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Mutations in ACTA2, encoding the smooth muscle cell (SMC)-specific isoform of α-actin (α-SMA), cause thoracic aortic aneurysms and dissections and occlusive vascular diseases, including early onset coronary artery disease and stroke. We have shown that occlusive arterial lesions in patients with heterozygous ACTA2 missense mutations show increased numbers of medial or neointimal SMCs. The contribution of SMC hyperplasia to these vascular diseases and the pathways responsible for linking disruption of α-SMA filaments to hyperplasia are unknown. Here, we show that the loss of Acta2 in mice recapitulates the SMC hyperplasia observed in ACTA2 mutant SMCs and determine the cellular pathways responsible for SMC hyperplasia. Acta2(-/-) mice showed increased neointimal formation following vascular injury in vivo, and SMCs explanted from these mice demonstrated increased proliferation and migration. Loss of α-SMA induced hyperplasia through focal adhesion (FA) rearrangement, FA kinase activation, re-localization of p53 from the nucleus to the cytoplasm and increased expression and ligand-independent activation of platelet-derived growth factor receptor beta (Pdgfr-β). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-β activation and Acta2(-/-) SMC proliferation in vitro and neointimal formation with vascular injury in vivo. Loss of α-SMA leads to SMC hyperplasia in vivo and in vitro through a mechanism involving FAK, p53 and Pdgfr-β, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.

The ability to measure real-time mechanosensitive events at the subcellular level in response to discrete mechanical stimulation is a critical component in understanding mechanically-induced cellular remodeling. Vascular smooth muscle cells (VSMC) were transfected with RhoA constructs (wild type, dominant negative or constitutively active) or treated with ML-7 to induce specific cytoskeletal tension characteristics prior to mechanical stimulation. Tensile stress was applied to live VSMC using an atomic force microscope probe functionalized with extracellular matrix (ECM) proteins. The ECM induces selective integrin activation and focal adhesion formation, enabling direct manipulation of cortical actin through an active ECM-integrin-actin linkage. Therefore, locally induced mechanosensitive events triggered downstream activation of intracellular signaling pathways responsible for actin and focal adhesion remodeling throughout the cell. Integration of mechanical stimulation with simultaneous fluorescence imaging by spinning-disk confocal and total internal reflection fluorescence microscopy enabled visualization and quantification of molecular dynamic events at the sub-cellular level in real-time. Results provide evidence that the pre-existing cytoskeletal tension affects the actomyosin apparatus which in turn coordinates the ability of the cell to adapt to the externally applied stress. RhoA activation induced high cytoskeletal tension that correlated with increased stress fiber formation, cell stiffness, integrin activation and myosin phosphorylation. In contrast, blocking Rho-kinase or myosin function was characterized by low cytoskeletal tension with a decreased level of stress fiber formation, lower cell stiffness and integrin activation. Our findings show that VSMC sense and adapt to physical microenvironmental changes by a coordinated response of the actomyosin apparatus necessary to establish a new homeostatic state.

The crosstalk between cells and their microenvironment enables cellular adaptation to external mechanical cues through the remodeling of cytoskeletal structures and cell-matrix adhesions to ensure normal cell function. This study investigates the relationship between the cytoskeletal tension and integrin α5β1 adhesion strength to the matrix (i.e. fibronectin) in the context of RhoA-Src crosstalk. Integration of atomic force microscopy (AFM) with total internal reflection fluorescence and spinning-disk confocal microscopy enabled acquisition of complementary structural and functional measurements on live vascular smooth muscle cells expressing RhoA and c-Src variants (wild-type, dominant negative, constitutively active). Single ligand-receptor interaction measurements performed with AFM probes functionalized with fibronectin showed that RhoA and c-Src activation have different effects on cytoskeletal tension development, inducing two distinct force-stiffness functional regimes for α5β1-integrin binding to fibronectin. Moreover, fluorescence measurements showed that c-Src activation had a modest effect on actin morphology, while RhoA significantly modulated stress fiber formation. In addition, c-Src was associated with regulation of myosin light chain (MLC) phosphorylation, suggesting a c-Src-dependent modulation of RhoA pathway through activation of downstream effectors. Therefore, c-Src may be a possible component of cytoskeletal tension regulation through MLC activation. Our findings suggest that Src and RhoA coordinate a regulatory network that determines cytoskeletal tension through activation of actomyosin contractility. In turn, the cytoskeletal tension state modulates integrin α5β1-fibronectin adhesion force.

