Human heat shock 27-kDa protein 1 (HSPB1)/heat shock protein (Hsp) 27 is a small heat shock protein which is thought to have several roles within the cell. One of these roles includes regulating actin filament dynamics in cell movement, since Hsp27 has previously been found to inhibit actin polymerization in vitro. In this study, the role of Hsp27 in regulating actin filament dynamics is further investigated. Hsp27 protein levels were reduced using siRNA in SW480 cells, a human colon cancer cell line. An in vitro wound closure assay showed that cells with knocked down Hsp27 levels were unable to close wounds, indicating that this protein is involved in regulating cell motility. Immunoprecipitation pull down assays were done, to observe if and when Hsp27 and actin are in the same complex within the cell, before and after heat shock. At all time points tested, Hsp27 and actin were present in the same cell lysate fraction. Lastly, indirect immunostaining was done before and after heat shock to evaluate Hsp27 and actin interaction in cells. Hsp27 and actin showed colocalization before heat shock, little association 3 h after heat shock, and increased association 24 h after heat shock. Cytoprotection was observed as early as 3 h after heat shock, yet cells were still able to move. These results show that Hsp27 and actin are in the same complex in cells and that Hsp27 is important for cell motility.
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Many thanks to Dr. Robert Tanguay for providing Hsp27 antibodies, Dr. Charles Giardina for technical assistance for molecular biology assays, and to Dr. Adam Zweifach for Western blot imaging assistance. This work was supported by the National Science Foundation Grants MCB-0114231 and MCB-0724147 to J.L. This work was funded by the University of Connecticut Research Foundation (grant 447393 to L.E.H.).
Electronic supplementary material
Supplementary materials are available.
The supplementary movie AfterHS. This movie was made at 3 h after heat shock of human SW480-fHsp27 cells. Note the increased lamellar dynamics compared with non heat-shocked cells shown in Supplement 2 and the location of the GFP-fHsp27 proteins. (MOV 800KB)
The supplementary movie BeforeHS of non heat-shocked human SW480-fHsp27 cells. (MOV 560KB)
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Doshi, B.M., Hightower, L.E. & Lee, J. The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock. Cell Stress and Chaperones 14, 445–457 (2009). https://doi.org/10.1007/s12192-008-0098-1
- Cell motility
- Heat shock