Echinoderms are the deuterostome group with the most striking capacity to regenerate lost body parts. In particular, members of the class Holothuroidea are able to regenerate most of their internal organs following a typical evisceration process. Such formation of new viscera in an adult organism provides a unique model to study the process of organogenesis. We have studied this process in the sea cucumber Holothuria glabberrima by describing the spatial and temporal pattern of cellular events that occur during intestine regeneration following chemically induced evisceration. Regeneration begins as a thickening of the mesenteries that supported the autotomized organs to the body wall. The mesenterial thickening consists of tissues where most of the cellular populations found in the normal intestine are already present. However, the cell numbers differ, particularly those of hemocytes and amoebocytes, suggesting that some of these cells play an important role in the formation of the solid rod of hypertrophic mesentery that characterizes the intestinal primordia. The appearance of the luminal epithelium, together with the formation of the lumen, occurs during the second week of regeneration by proliferation and extensive migration of cells from the esophagus and cloacal ends into the thickenings. At this stage all tissue layers are present, but it takes an additional week for them to exhibit the proportions typical of the normal organ. Cell division, as determined by BrdU labeling, mainly occurs in the coelomic epithelia of the hypertrophic mesentery and in the regenerating luminal epithelium. Our study provides evidence that the process of new organ formation in holothurians can be described as an intermediate process showing characteristics of both epimorphic and morphallactic phenomena. J. Exp. Zool. 281:288–304, 1998. © 1998 Wiley‐Liss, Inc.

Small cell lung cancer (SCLC) is a devastating disease because of its tendency to early invasion and refractory relapse after initial treatment response. These aggressive traits have been associated with phenotypic heterogeneity, which however remains incompletely understood. To fill this knowledge gap, we inferred a set of 33 transcription factors (TFs) associated with gene signatures of the known neuroendocrine/epithelial (NE) and non-neuroendocrine/mesenchymal-like (ML) SCLC phenotypes. The topology of this SCLC TF network was derived from prior knowledge and simulated using Boolean modeling. These simulations predicted that the network settles into attractors (TF expression patterns) correlated with NE or ML phenotypes, suggesting that TF network dynamics underlie emergence of heterogeneous SCLC phenotypes in an epigenetic landscape. However, several cell lines and patient samples did not correlate with either the NE or ML attractors. Flow cytometry indicated that single cells within these cell lines simultaneously express surface markers of both NE and ML differentiation, revealing existence of a “hybrid” phenotype. Upon exposure to standard-of-care cytotoxic drugs or epigenetic modifiers, NE and ML cell populations converged toward the hybrid state, suggesting a possible escape route from treatment. Our findings indicate that SCLC phenotypic heterogeneity can be specified dynamically by attractor states of a master regulatory TF network. Thus, SCLC heterogeneity may be best understood as states within an epigenetic landscape. Understanding phenotypic transitions within this landscape could provide insights to clinical applications.

