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. 2013 Jul 10;8(7):e68978.
doi: 10.1371/journal.pone.0068978. Print 2013.

Cell stress promotes the association of phosphorylated HspB1 with F-actin

Joseph P Clarke  1 , Karen M Mearow
Affiliations

Cell stress promotes the association of phosphorylated HspB1 with F-actin

Joseph P Clarke et al. PLoS One. .

Abstract

Previous studies have suggested that the small heat shock protein, HspB1, has a direct influence on the dynamics of cytoskeletal elements, in particular, filamentous actin (F-actin) polymerization. In this study we have assessed the influence of HspB1 phosphorylation on its interaction(s) with F-actin. We first determined the distribution of endogenous non-phosphorylated HspB1, phosphorylated HspB1 and F-actin in neuroendocrine PC12 cells by immunocytochemistry and confocal microscopy. We then investigated a potential direct interaction between HspB1 with F-actin by precipitating F-actin directly with biotinylated phalloidin followed by Western analyses; the reverse immunoprecipitation of HspB1 was also carried out. The phosphorylation influence of HspB1 in this interaction was investigated by using pharmacologic inhibition of p38 MAPK. In control cells, HspB1 interacts with F-actin as a predominantly non-phosphorylated protein, but subsequent to stress there is a redistribution of HspB1 to the cytoskeletal fraction and a significantly increased association of pHspB1 with F-actin. Our data demonstrate HspB1 is found in a complex with F-actin both in phosphorylated and non-phosphorylated forms, with an increased association of pHspB1 with F-actin after heat stress. Overall, our study combines both cellular and biochemical approaches to show cellular localization and direct demonstration of an interaction between endogenous HspB1 and F-actin using methodolgy that specifically isolates F-actin.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Cellular localization of HspB1 and F-actin in control PC12 cells.
Low passage PC12 cells were cultured on collagen-coated coverslips, and processed for immunocytochemistry using antibodies directed against total HspB1 (Stressgen SPA801) or phospho-S15-HspB1 followed by fluorescently tagged secondary antibodies; F-actin was labelled using Alexa-488-phalloidin. Panel A-D: PC12 cells visualized with confocal microscopy; images represent the z-stack projections. A – HspB1 (blue); B- phalloidin (green); C-pS15-HspB1 (magenta); D- merged image. Panels E-K, L-R: three-dimensional rendering of immunostained surfaces as compiled by Imaris software of cells (boxes in D) selected from the low power view. E, L -HspB1 (blue); F, M - Phalloidin (green); G, N - pHspB1(magenta); H, O - all 3 together; I, P - HspB1+pHspB1; J, Q – HspB1+Phalloidin; K, R - pHspB1+Phalloidin. Scale bar –50 µm, D; 10 µm, lower panels.
Figure 2
Figure 2. Cellular localization of HspB1 and F-actin in PC12 cells subsequent to a heat shock.
Low passage PC12 cells were cultured on collagen-coated coverslips and were subjected to a 30 min heat shock (HS) followed by immediate fixation, and processing for immunocytochemistry using antibodies directed against total HspB1 or phospho-S15-HspB1 followed by fluorescently tagged secondary antibodies; F-actin was labelled using Alexa-488-phalloidin. Panel A-D: PC12 cells visualized with confocal microscopy; images represent the z-stack projections. A – HspB1 (blue); B – phalloidin (green); C-pS15-HspB1 (magenta); D– merged image. Panels E–K, L–R: three-dimensional rendering of immunostained surfaces as compiled by Imaris software of cells (boxes in D) selected from the low power view. E, L -HspB1 (blue); F, M - Phalloidin (green); G, N – pHspB1(magenta); H, O – all 3 together; I, P - HspB1+pHspB1; J, Q – HspB1+Phalloidin; K, R – pHspB1+Phalloidin. Scale bar –10 µm.
Figure 3
Figure 3. Cellular localization of HspB1 and F-actin in PC12 cells subsequent to a heat shock in the presence of a p38MAPK inhibitor.
Low passage PC12 cells were cultured on collagen-coated coverslips, incubated with 10 µM SB203580 for 1 hr prior to and during a 30 min heat shock (HS), followed by immediate fixation and processing for immunocytochemistry using antibodies directed against total HspB1 (Stressgen SPA801) or phospho-S15-HspB1 followed by fluorescently tagged secondary antibodies; F-actin was labelled using Alexa-488-phalloidin. Panel A–D: PC12 cells visualized with confocal microscopy; images represent the z-stack projections. A – HspB1 (blue); B- phalloidin (green); C-pS15-HspB1 (magenta); D- merged image. Panels E–K, L–R: three-dimensional rendering of immunostained surfaces as compiled by Imaris software of cells (boxes in D) selected from the low power view. E, L -HspB1 (blue); F, M - Phalloidin (green); G, N – pHspB1(magenta); H, O – all 3 together; I, P – HspB1+pHspB1; J, Q – HspB1+Phalloidin; K, R - pHspB1+Phalloidin. Scale bar –10 µm.
