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Mouse Keratinocytes Without Keratin Intermediate Filaments Demonstrate Substrate Stiffness Dependent Behaviors

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Abstract

Introduction

Traditionally thought to serve active vs. passive mechanical functions, respectively, a growing body of evidence suggests that actin microfilament and keratin intermediate filament (IF) networks, together with their associated cell–cell and cell–matrix anchoring junctions, may have a large degree of functional interdependence. Therefore, we hypothesized that the loss of keratin IFs in a knockout mouse keratinocyte model would affect the kinematics of colony formation, i.e., the spatiotemporal process by which individual cells join to form colonies and eventually a nascent epithelial sheet.

Methods

Time-lapse imaging and deformation tracking microscopy was used to observe colony formation for both wild type (WT) and keratin-deficient knockout (KO) mouse keratinocytes over 24 h. Cells were cultured under high calcium conditions on collagen-coated substrates with nominal stiffnesses of ~ 1.2 kPa (soft) and 24 kPa (stiff). Immunofluorescent staining of actin and selected adhesion proteins was also performed.

Results

The absence of keratin IFs markedly affected cell morphology, spread area, and cytoskeleton and adhesion protein organization on both soft and stiff substrates. Strikingly, an absence of keratin IFs also significantly reduced the ability of mouse keratinocytes to mechanically deform the soft substrate. Furthermore, KO cells formed colonies more efficiently on stiff vs. soft substrates, a behavior opposite to that observed for WT keratinocytes.

Conclusions

Collectively, these data are strongly supportive of the idea that an interdependence between actin microfilaments and keratin IFs does exist, while further suggesting that keratin IFs may represent an important and under-recognized component of keratinocyte mechanosensation and the force generation apparatus.

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Acknowledgments

Support of this work was provided by the National Science Foundation (National Science Foundation CAREER CMMI 1452728) and the Carver Charitable Trust #14-4384 and #18-5045. In addition, J.C.S. acknowledges the Dermatology Foundation for their support of this work through a career development award. Work in the Magin lab is supported by the DFG (German Research Council; MA1316-15, MA1316-17, MA1316-19, MA1316-21, INST 268/230-1).

Conflict of interest

Hoda Zarkoob, Sathivel Chinnathambi, Spencer A. Halberg, John C. Selby, Thomas M. Magin, and Edward A. Sander declare that they have no conflict of interest.

Ethical Standards

No human or animal studies or were carried out by the authors for this article.

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Authors and Affiliations

  1. Department of Biomedical Engineering, College of Engineering, University of Iowa, 5629 Seamans Center, Iowa City, IA, USA

    Hoda Zarkoob, Sathivel Chinnathambi, Spencer A. Halberg & E. A. Sander

  2. Department of Dermatology, Carver College of Medicine, University of Iowa, Iowa City, IA, USA

    John C. Selby

  3. Division of Cell and Developmental Biology and SIKT, Institute of Biology, University of Leipzig, 04103, Leipzig, Germany

    Thomas M. Magin

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  1. Hoda Zarkoob
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Correspondence to E. A. Sander.

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Associate Editor Michael R. King oversaw the review of this article.

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Zarkoob, H., Chinnathambi, S., Halberg, S.A. et al. Mouse Keratinocytes Without Keratin Intermediate Filaments Demonstrate Substrate Stiffness Dependent Behaviors. Cel. Mol. Bioeng. 11, 163–174 (2018). https://doi.org/10.1007/s12195-018-0526-y

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