The mechanism of signal transduction by the GPI anchored Prod 1 protein

Salamanders such as the red spotted newt Notophthalamus viridescens and the axolotl Ambystoma mexicanum regenerate a number of anatomical structures following injury. Prod1 is believed to guide patterning processes operating during limb regeneration, however the molecular mechanism through which it operates is unclear. Being glycosylphosphatidylinositol (GPI) anchored, Prod1 does not make direct contact with the cytoplasm, raising questions as to how it functions in the transfer of information across the cell membrane. The transmembrane epidermal growth factor receptor was shown to associate with Prod1, initiating MAPK signalling and resulting in the induction of matrix metalloprotease 9 expression (MMP9). MMP9 is known to be rapidly upregulated in the hours following amputation in the wound epithelium, a structure essential for regeneration formed by the migration of epidermal cells across the surface of the amputation plane. Patches of newt limb skin explanted into culture were used as a model for this process. A sheet of cells expressing MMP9 was seen to migrate out from skin patches, and this was shown to be sensitive to MMP inhibitors. Further to this, upregulation of MMP9 was seen to occur in the dermis of explanted skin patches, a layer of the skin known to be instructive to the patterning of the limb. The relationship of Prod1s structure to its MMP9 inducing function was investigated through the creation of a series of point mutants, and it was shown that amino acids located on the α-helix of the protein were essential for this function. Axolotl Prod1 lacks a GPI anchor, however despite the requirement of newt Prod1 for GPI anchorage in order to induce MMP9 expression in either newt or axolotl cells, axolotl Prod1 was fully functional in cells from either species. There was some indication that amino acids on the α-helix may confer this ability to axolotl Prod1

Questions to Ask Before You Join a Club

Despite the recent flurry of large transactions in which a consortium of private equity firms have teamed up to make joint bids and acquisitions, “club deals” themselves are not breaking news. In fact, they have been a staple of small- and middle-sized private equity M&A transactions for years. Recently, however, there has been a growing trend toward large club deals with enterprise values over $1 billion.1 Due to their size, complexity and, often, international dimension, these transactions have generated considerable attention in the business press and have prompted much discussion among private equity professionals and the limited partners whose money they manage

Questions to Ask Before You Join a Club

Despite the recent flurry of large transactions in which a consortium of private equity firms have teamed up to make joint bids and acquisitions, “club deals” themselves are not breaking news. In fact, they have been a staple of small- and middle-sized private equity M&A transactions for years. Recently, however, there has been a growing trend toward large club deals with enterprise values over $1 billion.1 Due to their size, complexity and, often, international dimension, these transactions have generated considerable attention in the business press and have prompted much discussion among private equity professionals and the limited partners whose money they manage

A new trick for an old lipid.

Cholesterol can regulate the Hedgehog signalling pathway by directly binding to a receptor on the cell surface

Sox2 levels regulate the chromatin occupancy of WNT mediators in epiblast progenitors responsible for vertebrate body formation

WNT signalling has multiple roles. It maintains pluripotency of embryonic stem cells, assigns posterior identity in the epiblast and induces mesodermal tissue. Here we provide evidence that these distinct functions are conducted by the transcription factor SOX2, which adopts different modes of chromatin interaction and regulatory element selection depending on its level of expression. At high levels, SOX2 displaces nucleosomes from regulatory elements with high-affinity SOX2 binding sites, recruiting the WNT effector TCF/β-catenin and maintaining pluripotent gene expression. Reducing SOX2 levels destabilizes pluripotency and reconfigures SOX2/TCF/β-catenin occupancy to caudal epiblast expressed genes. These contain low-affinity SOX2 sites and are co-occupied by T/Bra and CDX. The loss of SOX2 allows WNT-induced mesodermal differentiation. These findings define a role for Sox2 levels in dictating the chromatin occupancy of TCF/β-catenin and reveal how context-specific responses to a signal are configured by the level of a transcription factor

Ptch1 and Gli regulate Shh signalling dynamics via multiple mechanisms.

In the vertebrate neural tube, the morphogen Sonic Hedgehog (Shh) establishes a characteristic pattern of gene expression. Here we quantify the Shh gradient in the developing mouse neural tube and show that while the amplitude of the gradient increases over time, the activity of the pathway transcriptional effectors, Gli proteins, initially increases but later decreases. Computational analysis of the pathway suggests three mechanisms that could contribute to this adaptation: transcriptional upregulation of the inhibitory receptor Ptch1, transcriptional downregulation of Gli and the differential stability of active and inactive Gli isoforms. Consistent with this, Gli2 protein expression is downregulated during neural tube patterning and adaptation continues when the pathway is stimulated downstream of Ptch1. Moreover, the Shh-induced upregulation of Gli2 transcription prevents Gli activity levels from adapting in a different cell type, NIH3T3 fibroblasts, despite the upregulation of Ptch1. Multiple mechanisms therefore contribute to the intracellular dynamics of Shh signalling, resulting in different signalling dynamics in different cell types

Statistically derived geometrical landscapes capture principles of decision-making dynamics during cell fate transitions

Fate decisions in developing tissues involve cells transitioning between discrete cell states, each defined by distinct gene expression profiles. The Waddington landscape, in which the development of a cell is viewed as a ball rolling through a valley filled terrain, is an appealing way to describe differentiation. To construct and validate accurate landscapes, quantitative methods based on experimental data are necessary. We combined principled statistical methods with a framework based on catastrophe theory and approximate Bayesian computation to formulate a quantitative dynamical landscape that accurately predicts cell fate outcomes of pluripotent stem cells exposed to different combinations of signaling factors. Analysis of the landscape revealed two distinct ways in which cells make a binary choice between one of two fates. We suggest that these represent archetypal designs for developmental decisions. The approach is broadly applicable for the quantitative analysis of differentiation and for determining the logic of developmental decisions

Cholesterol activates the G-protein coupled receptor Smoothened to promote Hedgehog signaling

Cholesterol is necessary for the function of many G-protein coupled receptors (GPCRs). We find that cholesterol is not just necessary but also sufficient to activate signaling by the Hedgehog (Hh) pathway, a prominent cell-cell communication system in development. Cholesterol influences Hh signaling by directly activating Smoothened (SMO), an orphan GPCR that transmits the Hh signal across the membrane in all animals. Unlike most GPCRs, which are regulated by cholesterol through their heptahelical transmembrane domains, SMO is activated by cholesterol through its extracellular cysteine-rich domain (CRD). Residues shown to mediate cholesterol binding to the CRD in a recent structural analysis also dictate SMO activation, both in response to cholesterol and to native Hh ligands. Our results show that cholesterol can initiate signaling from the cell surface by engaging the extracellular domain of a GPCR and suggest that SMO activity may be regulated by local changes in cholesterol abundance or accessibility