Figure 4
Figure 4. HspB1 is phosphorylated and redistributed to the cytoskeletal fraction during cellular stress.
PC12 cells were treated as described in the Methods, subsequently lysed and separated by cellular fractionation into Triton X-100 soluble (cytosolic; lysate) and Triton X-100 insoluble (cytoskeletal; pellet) samples, or were left as crude total protein samples. Untreated cells (Lanes 1–3); treated with 10 µM SB203580 (Lanes 4–6); heat shocked cells (Lanes 7–9); cells treated with inhibitor and heat shock (Lanes 10–12). Western blots were sequentially probed with antibodies to pS15-HspB1, pS86-HspB1, HspB1 and actin; blots were stripped after each probe and prior to the next. Note the increased amount of HspB1 in the cytoskeletal fraction after HS, as well as the increased amount of phosphorylated HspB1.
Figure 5
Figure 5. Specificity of the Phalloidin pull-down for F-actin.
In vitro preparations (2.5 or 5 µg) of globular actin (G-actin) and filamentous actin (F-actin) were prepared using an in vitro actin–binding assay kit (Cytoskeleton) and incubated with 5 µg of biotinylated-phalloidin as detailed in the Methods section. The resulting solutions were separated into supernatant (non-phalloidin interacting) and pull-down (phalloidin interacting) samples and assayed by Western blotting. Additionally, untreated G- or F-actin samples were also probed. Note that biotinylated-phalloidin selectively precipitates F-actin, but not G-actin, and that this pull-down is reasonably efficient.
Figure 6
Figure 6. HspB1 is associated with F-actin.
Representative blots showing precipitation of F-actin complexes (with biotinylated phalloidin, A–B) and IPs of HspB1 (C–D) from total cellular lysates (A, C) or the cytoskeletal pellet fraction (B, D). PC12 cells were treated as described in the Methods. Cell cultures were exposed to 10 µM SB203580 for 1 hr, after which cultures were either incubated for an additional 30 mins, or stressed with heat shock at 42°C for 30 mins; control cells were not treated. Immediately after treatments, cells were collected, lysed with actin stabilization buffer; samples were separated for analysis as total cell lysate or further fractionated into the TritonX-100 insoluble cytoskeletal pellet. Samples were incubated with biotinylated-phalloidin followed by precipitation of the captured complexes with streptavidin-linked magnetic beads. Precipitated fractions were then subjected to SDS-PAGE and sequentially immunoblotted to detect pHspB1, total HspB1 and actin. Pulldown of F-actin also captures HspB1 in both the cell lysates (A) and the cytoskeletal fraction (B); similarly the HspB1 IPs also bring down actin and this is enhanced in the cytoskeletal fraction.
Figure 7
Figure 7. Densitometric analyses of the amounts of F-actin and HspB1 in the precipitated complexes.
Western blots were scanned and densitometric analysis of bands carried out using Image J and a calibrated gray scale standard. The bars are the mean ROD (±SEM) of paired values for Actin, HspB1 (Hsp), pS86-HspB1 (S86) and pS15-HspB1 (S15) from each of 3 separate experiments for the cytoskeletal fraction samples (pellet) and from 2 separate experiments for total cell lysates. Panel A – Phalloidin pulldown from total cell lysates; B – Phalloidin pulldown from the cytoskeletal fraction; C – HspB1 IP from total cell lysates; D – HspB1 IP from the cytoskeletal fraction. Norm – control; HS – Heat Shock; HS+SB – Heat shock+SB203580. ** p<0.01; *** p<0.001.
Figure 8
Figure 8. Changes in the relative amounts of the association of F-actin and HspB1.
Densitometric data of F-actin and HspB1 expression in pulldowns presented as ratios of HspB1:F-actin or Actin:HspB1, as well as the pHspB1:F-actin or pHspB1:HspB1. Panel A - ratios for Phalloidin pulldown from total cell lysate; B - ratios for HspB1 IP from total cell lysate; C - ratios for Phalloidin pulldown from cytoskeletal fraction (pellet); D – ratios for HspB1 from the cytoskeletal fraction. ** p<0.01.
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This work was supported by an NSERC Discovery Grant to KMM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.