Organisms respond to circumstances threatening the cellular protein homeostasis by activation of heat-shock transcription factors (HSFs), which play important roles in stress resistance, development, and longevity. Of the four HSFs in vertebrates (HSF1-4), HSF1 is activated by stress, whereas HSF2 lacks intrinsic stress responsiveness. The mechanism by which HSF2 is recruited to stress-inducible promoters and how HSF2 is activated is not known. However, changes in the HSF2 expression occur, coinciding with the functions of HSF2 in development. Here, we demonstrate that HSF1 and HSF2 form heterotrimers when bound to satellite III DNA in nuclear stress bodies, subnuclear structures in which HSF1 induces transcription. By depleting HSF2, we show that HSF1-HSF2 heterotrimerization is a mechanism regulating transcription. Upon stress, HSF2 DNA binding is HSF1 dependent. Intriguingly, when the elevated expression of HSF2 during development is mimicked, HSF2 binds to DNA and becomes transcriptionally competent. HSF2 activation leads to activation of also HSF1, revealing a functional interdependency that is mediated through the conserved trimerization domains of these factors. We propose that heterotrimerization of HSF1 and HSF2 integrates transcriptional activation in response to distinct stress and developmental stimuli.

Heat shock transcription factor 1 (HSF1) is the main regulator of the stress response that triggers the transcription of several genes encoding heat shock proteins (Hsps). Hsps act as molecular chaperones involved in protein folding, stability, and trafficking. HSF1 is highly expressed in oocytes and Hsf1 knock-out in mice revealed that in the absence of stress this factor plays an important role in female reproduction. We previously reported that Hsf1(-/-) females produce oocytes but no viable embryos. Consequently, we asked whether oocytes require HSF1 to regulate a particular set of Hsps necessary for them to develop. We find that Hsp90alpha (Hspaa1) is the major HSF1-dependent chaperone inasmuch as Hsf1 knock-out resulted in Hsp90-depleted oocytes. These oocytes exhibited delayed germinal vesicle breakdown (or G(2)/M transition), partial meiosis I block, and defective asymmetrical division. To probe the role of Hsp90alpha in this meiotic syndrome, we analyzed meiotic maturation in wild-type oocytes treated with a specific inhibitor of Hsp90, 17-allylamino-17-demethoxy-geldanamycin, and observed similar defects. At the molecular level we showed that, together with these developmental anomalies, CDK1 and MAPK, key meiotic kinases, were significantly disturbed. Thus, our data demonstrate that HSF1 is a maternal transcription factor essential for normal progression of meiosis.

Glandular epithelial cells differentiate into complex multicellular or acinar structures, when embedded in three-dimensional (3D) extracellular matrix. The spectrum of different multicellular morphologies formed in 3D is a sensitive indicator for the differentiation potential of normal, non-transformed cells compared to different stages of malignant progression. In addition, single cells or cell aggregates may actively invade the matrix, utilizing epithelial, mesenchymal or mixed modes of motility. Dynamic phenotypic changes involved in 3D tumor cell invasion are sensitive to specific small-molecule inhibitors that target the actin cytoskeleton. We have used a panel of inhibitors to demonstrate the power of automated image analysis as a phenotypic or morphometric readout in cell-based assays. We introduce a streamlined stand-alone software solution that supports large-scale high-content screens, based on complex and organotypic cultures. AMIDA (Automated Morphometric Image Data Analysis) allows quantitative measurements of large numbers of images and structures, with a multitude of different spheroid shapes, sizes, and textures. AMIDA supports an automated workflow, and can be combined with quality control and statistical tools for data interpretation and visualization. We have used a representative panel of 12 prostate and breast cancer lines that display a broad spectrum of different spheroid morphologies and modes of invasion, challenged by a library of 19 direct or indirect modulators of the actin cytoskeleton which induce systematic changes in spheroid morphology and differentiation versus invasion. These results were independently validated by 2D proliferation, apoptosis and cell motility assays. We identified three drugs that primarily attenuated the invasion and formation of invasive processes in 3D, without affecting proliferation or apoptosis. Two of these compounds block Rac signalling, one affects cellular cAMP/cGMP accumulation. Our approach supports the growing needs for user-friendly, straightforward solutions that facilitate large-scale, cell-based 3D assays in basic research, drug discovery, and target validation.

Targeting of the Hsp function in tumor cells is currently being assessed as potential anticancer therapy. An improved understanding of the molecular signals that trigger or attenuate the stress protein response is essential for advances to be made in this field. The present study provides evidence that the membrane fluidizer benzyl alcohol (BA), a documented nondenaturant, acts as a chaperone inducer in B16(F10) melanoma cells. It is demonstrated that this effect relies basically on heat shock transcription factor 1 (HSF1) activation. Under the conditions tested, the BA-induced Hsp response involves the up-regulation of a subset of hsp genes. It is shown that the same level of membrane fluidization (estimated in the core membrane region) attained with the closely analogous phenethyl alcohol (PhA) does not generate a stress protein signal. BA, at a concentration that activates heat shock genes, exerts a profound effect on the melting of raft-like cholesterol-sphingomyelin domains in vitro , whereas PhA, at a concentration equipotent with BA in membrane fluidization, has no such effect. Furthermore, through the in vivo labeling of melanoma cells with a fluorescein labeled probe that inserts into the cholesterol-rich membrane domains [fluorescein ester of polyethylene glycol-derivatized cholesterol (fPEG-Chol)], we found that, similarly to heat stress per se , BA, but not PhA, initiates profound alterations in the plasma membrane microdomain structure. We suggest that, apart from membrane hyperfluidization in the deep hydrophobic region, a distinct reorganization of cholesterol-rich microdomains may also be required for the generation and transmission of stress signals to activate hsp genes.