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The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time. Nobis et al. generated a RhoA-FRET biosensor mouse to characterize and quantify the spatiotemporal distribution of RhoA activity in native mammalian tissues in vivo during development and disease progression. They show that RhoA activity is tightly regulated during various normal biological processes and is co-opted in disease settings, such as invasive breast and pancreatic cancers. Refereed/Peer-reviewed

Tumors and associated stroma manifest mechanical properties that promote cancer. Mechanosensation of tissue stiffness activates the Rho/ROCK pathway to increase actomyosin-mediated cellular tension to re-establish force equilibrium. To determine how actomyosin tension affects tissue homeostasis and tumor development, we expressed conditionally active ROCK2 in mouse skin. ROCK activation elevated tissue stiffness via increased collagen. beta-catenin, a key element of mechanotranscription pathways, was stabilized by ROCK activation leading to nuclear accumulation, transcriptional activation, and consequent hyperproliferation and skin thickening. Inhibiting actomyosin contractility by blocking LIMK or myosin ATPase attenuated these responses, as did FAK inhibition. Tumor number, growth, and progression were increased by ROCK activation, while ROCK blockade was inhibitory, implicating actomyosin-mediated cellular tension and consequent collagen deposition as significant tumor promoters. [KEYWORDS: Actomyosin/ physiology, Animals, Biomechanics, Cell Proliferation, Cells, Cultured, Epidermis/ pathology, Humans, Hyperplasia, Mice, Papilloma/etiology, Signal Transduction, Skin Neoplasms/ etiology, beta Catenin/ physiology, rho-Associated Kinases/analysis/genetics/physiology]

Reductive amination is a common technique for the derivatisation of reducing carbohydrates, thereby providing appropriate chromophores or fluorophores to overcome native detection deficiencies. Rarely, however, is the issue of labelling efficiency addressed for substrates larger than monosaccharides. Utilising a variety of radiolabelled synthetic maltooligosaccharides, we now present data on the APTS labelling efficiency for substrates up to an average degree of polymerization (dp) of 135. The labelling reaction was found to be highly reproducible and independent of average chain length between dp 3 and dp 135, with an average efficiency of 80%. Glucose (95%) and maltose (88%) were labelled more efficiently. In addition to this work, electrophoretic methodologies have been developed to aid the characterization of APTS-labelled oligosaccharide distributions across a wide range of chain lengths. Fluorescent imaging of polyacrylamide slab gels provides flexibility of gel format, and conditions that can be adapted to the resolution and quantification of short oligosaccharide populations (less than dp 30) or to enable the observation of polysaccharides. A capillary gel electrophoretic method was developed using laser–induced fluorescence (LIF) detection to fully resolve and quantify maltooligosaccharides up to approximately dp 100, a technique that finds particular use in the analysis of oligosaccharide distributions obtained from isoamylase debranching of the amylopectin component of starch. A comparison of data reproducibility across a range of chain lengths established the superiority of results obtained by the capillary gel electrophoretic method over a previously reported method involving DNA sequencer-mediated electrophoretic separation.

Background Mast cells have gained notoriety based on their detrimental contributions to IgE-mediated allergic disorders. Although mast cells express the vitamin D receptor (VDR), it is not clear to what extent 1α,25-dihydroxyvitamin D 3 (1α,25[OH] 2 D 3 ) or its predominant inactive precursor metabolite in the circulation, 25-hydroxyvitamin D 3 (25OHD 3 ), can influence IgE-mediated mast cell activation and passive cutaneous anaphylaxis (PCA) in vivo . Objective We sought to assess whether the vitamin D 3 metabolites 25OHD 3 and 1α,25(OH) 2 D 3 can repress IgE-dependent mast cell activation through mast cell–25-hydroxyvitamin D-1α-hydroxylase (CYP27B1) and mast cell–VDR activity. Methods We measured the extent of vitamin D 3 suppression of IgE-mediated mast cell degranulation and mediator production in vitro , as well as the vitamin D 3 –induced curtailment of PCA responses in WBB6F 1 - Kit W/W-v or C57BL/6J- Kit W-sh/W-sh mice engrafted with mast cells that did or did not express VDR or CYP27B1. Results Here we show that mouse and human mast cells can convert 25OHD 3 to 1α,25(OH) 2 D 3 through CYP27B1 activity and that both of these vitamin D 3 metabolites suppressed IgE-induced mast cell–derived proinflammatory and vasodilatory mediator production in a VDR-dependent manner in vitro . Furthermore, epicutaneously applied vitamin D 3 metabolites significantly reduced the magnitude of skin swelling associated with IgE-mediated PCA reactions in vivo ; a response that required functional mast cell–VDRs and mast cell–CYP27B1. Conclusion Taken together, our findings provide a mechanistic explanation for the anti-inflammatory effects of vitamin D 3 on mast cell function by demonstrating that mast cells can actively metabolize 25OHD 3 to dampen IgE-mediated mast cell activation in vitro and in vivo .

In this issue of Cell Stem Cell, Chen et al. (2010) and Ohgushi et al. (2010) report that Rho-mediated actomyosin contractility underlies the proclivity of human embryonic stem cells to undergo apoptosis upon dissociation or during low-density propagation, a property that challenges their in vitro isolation and manipulation.