). The LIS antibody labels espin 2B in the transfected cells (A), but not espin 3B (D, E), despite an abundance of espin 3B-induced cytoplasmic F-actin bundles (E). Scale bars, 25 μm. F, Identical Western blots of purified recombinant espin isoforms labeledwith whole (left panel) or LIS (right panel) antibody. Note that the LIS antibody labels espins 2A and 2B, but not espins 3A and 3B, and also detects some minor, proteolytic breakdown products bearing C-terminal truncations that are not detected by whole antibody at this exposure. G,H, Immunofluorescence detection of espin 1 in the Sertoli cell junctions observed in consecutive sections of rat testis using diluted, whole (G), or LIS (H) antibody (red). Note that the labeling intensities obtained for the Sertoli cell junctions when using these two antibodies at these dilutions match closely, even when comparing seminiferous tubules in different stages of spermatogenesis. Scale bar, 100 μm. I-P, Comparisons of the immunofluorescence intensities obtained when labeling consecutive tissue sections of sensory tissues with whole or LIS antibody at the dilutions giving equal intensities on testis sections (G,H). I,J, Rat VNO sections double labeled with whole (I) or LIS (J) antibody (red) and IP₃R3 antibody (green). Note reduction of labeling of neuronal dendritic microvilli (arrowheads) and loss of labeling of SCC microvilli (I, arrows) with LIS antibody (J). Scale bar, 25 μm. K-M, Mouse cochlear sections double labeled with whole (K) or LIS (L,M) antibody (red) and calretinin antibody (green). Note loss of labeling in the stereocilia of inner hair cells (arrows) and outer hair cells (OHC) (K, arrowheads) with LIS antibody (L, M). M, Higher-magnification view of three inner hair cells showing absence of staining with the LIS antibody in the stereocilia, which are positioned just above the cuticular plate (asterisks). Scale bars: L, 25 μm; M, 10 μm. N-P, Mouse utricular macula sections labeled with whole (N) or LIS (O,P) antibody (red). Note reduction in labeling of the stereocilia of vestibular hair cells (HC) with the LIS antibody (O). P, Higher-magnification view of the collections of vestibular hair cell stereocilia labeled with LIS antibody. Scale bars: O, 50 μm; P, 10 μm. Table (bottom right) summarizes results comparing the immunofluorescence intensity obtained with the LIS antibody with that obtained with the whole espin antibody at the dilutions giving equal intensities on testis sections. ++, No difference; +, decreased; -, not detected. HC, Hair cell.

Interactions of the espin 3 isoforms with actin. A, Sedimentation actin-bundling assay. The bundling of actin filaments by espin 3B (triangles, red) shows a concentration dependence similar to that for espin 2B (circles, blue) and is not changed significantly when 20 μm CaCl₂ (square, black) is substituted for 1 mm EGTA in the assay. B, Microvillar PAB elongation assay. GFP-espin 3B (green) is efficiently targeted to microvilli in differentiated LLC-PK1-CL4cells and causes the microvilli and their PABs to grow dramatically longer than those in neighboring control cells. The microvillar PABs are visualized by labeling for F-actin with Texas Red-phalloidin (red). Note colocalization (yellow) of GFP-espin 3B and F-actin in the long microvilli. Scale bar, 10 μm. C, Pull-down assay for binding actin monomer by espin 3 isoforms with (+) or without (-) the WH2 domain. Beads loaded with GST-conjugates of espin 3A (GST-3A) and espin 3B (GST-3B) bind G-actin, and the binding is reduced dramatically by deletion of the WH2 domain (duplicate samples are shown). GST, Control beads loaded with GST alone. D, E, Pyrene-actin polymerization assay. A.U., Arbitrary units. Molar ratios of designated espin isoform to actin monomer are shown at right. Black curves indicate actin alone. D, Espin 3B (red curves) inhibits the polymerization of pyrene-actin in a concentration-dependent manner. E, Espin 2B (blue curves) does not inhibit actin polymerization but causes a modest, concentration-dependent stimulation at high molar ratios. The inhibitory effect observed with espin 3A at a molar ratio of 0.7 (0.7^*) is shown for comparison (red curve). F, Calculated relative nucleation rates for different molar ratios of espin 3B (triangles, red), espin 2B (circles, blue), and espin 4 (square, red). Note that espin 3B and espin 4 inhibit nucleation, but espin 2B does not.

Interactions of the espin 3 isoforms with ligands for proline-rich peptides. A, Pull-down assay for binding the designated espin isoform to beads loaded with GST-IRSp53 SH3 domain or control beads loaded with GST. Fifty percent of the espin isoform added to the assay is shown in the left panel. Espin 2B binds to the IRSp53 SH3 domain beads, but espin 3A and espin 3B do not. B, Pull-down assay for binding the designated espin 3 isoform, with (+) or without (-) the eight consecutive proline residues (P₈) in their single proline-rich peptide, to beads loaded with GST-profilin IIa, GST-profilin I, or GST. Fifty percent of the espin isoform added to the assay is shown in the left panel. The espin 3 isoforms bind better to profilin IIa than profilin I, and the binding is reduced dramatically after deletion of the eight prolines.