In Situ Quantification of Living Cell Adhesion Forces: Single Cell Force Spectroscopy with a Nanotweezer

Xie, Hui; Yin, Munan; Rong, Weibin; Sun, Lining

doi:10.1021/la500045q

Cited by 29 publications

(14 citation statements)

References 39 publications

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“…To see this figure in color, go online. 90% for dual) in agreement with other recent studies (48,52), bias again did not significantly affect total force at detachment (Fig. 5 v).…”

Section: Iv)supporting

confidence: 93%

Nanonet Force Microscopy for Measuring Cell Forces

Sheets

Wang

Zhao

et al. 2016

Biophysical Journal

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The influence of physical forces exerted by or felt by cells on cell shape, migration, and cytoskeleton arrangement is now widely acknowledged and hypothesized to occur due to modulation of cellular inside-out forces in response to changes in the external fibrous environment (outside-in). Our previous work using the non-electrospinning Spinneret-based Tunable Engineered Parameters' suspended fibers has revealed that cells are able to sense and respond to changes in fiber curvature and structural stiffness as evidenced by alterations to focal adhesion cluster lengths. Here, we present the development and application of a suspended nanonet platform for measuring C2C12 mouse myoblast forces attached to fibers of three diameters (250, 400, and 800 nm) representing a wide range of structural stiffness (3-50 nN/μm). The nanonet force microscopy platform measures cell adhesion forces in response to symmetric and asymmetric external perturbation in single and cyclic modes. We find that contractility-based, inside-out forces are evenly distributed at the edges of the cell, and that forces are dependent on fiber structural stiffness. Additionally, external perturbation in symmetric and asymmetric modes biases cell-fiber failure location without affecting the outside-in forces of cell-fiber adhesion. We then extend the platform to measure forces of (1) cell-cell junctions, (2) single cells undergoing cyclic perturbation in the presence of drugs, and (3) cancerous single-cells transitioning from a blebbing to a pseudopodial morphology.

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“…To see this figure in color, go online. 90% for dual) in agreement with other recent studies (48,52), bias again did not significantly affect total force at detachment (Fig. 5 v).…”

Section: Iv)supporting

confidence: 93%

Nanonet Force Microscopy for Measuring Cell Forces

Sheets

Wang

Zhao

et al. 2016

Biophysical Journal

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“…We attributed the tensile anisotropy to the aligned intercellular interactions spanning the length of the construct; for reference, the detachment force required to separate a pair of myoblasts is ≈12 nN after just 10 min of adhesion. [16] We next evaluated the differentiation state of the tissue after 4 d using quantitative polymerase chain reaction (qPCR) and immunostaining, in which we observed upregulation of myogenic regulatory factors (MYOG, MRF4) and positive staining for contractile proteins ( α -myosin skeletal fast, tropomyosin) (Figure S11, Supporting Information).…”

mentioning

confidence: 99%

Engineering Anisotropic Muscle Tissue using Acoustic Cell Patterning

et al. 2018

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Tissue engineering has offered unique opportunities for disease modeling and regenerative medicine; however, the success of these strategies is dependent on faithful reproduction of native cellular organization. Here, it is reported that ultrasound standing waves can be used to organize myoblast populations in material systems for the engineering of aligned muscle tissue constructs. Patterned muscle engineered using type I collagen hydrogels exhibits significant anisotropy in tensile strength, and under mechanical constraint, produced microscale alignment on a cell and fiber level. Moreover, acoustic patterning of myoblasts in gelatin methacryloyl hydrogels significantly enhances myofibrillogenesis and promotes the formation of muscle fibers containing aligned bundles of myotubes, with a width of 120–150 µm and a spacing of 180–220 µm. The ability to remotely pattern fibers of aligned myotubes without any material cues or complex fabrication procedures represents a significant advance in the field of muscle tissue engineering. In general, these results are the first instance of engineered cell fibers formed from the differentiation of acoustically patterned cells. It is anticipated that this versatile methodology can be applied to many complex tissue morphologies, with broader relevance for spatially organized cell cultures, organoid development, and bioelectronics.

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“…AFM imaging and measurements were generally taken within 1 h after blocking. The spring constant of the cantilever was calibrated using the Sader method in air 59 . The cantilever spring constant varied between 0.10-0.15 N/m.…”

Section: Atomic Force Microscopy (Afm)mentioning

confidence: 99%

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis

Drelich

Chang

et al. 2017

Preprint

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Rationale: To maintain vascular patency, endothelial cells (ECs) actively regulate hemostasis. Among the myriad of pathways by which they control both fibrin formation and fibrinolysis is EC expression of annexin A2 (ANXA2) in a heterotetrameric complex with S100A10 [(ANXA2-S100A10)2]. This complex is a well-recognized endothelial surface platform for the activation of plasminogen by tissue plasminogen activator. A noteworthy advance in this field came about when it was shown that the cAMP pathway is linked to the regulation of (ANXA2-S100A10)2 in ECs. Objective: These findings prompted us to determine whether a druggable target, namely the exchange protein directly activated by cAMP (EPAC) pathway, plays a role in vascular luminal fibrinolysis. Methods and Results: Taking advantage of our Epac1-null mouse model, we found that depletion of Epac1 results in fibrin deposition, fibrinolytic dysfunction, and decreased endothelial surface ANXA2 in mice, which are similar to phenomena discovered in ANXA2-null and S100A10-null mice. We observed upregulation of EPAC1 and downregulation of fibrin in endocardial tissues beneath atrial mural thrombi in humans. Of note, our thrombosis model revealed that dysfunction of fibrinolysis in EPAC1-null mice can be ameliorated by recombinant ANXA2. Furthermore, we demonstrated that suppression of EPAC1 using a small-molecule inhibitor (ESI09) reduces the expression of ANXA2 in lipid rafts and impedes ANXA2 association with S100A10. Endothelial apical surface expression of both ANXA2 and S100A10 were markedly decreased in ESI09-treated ECs, which was corroborated by results from a nanoforce spectroscopy study. Moreover, inactivation of EPAC1 decreases tyrosine 23 phosphorylation of ANXA2 in the cell membrane compartment. Conclusions: Our data reveal a novel role for EPAC1 in vascular fibrinolysis, by showing that EPAC1 is responsible for the translocation of ANXA2 to the EC surface. This process promotes conversion of plasminogen to plasmin, thereby enhancing local fibrinolytic activity.

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In Situ Quantification of Living Cell Adhesion Forces: Single Cell Force Spectroscopy with a Nanotweezer

Cited by 29 publications

References 39 publications

Nanonet Force Microscopy for Measuring Cell Forces

Nanonet Force Microscopy for Measuring Cell Forces

Engineering Anisotropic Muscle Tissue using Acoustic Cell Patterning

Exchange protein directly activated by cAMP plays a critical role in regulation of vascular fibrinolysis

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