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Insights into the Roles of Non-Muscle Myosin IIA in Human Keratinocyte Migration

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Abstract

Epidermal cell migration is a key factor in wound healing responses, regulated by the F-actin–myosin II systems. Previous reports have established the importance of non-muscle myosin II (NMII) in regulating cell migration. However, the role of NMII in primary human keratinocytes has not been investigated. In this study, we used a microfabrication-based two-dimensional migration assay to examine the role of NMII in keratinocyte migration. We developed confluent cell islands of various sizes (0.025–0.25 mm2) and quantified migration as Fold Increase in island area over time. We report here that NMII was expressed and activated in migrating keratinocytes. Inhibition of NMIIA motor activity with blebbistatin increased migration significantly in all cell island sizes in six hours compared to control. Inhibition of Rho-kinase by Y-27632 did not alter migration while inhibition of myosin light chain kinase by ML-7 suppressed migration significantly in six hours. Both blebbistatin and Y-27632 induced formation of large membrane ruffles and elongated tails in keratinocytes. In contrast, ML-7 blocked cell spreading, resulting in a rounded morphology. Taken together, these data suggest that NMIIA decreases migration in keratinocytes, but the mechanism may be differentially regulated by upstream kinases.

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Acknowledgments

We thank Drs. Venkaiah Betapudi and James Crish (Cleveland Clinic Foundation) for generous assistance during the early stages of immunocytochemistry. We are grateful to Drs. Judith Drazba and John Peterson (Cleveland Clinic Foundation) for confocal microscopy support. The above work was funded in part by grants EB0062033 (H.B.) and GM0077224 (T.T.E.), from the National Institutes of Health.

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Authors and Affiliations

  1. Department of Biomedical Engineering, Case Western Reserve University, 111C A.W. Smith Building, Cleveland, OH, 44106-7217, USA

    Saheli Sarkar & Harihara Baskaran

  2. Department of Chemical Engineering, Case Western Reserve University, Cleveland, OH, 44106, USA

    Harihara Baskaran

  3. Department of Cell Biology, Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195, USA

    Thomas Egelhoff

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  1. Saheli Sarkar
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  2. Thomas Egelhoff
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Correspondence to Harihara Baskaran.

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Figure S1

RLC phosphorylation in keratinocyte island. A typical 0.025 mm2 cell island was stained for pRLC expression (green) and F-actin (red). RLC phosphorylation was observed primarily at the island edges and at cell margins not in contact with other cells. Scale bar: 20 μm (1,438 KB TIF)

Figure S2

Expression of NMIIB in keratinocytes. Keratinocytes plated on fibronectin were allowed to migrate for 6 h. Cells were double-stained for NMIIB (green) and F-actin (red). Cell nuclei were stained with DAPI (blue). NMIIB was expressed at the cell periphery. Scale bar: 20 μm (1,325 KB TIF)

Figure S3

Quantification of island migration on untreated surfaces in the presence of inhibitors. Confluent cell islands were treated with blebbistatin (30 μM), ML-7 (10 μM), or Y-27632 (5 μM) for 6 h. Migration is shown as normalized Fold Increase in island area after 6 h. Blebbistatin induced significant increase in keratinocytes migration (p < 0.05). TC: Tissue Culture (2,168 KB TIF)

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Sarkar, S., Egelhoff, T. & Baskaran, H. Insights into the Roles of Non-Muscle Myosin IIA in Human Keratinocyte Migration. Cel. Mol. Bioeng. 2, 486–494 (2009). https://doi.org/10.1007/s12195-009-0094-2

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