Cells require optimal substrate stiffness for normal function and differentiation. The mechanisms for sensing matrix rigidity and durotaxis, however, are not clear. Here we showed that control, Shp2-/-, integrin beta1-/-, and talin1-/- cell lines all spread to a threefold greater area on fibronectin (FN)-coated rigid polyacrylamide surfaces than soft. In contrast, RPTPalpha-/- cells spread to the same area irrespective of rigidity on FN surfaces but spread 3x greater on rigid collagen IV-coated surfaces than soft. RPTPalpha and alphavbeta3 integrins were shown previously to be colocalized at leading edges and antibodies to alphavbeta3 blocked FN rigidity sensing. When FN beads were held with a rigid laser trap at the leading edge, stronger bonds to the cytoskeleton formed than when held with a soft trap; whereas back from the leading edge and in RPTPalpha-/- cells, weaker bonds were formed with both rigid and soft laser traps. From the rigidity of the trap, we calculate that a force of 10 pN generated in 1 s is sufficient to activate the rigidity response. We suggest that RPTPalpha and alphavbeta3 at the leading edge are critical elements for sensing FN matrix rigidity possibly through SFK activation at the edge and downstream signaling.

(2015) A rapid and high content assay that measures cyto-ID-stained autophagic compartments and estimates autophagy flux with potential clinical applications, Autophagy, 11:3, 560-572, DOI: 10.1080/15548627.2015 T he lack of a rapid and quantitative autophagy assay has substantially hindered the development and implementation of autophagy-targeting therapies for a variety of human diseases. To address this critical issue, we developed a novel autophagy assay using the newly developed Cyto-ID fluorescence dye. We first verified that the Cyto-ID dye specifically labels autophagic compartments with minimal staining of lysosomes and endosomes. We then developed a new Cyto-ID fluorescence spectrophotometric assay that makes it possible to estimate autophagy flux based on measurements of the Cyto-ID-stained autophagic compartments. By comparing to traditional autophagy approaches, we found that this assay yielded a more sensitive, yet less variable, quantification of the stained autophagic compartments and the estimate of autophagy flux. Furthermore, we tested the potential application of this autophagy assay in high throughput research by integrating it into an RNA interference (RNAi) screen and a small molecule screen. The RNAi screen revealed WNK2 and MAP3K6 as autophagy-modulating genes, both of which inhibited the MTOR pathway. Similarly, the small molecule screen identified sanguinarine and actinomycin D as potent autophagy inducers in leukemic cells. Moreover, we successfully detected autophagy responses to kinase inhibitors and chloroquine in normal or leukemic mice using this assay. Collectively, this new Cyto-ID fluorescence spectrophotometric assay provides a rapid, reliable quantification of autophagic compartments and estimation of autophagy flux with potential applications in developing autophagy-related therapies and as a test to monitor autophagy responses in patients being treated with autophagymodulating drugs.

This review will focus on two elements that are essential for functional arterial regeneration in vitro: the mechanical environment and the bioreactors used for tissue growth. The importance of the mechanical environment to embryological development, vascular functionality, and vascular graft regeneration will be discussed. Bioreactors generate mechanical stimuli to simulate the biomechanical environment of the arterial system. This system has been used to reconstruct arterial grafts with appropriate mechanical strength for implantation by controlling the chemical and mechanical environments in which the grafts are grown. Bioreactors are powerful tools to study the effect of mechanical stimuli on extracellular matrix (ECM) architecture and the mechanical properties of engineered vessels. Hence biomimetic systems enable us to optimize chemo-biomechanical culture conditions to regenerate engineered vessels with physiological properties similar to those of native arterial vessels. In addition, this review will introduce and examine various approaches and techniques that have been used to engineer biologically-based vascular grafts, including collagen-based grafts, fibrin-gel grafts, cell sheet engineering, biodegradable polymers, and decellularization of native vessels.