A system consisting of five purified proteins: Hsp90, Hsp70, Hop, Hsp40, and p23, acts as a machinery for assembly of glucocorticoid receptor (GR)⅐Hsp90 heterocomplexes. Hop binds independently to Hsp90 and to Hsp70 to form a Hsp90⅐Hop⅐Hsp70⅐Hsp40 complex that is sufficient to convert the GR to its steroid binding form, and this four-protein complex will form stable GR⅐Hsp90 heterocomplexes if p23 is added to the system (Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B. (1998) J. Biol. Chem. 273, 7358 -7366). Hop has been considered essential for the formation of receptor⅐Hsp90 heterocomplexes and GR folding. Here we use Hsp90 and Hsp70 purified free of all traces of Hop and Hsp40 to show that Hop is not required for GR⅐Hsp90 heterocomplex assembly and activation of steroid binding activity. Rather, Hop enhances the rate of the process. We also show that Hsp40 is not essential for GR folding by the five-protein system but enhances a process that occurs less effectively when it is not present. By carrying out assembly in the presence of radiolabeled steroid to bind to the GR as soon as it is converted to the steroid binding state, we show that the folding change is brought about by only two essential components, Hsp90 and Hsp70, and that Hop, Hsp40, and p23 act as nonessential co-chaperones.The steroid receptors are recovered from cells as multiprotein heterocomplexes containing a dimer of Hsp90, substochiometric amounts of Hsp70, 1 an acidic 23-kDa protein, p23, and a tetratricopeptide repeat domain protein, such as immunophilin or protein phosphatase 5 (for review see Refs. 1 and 2). The steroid receptor⅐Hsp90 heterocomplexes can be formed under cell-free conditions by incubating the immunoadsorbed proteins with reticulocyte lysate (3, 4). Inasmuch as the glucocorticoid receptor (GR) 2 must be associated with Hsp90 for it to have steroid binding activity (5), incubation of Hsp90-free GR with reticulocyte lysate results in generation of steroid binding activity in direct proportion to the number of GR⅐Hsp90 heterocomplexes that are assembled (6). Hsp90 binds directly to the ligand-binding domain (LBD) of the GR (1), and complexing with Hsp90 also opens up both thiol moieties (7) and trypsin cleavage sites (8, 9) in the LBD to attack by a thiol-derivatizing agent and the protease. These biochemical observations, coupled with data derived from GR mutants (10), support the idea (6, 11) that the Hsp90 heterocomplex assembly system in reticulocyte lysate directs an ATP-dependent partial unfolding of the GR LBD, thus opening the hydrophobic steroid-binding cleft to access by steroid.The heterocomplex assembly system has been reconstituted (12-16), and five proteins, including Hsp90, Hsp70, Hop (60-kDa Hsp organizer protein), Hsp40, and p23, participate in the ATP/Mg 2ϩ -dependent and K ϩ -dependent assembly process (for review of heterocomplex assembly see Refs. 17 and 18). Besides Hsp90 itself, the only component of this Hsp90-based chaperone system proven to be essential for both GR⅐Hsp90 heterocomplex...

Background: Classical in vitro wound-healing assays and other techniques designed to study cell migration and invasion have been used for many years to elucidate the various mechanisms associated with metastasis. However, many of these methods are limited in their ability to achieve reproducible, quantitative results that translate well in vivo. Such techniques are also commonly unable to elucidate single-cell motility mechanisms, an important factor to be considered when studying dissemination. Therefore, we developed and applied a novel in vitro circular invasion assay (CIA) in order to bridge the translational gap between in vitro and in vivo findings, and to distinguish between different modes of invasion.

FGD1 mutations result in Faciogenital Dysplasia (FGDY), an X-linked human disease that affects skeletal formation and embryonic morphogenesis. FGD1 and Fgd1, the mouse FGD1 ortholog, encode guanine nucleotide exchange factors (GEF) that specifically activate Cdc42, a Rho GTPase that controls the organization of the actin cytoskeleton. To further understand FGD1/Fgd1 signaling and begin to elucidate the molecular pathophysiology of FGDY, we demonstrate that Fgd1 directly interacts with cortactin and mouse actin-binding protein 1 (mAbp1), actin-binding proteins that regulate actin polymerization through the Arp2/3 complex. In yeast two-hybrid studies, cortactin and mAbp1 Src homology 3 (SH3) domains interact with a single Fgd1 SH3-binding domain (SH3-BD), and biochemical studies show that the Fgd1 SH3-BD directly binds to cortactin and mAbp1 in vitro. Immunoprecipitation studies show that Fgd1 interacts with cortactin and mAbp1 in vivo and that Fgd1 SH3-BD mutations disrupt binding. Immunocytochemical studies show that Fgd1 colocalizes with cortactin and mAbp1 in lamellipodia and membrane ruffles, and that Fgd1 subcellular targeting is dynamic. By using truncated cortactin proteins, immunocytochemical studies show that the cortactin SH3 domain targets Fgd1 to the subcortical actin cytoskeleton, and that abnormal Fgd1 localization results in actin cytoskeletal abnormalities and significant changes in cell shape and viability. Thus, this study provides novel in vitro and in vivo evidence that Fgd1 specifically and directly interacts with cortactin and mAbp1, and that these interactions play an important role in regulating the actin cytoskeleton and, subsequently, cell shape.