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1.
Prog Biophys Mol Biol ; 176: 67-81, 2022 12.
Article in English | MEDLINE | ID: mdl-36055517

ABSTRACT

Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases.


Subject(s)
Cartilage, Articular , Mechanotransduction, Cellular , Microscopy, Atomic Force/methods , Biophysics , Microscopy, Fluorescence
2.
Front Physiol ; 9: 856, 2018.
Article in English | MEDLINE | ID: mdl-30108507

ABSTRACT

Aging induces a progressive decline in vasoconstrictor responses in central and peripheral arteries. This study investigated the hypothesis that vascular smooth muscle (VSM) contractile function declines with age in soleus muscle feed arteries (SFA). Contractile function of cannulated SFA isolated from young (4 months) and old (24 months) Fischer 344 rats was assessed by measuring constrictor responses of denuded (endothelium removed) SFA to norepinephrine (NE), phenylephrine (PE), and angiotensin II (Ang II). In addition, we investigated the role of RhoA signaling in modulation of VSM contractile function. Structural and functional characteristics of VSM cells were evaluated by fluorescence imaging and atomic force microscopy (AFM). Results indicated that constrictor responses to PE and Ang II were significantly impaired in old SFA, whereas constrictor responses to NE were preserved. In the presence of a Rho-kinase inhibitor (Y27632), constrictor responses to NE, Ang II, and PE were significantly reduced in young and old SFA. In addition, the age-group difference in constrictor responses to Ang II was eliminated. ROCK1 and ROCK2 content was similar in young and old VSM cells, whereas pROCK1 and pROCK2 were significantly elevated in old VSM cells. Aging was associated with a reduction in smooth muscle α-actin stress fibers and recruitment of proteins to cell-matrix adhesions. Old VSM cells presented an increase in integrin adhesion to the matrix and smooth muscle γ-actin fibers that was associated with increased cell stiffness. In conclusion, our results indicate that VSM contractile function declined with age in SFA. The decrement in contractile function was mediated in part by RhoA/ROCK signaling. Upregulation of pROCK in old VSM cells was not able to rescue contractility in old SFA. Collectively, these results indicate that changes at the VSM cell level play a central role in the reduced contractile function of aged SFA.

3.
Integr Biol (Camb) ; 6(8): 743-54, 2014 Aug.
Article in English | MEDLINE | ID: mdl-24984203

ABSTRACT

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 α5ß1 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 α5ß1-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 α5ß1-fibronectin adhesion force.


Subject(s)
Cytoskeleton/metabolism , rhoA GTP-Binding Protein/metabolism , src-Family Kinases/metabolism , Actins/chemistry , Animals , Cell Adhesion , Fibronectins/metabolism , Fluorescent Dyes/chemistry , Integrin alpha5beta1/metabolism , Ligands , Microscopy, Atomic Force , Microscopy, Confocal , Phosphorylation , Pressure , Rats , Stress, Mechanical
4.
Hum Mol Genet ; 22(15): 3123-37, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23591991

ABSTRACT

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-ß). Disruption of α-SMA in wild-type SMCs also induced similar cellular changes. Imatinib mesylate inhibited Pdgfr-ß 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-ß, supporting the hypothesis that SMC hyperplasia contributes to occlusive lesions in patients with ACTA2 missense mutations.


Subject(s)
Actins/metabolism , Focal Adhesion Protein-Tyrosine Kinases/metabolism , Myocytes, Smooth Muscle/metabolism , Myocytes, Smooth Muscle/pathology , Receptor, Platelet-Derived Growth Factor beta/metabolism , Tumor Suppressor Protein p53/metabolism , Actins/genetics , Animals , Cell Movement/genetics , Cell Nucleus/metabolism , Cell Proliferation , Enzyme Activation , Hyperplasia , Mice , Mice, Knockout , Models, Biological , Phenotype , Protein Transport , Reactive Oxygen Species/metabolism
5.
J Cell Sci ; 126(Pt 7): 1637-49, 2013 Apr 01.
Article in English | MEDLINE | ID: mdl-23444376

ABSTRACT

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 α5ß1-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. By 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 crucial processes in development and disease.


Subject(s)
Actin Cytoskeleton/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Cell Movement/physiology , Cell Polarity/physiology , Oncogene Proteins/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Blotting, Western , Cell Adhesion/genetics , Cell Adhesion/physiology , Cell Line , Cell Movement/genetics , Cell Polarity/genetics , Focal Adhesions/metabolism , Humans , Integrin alpha5beta1/metabolism , Mice , Microscopy, Atomic Force , Microscopy, Fluorescence , NIH 3T3 Cells , Oncogene Proteins/genetics
6.
Integr Biol (Camb) ; 4(6): 615-27, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22546924

ABSTRACT

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.


Subject(s)
Cytoskeleton/physiology , Extracellular Matrix/physiology , Focal Adhesions/physiology , Integrins/physiology , Muscle, Smooth, Vascular/physiology , rhoA GTP-Binding Protein/physiology , Actomyosin/physiology , Animals , Cell Communication , Extracellular Matrix Proteins/physiology , Microscopy, Atomic Force , Microscopy, Confocal , Microscopy, Fluorescence , Muscle, Smooth, Vascular/cytology , Myocytes, Smooth Muscle/cytology , Myocytes, Smooth Muscle/physiology , Rats , Stress, Mechanical
7.
Biosens Bioelectron ; 26(1): 289-94, 2010 Sep 15.
Article in English | MEDLINE | ID: mdl-20650629

ABSTRACT

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 24h. 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.


Subject(s)
Aluminum Oxide/chemistry , Anti-Bacterial Agents/analysis , Biosensing Techniques/instrumentation , Microfluidic Analytical Techniques/instrumentation , Nanostructures/chemistry , Nanotechnology/instrumentation , Optical Devices , Ultrafiltration/instrumentation , Equipment Design , Equipment Failure Analysis , Nanostructures/ultrastructure
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