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First published online 21 February 2006
doi: 10.1242/jcs.02825

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Identification of palladin isoforms and characterization of an isoform-specific interaction between Lasp-1 and palladin

¹ Department of Cell and Molecular Physiology, University of North Carolina School of Medicine, Chapel Hill, NC 27599-7545, USA
² Neuroscience Center, University of North Carolina School of Medicine, Chapel Hill, NC 27599, USA

View larger version (28K): [in a new window]   Fig. 1. Multiple palladin isoforms are transcribed from one gene. (A) Schematic representation of the gene structure of palladin. The murine palladin gene comprises at least 24 exons over 390 kb on chromosome 8 (Palld; cytoband B3.1). Transcripts are initiated from one of three nested promoters, which are indicated with arrowheads. (B) Schematic representation of the largest palladin isoform (200 kDa), which is transcribed from the most 5' promoter. The two proline-rich regions have been designated PR1 and PR2. (C) Schematic representation of mRNA splicing patterns for observed isoforms. Translation initiation and termination sites have been aligned with the above peptide sequence for the 200 kDa isoform. (D) Schematic representation of palladin isoforms. The 200 kDa, 140 kDa and 90 kDa isoforms are the primary products of the palladin gene and have been detected by immunoblotting. They share binding sequences for ezrin, -actinin, Ena/VASP family members and profilin. The 140 kDa and 200 kDa isoforms also encode two additional FPPPP motifs predicted to interact with Ena/VASP family members. Two other murine isoforms, designated N-terminus and C-terminus, have been reported at the cDNA level. Expression of the N-terminus isoform has not yet been directly confirmed.

View larger version (35K): [in a new window]   Fig. 2. Immunoblot of larger palladin isoforms. (A) Tissues were dissected from a neonatal mouse and immunoblotted with the polyclonal antibody a-4IgNT that detects the N-terminal extension of the 200 kDa and 140 kDa palladin isoforms. This antibody does not crossreact with the 90 kDa isoform. Similar to the reported expression of the 90 kDa isoform, the 140 kDa isoform of palladin is expressed in most neonatal mouse tissues, whereas the 200 kDa isoform is predominantly restricted to heart, muscle and bone. (B) The 140 kDa isoform is considerately downregulated in many adult tissues, but is abundant in tissues rich in smooth muscle, such as stomach and uterus. The 200 kDa isoform is expressed in striated muscle of the adult mouse. (C) Vector-driven expression of the putative 140 kDa palladin nucleotide sequence in COS-7 cells results in the translation of an immunoreactive band with the same apparent molecular mass observed for endogenous protein in astrocytes. Astrocytes express relatively high levels of the 140 kDa isoform of palladin. Immunodetection with a previously characterized monoclonal (left blot) that reacts with the 90 kDa, 140 kDa and 200 kDa isoforms demonstrates that the 90 kDa and 140 kDa isoforms are expressed in cultured astrocytes. The polyclonal antibody a-4IgNT (right blot) specifically detects the 140 kDa isoform in these cells.

View larger version (94K): [in a new window]   Fig. 3. Immunolocalization of palladin isoforms in cultured astrocytes. The myc-tagged, 90 kDa isoform of palladin was transfected into embryonic rat astrocytes with adenovirus. (D-F) Expressed 90 kDa palladin was detected 12 hours later with a monoclonal antibody directed against the N-terminal myc-epitope tag. (A-C) The endogenous 140 kDa isoform of palladin was detected with the polyclonal antibody a-4IgNT. The two isoforms strongly colocalize. Both isoforms are detected periodically along stress fibers (B and E) and both localize to cell-cell junctions (C and F). Bars, 50 µm.

