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.

FGD1, the gene responsible for the inherited disease faciogenital dysplasia, encodes a guanine nucleotide exchange factor (GEF) that specifically activates the p21 GTPase Cdc42. In order, FGD1 is composed of a proline-rich N-terminal region, adjacent GEF and pleckstrin homology (PH) domains, a FYVE-finger domain and a second C-terminal PH domain (PH2), structural motifs involved in signaling and subcellular localization. Fgd1, the mouse FGD1 ortholog, is expressed in regions of active bone formation within osteoblasts and in the osteoblast-like cell line MC3T3-E1, a finding consistent with its role in skeletal formation. Here, we use subcellular fractionation studies to show that endogenous Fgd1 protein is localized in the cytosolic and Golgi and plasma membrane fractions of mouse calvarial cells. Immunocytochemical studies performed with osteoblast-like MC3T3-E1 cells and other mammalian cell lines confirm the localization of Fgd1 and show that the proline-rich N-terminal region is necessary and sufficient for Fgd1 subcellular localization to the plasma membrane and Golgi complex. In contrast, the FYVE-finger and PH2 domains do not appear to direct the localization of Fgd1 or the activation of Cdc42. In addition, microinjection studies indicate that the N-terminal Fgd1 domain inhibits filopodia formation, suggesting that this region down-regulates GEF function. These results characterize the function of the Fgd1 domains for both protein localization and Cdc42 activation and indicate that the Fgd1 Cdc42GEF protein is involved in the regulation of Cdc42 activity at the subcortical actin cytoskeleton and Golgi complex.

BackgroundOncogenic mechanisms in small-cell lung cancer remain poorly understood leaving this tumor with the worst prognosis among all lung cancers. Unlike other cancer types, sequencing genomic approaches have been of limited success in small-cell lung cancer, i.e., no mutated oncogenes with potential driver characteristics have emerged, as it is the case for activating mutations of epidermal growth factor receptor in non-small-cell lung cancer. Differential gene expression analysis has also produced SCLC signatures with limited application, since they are generally not robust across datasets. Nonetheless, additional genomic approaches are warranted, due to the increasing availability of suitable small-cell lung cancer datasets. Gene co-expression network approaches are a recent and promising avenue, since they have been successful in identifying gene modules that drive phenotypic traits in several biological systems, including other cancer types.ResultsWe derived an SCLC-specific classifier from weighted gene co-expression network analysis (WGCNA) of a lung cancer dataset. The classifier, termed SCLC-specific hub network (SSHN), robustly separates SCLC from other lung cancer types across multiple datasets and multiple platforms, including RNA-seq and shotgun proteomics. The classifier was also conserved in SCLC cell lines. SSHN is enriched for co-expressed signaling network hubs strongly associated with the SCLC phenotype. Twenty of these hubs are actionable kinases with oncogenic potential, among which spleen tyrosine kinase (SYK) exhibits one of the highest overall statistical associations to SCLC. In patient tissue microarrays and cell lines, SCLC can be separated into SYK-positive and -negative. SYK siRNA decreases proliferation rate and increases cell death of SYK-positive SCLC cell lines, suggesting a role for SYK as an oncogenic driver in a subset of SCLC.ConclusionsSCLC treatment has thus far been limited to chemotherapy and radiation. Our WGCNA analysis identifies SYK both as a candidate biomarker to stratify SCLC patients and as a potential therapeutic target. In summary, WGCNA represents an alternative strategy to large scale sequencing for the identification of potential oncogenic drivers, based on a systems view of signaling networks. This strategy is especially useful in cancer types where no actionable mutations have emerged.

Invadopodia are subcellular organelles thought to be critical for extracellular matrix (ECM) degradation and the movement of cells through tissues. Here we examine invadopodia generation, turnover, and function in relation to two structural aspects of the ECM substrates they degrade: cross-linking and fiber density. We set up a cellular automaton computational model that simulates ECM penetration and degradation by invadopodia. Experiments with denatured collagen (gelatin) were used to calibrate the model and demonstrate the inhibitory effect of ECM cross-linking on invadopodia degradation and penetration. Incorporation of dynamic invadopodia behavior into the model amplified the effect of cross-linking on ECM degradation, and was used to model feedback from the ECM. When the model was parameterized with spatial fibrillar dimensions that closely matched the organization, in real life, of native ECM collagen into triple-helical monomers, microfibrils, and macrofibrils, little or no inhibition of invadopodia penetration was observed in simulations of sparse collagen gels, no matter how high the degree of cross-linking. Experimental validation, using live-cell imaging of invadopodia in cells plated on cross-linked gelatin, was consistent with simulations in which ECM cross-linking led to higher rates of both invadopodia retraction and formation. Analyses of invadopodia function from cells plated on cross-linked gelatin and collagen gels under standard concentrations were consistent with simulation results in which sparse collagen gels provided a weak barrier to invadopodia. These results suggest that the organization of collagen, as it may occur in stroma or in vitro collagen gels, forms gaps large enough so as to have little impact on invadopodia penetration/degradation. By contrast, dense ECM, such as gelatin or possibly basement membranes, is an effective obstacle to invadopodia penetration and degradation, particularly when cross-linked. These results provide a novel framework for further studies on ECM structure and modifications that affect invadopodia and tissue invasion by cells.