Culture of and experiments with sea urchin embryo primary mesenchyme cells.

Skeletogenesis in the sea urchin embryo gives rise to a pair of intricate endoskeletal spicules. Deposition of these skeletal elements in the early larva is the outcome of a morphogenetic program that begins with maternal inputs in the early zygote and results in the specification of the large micromere-primary mesenchyme cell (PMC) lineage. PMCs are of considerable interest as a model system, not only to dissect the mechanism of specific developmental processes, but also to investigate their evolution and the unrivaled level of control over the formation of a graded, mechanically robust, yet single crystalline biomineral. The ability to study gene regulatory circuits, cellular behavior, signaling pathways, and molecular players involved in biomineralization is significantly boosted by the high level of autonomy of PMCs. In fact, in the presence of horse serum, micromeres differentiate into PMCs and produce spicules in vitro, separated from the embryonic milieu. PMC culture eliminates indirect effects that can complicate the interpretation of experiments in vivo, offers superior spatiotemporal control, enables PMC-specific readouts, and is compatible with most imaging and characterization techniques. In this chapter, we provide an updated protocol, based on the pioneering work by Okazaki and Wilt, for the isolation of micromeres and subsequent culture of PMCs, as well as protocols for fixation and staining for fluorescent microscopy, preparation of cell cultures for electron microscopy, and the isolation of RNA.

Hsp70 cochaperones HspBP1 and BAG-1M differentially regulate steroid hormone receptor function.

Hsp70 binding protein 1 (HspBP1) and Bcl2-associated athanogene 1 (BAG-1), the functional orthologous nucleotide exchange factors of the heat shock protein 70 kilodalton (Hsc70/Hsp70) chaperones, catalyze the release of ADP from Hsp70 while inducing different conformational changes of the ATPase domain of Hsp70. An appropriate exchange rate of ADP/ATP is crucial for chaperone-dependent protein folding processes. Among Hsp70 client proteins are steroid receptors such as the glucocorticoid receptor (GR), the mineralocorticoid receptor (MR), and the androgen receptor (AR). BAG-1 diversely affects steroid receptor activity, while to date the influence of HspBP1 on steroid receptor function is mostly unknown. Here, we compared the influence of HspBP1 and BAG-1M on Hsp70-mediated steroid receptor folding complexes and steroid receptor activity. Coimmunoprecipitation studies indicated preferential binding of Hsp40 and the steroid receptors to BAG-1M as compared to HspBP1. Furthermore, Hsp70 binding to the ligand-binding domain of GR was reduced in the presence of HspBP1 but not in the presence of BAG-1M as shown by pull-down assays. Reporter gene experiments revealed an inhibitory effect on GR, MR, and AR at a wide range of HspBP1 protein levels and at hormone concentrations at or approaching saturation. BAG-1M exhibited a transition from stimulatory effects at low BAG-1M levels to inhibitory effects at higher BAG-1M levels. Overall, BAG-1M and HspBP1 had differential impacts on the dynamic composition of steroid receptor folding complexes and on receptor function with important implications for steroid receptor physiology.

Recombinant sea urchin vascular endothelial growth factor directs single-crystal growth and branching in vitro.

Biomineralization in sea urchin embryos is a crystal growth process that results in oriented single-crystalline spicules with a complex branching shape and smoothly curving surfaces. Uniquely, the primary mesenchyme cells (PMCs) that construct the endoskeleton can be cultured in vitro. However, in the absence of morphogenetic cues secreted by other cells in the embryo, spicules deposited in PMC culture lack the complex branching behavior observed in the embryo. Herein we demonstrate that recombinant sea urchin vascular endothelial growth factor (rVEGF), a signaling molecule that interacts with a cell-surface receptor, induces spiculogenesis and controls the spicule shape in PMC culture. Depending on the rVEGF concentration, PMCs deposit linear, "h"- and "H"-shaped, or triradiate spicules. Remarkably, the change from linear to triradiate occurs with a switch from bidirectional crystal growth parallel to the calcite c axis to growth along the three a axes. This finding has implications for our understanding of how cells integrate morphogenesis on the multi-micrometer scale with control over lattice orientation on the atomic scale. The PMC model system is uniquely suited to investigate this mechanism and develop biotechnological approaches to single-crystal growth.

BAG-1 diversely affects steroid receptor activity.

