The role of Hsp27 and actin in the regulation of movement in human cancer cells responding to heat shock

Abstract

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.

Thiolutin inhibits endothelial cell adhesion by perturbing Hsp27 interactions with components of the actin and intermediate filament cytoskeleton

“…Using purified Hsp27 and actin, in vitro studies have shown that nonphosphorylated Hsp27 inhibits actin polymerization, while phosphorylated Hsp27 permits actin polymerization. However, subsequent studies have concluded that Hsp27 plays a more dynamic role in both the assembly and disassembly of actin filaments (During et al 2007;Doshi et al 2009). Employing a quantitative LC-MS analysis of Hsp27-associated proteins combined with thiolutin treatment to rapidly stimulate Hsp27 phosphorylation, we have now identified several components of the actin and intermediate filament cytoskeletons that differentially interact with phosphorylated and nonphosphorylated forms of Hsp27.…”

Structure and properties of chimeric small heat shock proteins containing yellow fluorescent protein attached to their C-terminal ends

“…Chimeras of HspB1, HspB5, HspB6, and HspB8 containing different fluorescent proteins (green, cyan, yellow, and citrine) fused to the N-terminal ends or to the C-terminal ends of sHsp were designed and successfully used in a number of investigations Abraham 2011, 2013;Borrelli et al 2002;Shelden et al 2002;Qian et al 2006;Fontaine et al 2005Fontaine et al , 2006Sun et al 2004Sun et al , 2007Doshi et al 2009). …”

“…For instance, chimeric HspB1 conferred stress protection in certain cell lines and this protection was comparable with that conferred by the wild-type HspB1 . Under stress conditions, both the wild-type HspB1 and its fluorescent chimeras migrate from cytosol to actin filaments and seem to be involved in protection of the (Doshi et al 2009;Clarke and Mearow 2013). Different stimuli can induce translocation of both the wildtype HspB1 and its fluorescent chimeras from the cytoplasm to the nuclei (Qian et al 2006).…”

“…Utilization of fluorescent proteins seems to be a very promising tool for this purpose (Chudakov et al 2010). A number of different fluorescent chimeras of sHsp have been designed and used for investigation of the intracellular location (Doshi et al 2009;Qian et al 2006;Borrelli et al 2002;Shelden et al 2002;Abraham 2011, 2013;Irobi et al 2004;Sun et al 2007) and formation of homooligomeric and heterooligomeric complexes of sHsp (Fontaine et al 2005;Fontaine et al 2006;Sun et al 2004;Datskevich et al 2012a). Utilization of fluorescent chimeras of sHsp has provided important and interesting information.…”

The role of small GTPase Rac1 in stress signaling

“…Non-phosphorylated, monomeric HSP25/27 can inhibit actin polymerization (Wettstein et al 2012). Phosphorylation of HSP25/27 stimulates the actin filament organizations following heat stress in order to prevent the aggregation of denatured actin (Pivovarova et al 2005;Doshi et al 2009;Clarke & Mearow 2013).…”