scholarly journals Integration of Magnetic Tweezers and Traction Force Microscopy for the Exploration of Matrix Rheology and Keratinocyte Mechanobiology: Model Force- and Displacement-Controlled Experiments

2020 ◽  
Author(s):  
Waddah I. Moghram ◽  
Pratibha Singh ◽  
Christian A. VandeLune ◽  
Edward A. Sander ◽  
John C. Selby

ABSTRACTIn this work we demonstrate the integration of magnetic tweezers (MT) with substrate deformation tracking microscopy (DTM) and traction force microscopy (TFM) for the investigation of extracellular matrix rheology and human epidermal keratinocyte mechanobiology in the context of human blistering skin diseases. Two model bead-on-gel experiments are described in which an MT device is used to apply a prescribed force or displacement waveform to a fibronectin-coated superparamagnetic bead attached to a type I collagen gel containing a layer of covalently attached red-fluorescent microspheres. Serial fast time-lapse DIC and epifluorescence image acquisitions are used to capture displacements of the bead and microspheres, respectively, in response to the applied force or displacement. Due to the large number of acquired images and the dynamic behavior of substrate microspheres observed during the experiment, new quantitative methods are developed for the tracking and filtering of microsphere displacement data, the selection of L2 regularization parameters used for TFM analysis, and the identification of time intervals within the overall image set that can be approximated as being subject to elastostatic conditions. Two major proof-of-concept applications are described in which integrated MT-DTM/TFM experiments are used to (i) estimate the elastic properties of a fibrillar type I collagen gel substrate and (ii) demonstrate how a force applied to a focal adhesion contact on the apical surface of a living keratinocyte is directly transmitted to basal cell-matrix anchoring junctions as observed by substrate deformations and incremental traction stresses that develop within the collagen subjacent to the cell.

1996 ◽  
Vol 109 (7) ◽  
pp. 1875-1889 ◽  
Author(s):  
A. Zuk ◽  
K.S. Matlin

A number of epithelia form tubulocysts in vitro when overlaid with type I collagen gel. Because collagen receptors are generally believed to be expressed on the basolateral domain, the mechanism by which collagen elicits this morphogenetic response from the apical surface is unclear. To investigate the role of beta 1 integrins, the major receptor family for collagen, in this process, we overlaid polarized monolayers of MDCK II cells grown on permeable supports with type I collagen gel and correlated integrin polarity with the polarity of other apical and basolateral membrane markers during tubulocyst formation. Polarized monolayers of one clone of MDCK II cells, referred to as Heidelberg MDCK, initially respond to collagen overlay by stratifying; within 48 hours, lumena develop between the cell layers giving rise to tubulocysts. Tight junctions remain intact during tubulocyst formation because transepithelial electrical resistance does not significantly change. Major alterations are observed, however, in the expression and localization of apical and basolateral membrane markers. beta 1 integrins are necessary for tubulocyst morphogenesis because a function-blocking antibody administered to the apical pole of the cells completely inhibits the formation of these structures. To determine how apical-cell collagen interactions elicit tubulocyst formation, we examined whether beta 1 integrins are mobilized to apical plasma membranes in response to collagen overlay. We found that in the absence of collagen, polarized monolayers of Heidelberg MDCK cells endogenously express on apical plasma membranes a small pool of the beta 1 family, including alpha 2 beta 1 and alpha 3 beta 1. Collagen overlay does not mobilize additional beta 1 integrins to apical domains. If beta 1 integrins are not already apically expressed, as in the C6 MDCK cell line (Schoenenberger et al. (1994) J. Cell Biol. 107, 527–541), beta 1 integrins are not directed apically and tubulocysts do not develop in response to collagen. Thus, interaction of beta 1 integrin pre-existing on apical plasma membranes of polarized epithelia with type I collagen gel is the mechanism by which apical application of collagen elicits the formation of tubulocysts. Depolarized integrins on apical plasma membranes of polarized epithelia may be relevant to the pathogenesis of disease and injury.


2000 ◽  
Vol 48 (6) ◽  
pp. 2028-2032 ◽  
Author(s):  
Yoshihiro Nomura ◽  
Shinzi Toki ◽  
Yasuhiro Ishii ◽  
Kunio Shirai

2008 ◽  
Vol 133 (2) ◽  
pp. 245-252 ◽  
Author(s):  
Yuji Hirano ◽  
Naoki Ishiguro ◽  
Masahiro Sokabe ◽  
Masaharu Takigawa ◽  
Keiji Naruse

Biomaterials ◽  
2006 ◽  
Vol 27 (1) ◽  
pp. 79-90 ◽  
Author(s):  
Laurent Galois ◽  
Sandrine Hutasse ◽  
Delphine Cortial ◽  
Cécile F. Rousseau ◽  
Laurent Grossin ◽  
...  

2010 ◽  
Vol 47 (2) ◽  
pp. 114-124 ◽  
Author(s):  
Shinsaku Togo ◽  
Tadashi Sato ◽  
Hisatoshi Sugiura ◽  
Xingqi Wang ◽  
Hesham Basma ◽  
...  

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