Part of the cellular and physiological functions of BAG-1 (Bcl-2-associated athanogene 1) has been ascribed to the ability of this hsp70 (heat-shock protein 70) co-chaperone to regulate steroid receptor activity. BAG-1 has been reported to inhibit the GR (glucocorticoid receptor) and stimulate the androgen receptor, but to leave the activity of the MR (mineralocorticoid receptor) unchanged. Given the high homology between the MR and GR, this disparity in the actions of BAG-1 is surprising. In the present study, we analysed the effect of BAG-1 on the activity of the closely related PR (progesterone receptor). Similarly to the GR, the transcriptional activity of the PR is inhibited by the long and middle isoforms of BAG-1, BAG-1L and BAG-1M, but not by the short isoform, BAG-1S. We found this inhibition to require the hsp70-binding domain of BAG-1. To shed light on the mechanisms that could explain BAG-1's differential actions on steroid receptors, we tested the binding of BAG-1M to the PR. Mutational analyses of the PR and BAG-1M revealed that the mode of interaction and BAG-1M-mediated inhibition of the PR differs from the reported scenario for the GR. Surprisingly, we also found binding of BAG-1M to the MR. In addition, BAG-1M was able to inhibit the transcriptional activity of the MR. These data entail a reappraisal of the physiological actions of BAG-1M on steroid receptor activity.

Differential impact of tetratricopeptide repeat proteins on the steroid hormone receptors.

BACKGROUND: Tetratricopeptide repeat (TPR) motif containing co-chaperones of the chaperone Hsp90 are considered control modules that govern activity and specificity of this central folding platform. Steroid receptors are paradigm clients of Hsp90. The influence of some TPR proteins on selected receptors has been described, but a comprehensive analysis of the effects of TPR proteins on all steroid receptors has not been accomplished yet. METHODOLOGY AND PRINCIPAL FINDINGS: We compared the influence of the TPR proteins FK506 binding proteins 51 and 52, protein phosphatase-5, C-terminus of Hsp70 interacting protein, cyclophillin 40, hepatitis-virus-B X-associated protein-2, and tetratricopeptide repeat protein-2 on all six steroid hormone receptors in a homogeneous mammalian cell system. To be able to assess each cofactor's effect on the transcriptional activity of on each steroid receptor we employed transient transfection in a reporter gene assay. In addition, we evaluated the interactions of the TPR proteins with the receptors and components of the Hsp90 chaperone heterocomplex by coimmunoprecipitation. In the functional assays, corticosteroid and progesterone receptors displayed the most sensitive and distinct reaction to the TPR proteins. Androgen receptor's activity was moderately impaired by most cofactors, whereas the Estrogen receptors' activity was impaired by most cofactors only to a minor degree. Second, interaction studies revealed that the strongly receptor-interacting co-chaperones were all among the inhibitory proteins. Intriguingly, the TPR-proteins also differentially co-precipitated the heterochaperone complex components Hsp90, Hsp70, and p23, pointing to differences in their modes of action. CONCLUSION AND SIGNIFICANCE: The results of this comprehensive study provide important insight into chaperoning of diverse client proteins via the combinatorial action of (co)-chaperones. The differential effects of the TPR proteins on steroid receptors bear on all physiological processes related to steroid hormone activity.

The molecular cell death machinery in the simple cnidarian Hydra includes an expanded caspase family and pro- and anti-apoptotic Bcl-2 proteins.

The fresh water polyp Hydra belongs to the phylum Cnidaria, which diverged from the metazoan lineage before the appearance of bilaterians. In order to understand the evolution of apoptosis in metazoans, we have begun to elucidate the molecular cell death machinery in this model organism. Based on ESTs and the whole Hydra genome assembly, we have identified 15 caspases. We show that one is activated during apoptosis, four have characteristics of initiator caspases with N-terminal DED, CARD or DD domain and two undergo autoprocessing in vitro. In addition, we describe seven Bcl-2-like and two Bak-like proteins. For most of the Bcl-2 family proteins, we have observed mitochondrial localization. When expressed in mammalian cells, HyBak-like 1 and 2 strongly induced apoptosis. Six of the Bcl-2 family members inhibited apoptosis induced by camptothecin in mammalian cells with HyBcl-2-like 4 showing an especially strong protective effect. This protein also interacted with HyBak-like 1 in a yeast two-hybrid assay. Mutation of the conserved leucine in its BH3 domain abolished both the interaction with HyBak-like 1 and the anti-apoptotic effect. Moreover, we describe novel Hydra BH-3-only proteins. One of these interacted with Bcl-2-like 4 and induced apoptosis in mammalian cells. Our data indicate that the evolution of a complex network for cell death regulation arose at the earliest and simplest level of multicellular organization, where it exhibited a substantially higher level of complexity than in the protostome model organisms Caenorhabditis and Drosophila.