Directional migration requires the coordination of cytoskeletal changes essential for cell polarization and adhesion turnover. Extracellular signals that alter tyrosine phosphorylation drive directional migration by inducing reorganization of the actin cytoskeleton. It is recognized that Nck is an important link between tyrosine phosphorylation and actin dynamics, however, the role of Nck in cytoskeletal remodeling during directional migration and the underlying molecular mechanisms remain largely undetermined. In this study, a combination of molecular genetics and quantitative live cell microscopy was used to show that Nck is essential in the establishment of front-back polarity and directional migration of endothelial cells. Time-lapse differential interference contrast and total internal reflection fluorescence microscopy showed that Nck couples the formation of polarized membrane protrusions with their stabilization through the assembly and maturation of cell-substratum adhesions. Measurements by atomic force microscopy showed that Nck also modulates integrin α5β1-fibronectin adhesion force and cell stiffness. Fluorescence resonance energy transfer imaging revealed that Nck depletion results in delocalized and increased activity of Cdc42 and Rac. In contrast, the activity of RhoA and myosin II phosphorylation were reduced by Nck knockdown. Thus, this study identifies Nck as a key coordinator of cytoskeletal changes that enable cell polarization and directional migration which are critical processes in development and disease.

We have developed a filter-chip and optical detection system for rapid antibiotic efficacy screening. The filter-chip consisted of a 1-mL reservoir and an anodic aluminum oxide (AAO) nanoporous membrane. Sample solution with liquid growth media, bacteria, and antibiotics was incubated in the reservoir for a specific period of time. The number of live bacteria on the surface of membrane was counted after the incubation with antibiotics and filtration. Using this biosensing system, we have demonstrated a 1-h antibiotic screening for patients’ clinical samples, significantly faster than the conventional antibiotic susceptibility tests that typically take more than 24 h. This rapid screening nature makes the filter-chip and detection system ideal for tailoring antibiotic treatment to individual patients by reducing the microbial antibiotic resistance, and improving the survival rate for patients suffering from postoperative infections.

RhoA and Src are considered potential modulators of the mechanical signaling that promotes actin polymerization and focal adhesion formation. Here, we performed adhesion force spectroscopy measurements on vascular smooth muscle cells (VSMC) expressing RhoA or Src (wild type, constitutively active or dominant negative mutants) using an atomic force microscope (AFM) tip functionalized with fibronectin. Integrin alpha5beta1-fibronectin adhesion force, local cell stiffness at the point of contact, and adhesion probability were determined. The results show that the mechanical properties of the VSMC as well as integrin-fibronectin adhesion force are modulated by RhoA and Src. Inhibition of RhoA activity induces low cytoskeletal tension and decreased integrin-fibronectin adhesion force. Also, VSMC expressing RhoA or Src dominant negative constructs were found to be the softest. Conversely, RhoA and Src activation increased alpha5beta1 integrin-fibronectin adhesion force and cell stiffness. To independently verify the effect of RhoA and Src on cytoskeletal changes, fluorescence imaging experiments were performed to quantify actin fibers (confocal imaging of cells co-expressing RhoA-GFP and actin-mRFP) or protein phosphorylation at focal adhesions (total internal reflection fluorescence imaging of cells co-expressing Src-mCherry and dSH2-YFP), respectively. Consistent with the AFM measurements, we observed an increase in actin fibers, and in protein phosphorylation at focal adhesion sites in cells expressing constitutively active RhoA and Src, respectively. In addition, these two proteins were maximally decreased by expression of dominant negative RhoA and Src. Taken together, these results show that RhoA and Src are critical modulators of the mechanical signaling cross-talk between the cell and the extracellular matrix.

Analysis of protein-protein or protein-DNA interaction in cells is indispensable for current basic cancer research and anti-cancer drug screening. However, it usually performed by conventional biochemical approaches, which require long process time and a large amount of samples. In this presentation, we will show the new application of Raster image Correlation spectroscopy (RICS) that can detect protein-protein, or protein-DNA interactions directly without the time-consuming biochemical process. As a result, this technique significantly reduces the analysis time from a few days to a few hours. As a proof of the concept, we investigated the effects of anti-cancer drugs, cisplatin and etoposide, on tumor-suppressor p53 protein dynamics in Hela cells. We measured the fast diffusion of GFP-tagged p53 in living Hela cells treated or untreated with each anti-cancer drug by RICS. After the drug treatment, the significant reductions of p53 mobility were observed compared to the one without drug treatment. Both cisplatin and etoposide induce DNA damage, and it has been shown that DNA damage stabilizes and activates p53, resulting in the formation of the DNA-p53 complex. Therefore, data obtained by RICS perfectly explain the status of p53 inside the cells. Together, these results suggest that RICS approach is a powerful tool to measure protein-protein or protein-DNA interactions in living cells. Since the small molecules disrupt specific protein-protein interactions are considered as promising drugs for targeted cancer therapy, our novel RICS system may serve as a powerful tool for future drug screening.