View larger version (68K): [in a new window]   Fig. 4. (A) Overexpression of palladin isoforms in COS-7 cells results in striking, but distinct, actin organization phenotypes. Low-magnification images of COS-7 cells stained for F-actin and exogenous palladin 24 hours after transfection of the 90 kDa or 140 kDa isoforms. The overexpression of the 90 kDa isoform results in the formation of unusually robust actin-cables, whereas overexpression of the 140 kDa isoform results in the assembly of compacted star-like F-actin arrays. In the merged images, phalloidin staining is shown in green and myc-epitope staining is in red. Bars, 50 µm. (B) High-magnification images of single cells from the same fields. Bars, 50 µm. (C) Bar graph, indicating the differences between phenotypes overexpressing the 90 kDa and 140 kDa isoforms. For each transfection, 100 cells were scored blindly and F-actin phenotypes were grouped into one of three classes: stars-like assemblies, large bundles and small bundles (examples are given in supplementary material, Fig. S3). Overexpression of the 140 kDa isoform leads to more pronounced, compacted F-actin phenotype than overexpression of the 90 kDa isoform.

View larger version (25K): [in a new window]   Fig. 5. (A) Alignment of the sequence conserved between myopalladin and palladin that mediates binding of the SH3 domain of nebulin. The poly-proline motif responsible for the nebulin and Lasp-1 interactions is indicated by an asterisk. (B) Alignment of the SH3 domain of nebulin with the SH3 domain of Lasp-1. The core SH3-domain sequence is 80% identical.

View larger version (15K): [in a new window]   Fig. 6. (A) Yeast two-hybrid analysis of interaction of Lasp-1 with palladin. (i) Schematic representations of constructs used for the yeast two-hybrid analysis. (ii) The strength of the interaction between bait and prey constructs, as indicated by cell growth and ß-galactosidase activity was scaled from no interaction (-) to strong interaction (+++). Yeast two-hybrid analysis indicates that Lasp-1 interacts through its SH3 domain with the poly-proline motif in the 140 kDa isoform, but not that in the 90 kDa isoform. A mutation to the putative poly-proline motif abrogates this interaction in the 140 kDa isoform. (B) HeLa cells express both the 140 kDa and 90 kDa isoforms of palladin. The SH3 domain of Lasp-1 co-precipitates the 140 kDa palladin. GST protein or the GST domain fused to the SH3 domain of Lasp-1 was incubated with HeLa-cell lysate and precipitated with glutathione Sepharose. Western blot analysis indicates that the SH3 domain of Lasp-1 specifically co-precipitates the 140 kDa isoform but not the 90 kDa isoform of palladin (lane, GST-SH3 bound).

View larger version (123K): [in a new window]   Fig. 7. Localization of Lasp-1 and 140 kDa palladin in astrocytes. The 140 kDa isoform of palladin was detected with the polyclonal 4IgNT antibody characterized in Fig. 2, and Lasp-1 was detected with the 3H8 monoclonal antibody. In the merged images, palladin staining is shown in green and Lasp-1 staining is shown in red. Both Lasp-1 and the 140 kDa isoform of palladin colocalize in stress fibers of astrocytes (A-F), although Lasp-1 exhibits a more pronounced localization to focal adhesions. In longer-term cultures of astrocytes that have formed monolayers of epithelial-like cells, both palladin and Lasp-1 are detected within cell-cell-contacts (G-I). Bars, 50 µm.

View larger version (59K): [in a new window]   Fig. 8. siRNA targeting of 140 kDa palladin results in loss of Lasp-1 from stress fibers. HeLa cells were transfected with either control siRNA or palladin siRNA specific for 140 kDa palladin. (A-C) Both 140 kDa palladin and Lasp-1 are detected in stress fibers (controls). Lasp-1 is also detected in peripheral adhesions. (D-F) Loss of 140 kDa palladin from these cells results in the loss of Lasp-1 recruitment to stress fibers, although it is still strongly detected in peripheral adhesions. When 140 kDa palladin is re-expressed in these cells by transfection of a construct in which GFP is conjugated to the 140 kDa isoform, Lasp-1 is observed to regain its localization to stress fibers (G-I). Higher magnification of Lasp-1 staining in these cells is provided in a, d, and g. Bars, 50 µm (A-I) and 10 µm (a, d and g).

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