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, 331 (2), 331-7

Determining the Mechanical Properties of Plectin in Mouse Myoblasts and Keratinocytes

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Determining the Mechanical Properties of Plectin in Mouse Myoblasts and Keratinocytes

Navid Bonakdar et al. Exp Cell Res.

Abstract

Plectin is the prototype of an intermediate filament (IF)-based cytolinker protein. It affects cells mechanically by interlinking and anchoring cytoskeletal filaments and acts as scaffolding and docking platform for signaling proteins to control cytoskeleton dynamics. The most common disease caused by mutations in the human plectin gene, epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), is characterized by severe skin blistering and progressive muscular dystrophy. Therefore, we compared the biomechanical properties and the response to mechanical stress of murine plectin-deficient myoblasts and keratinocytes with wild-type cells. Using a cell stretching device, plectin-deficient myoblasts exhibited lower mechanical vulnerability upon external stress compared to wild-type cells, which we attributed to lower cellular pre-stress. Contrary to myoblasts, wild-type and plectin-deficient keratinocytes showed no significant differences. In magnetic tweezer measurements using fibronectin-coated paramagnetic beads, the stiffness of keratinocytes was higher than of myoblasts. Interestingly, cell stiffness, adhesion strength, and cytoskeletal dynamics were strikingly altered in plectin-deficient compared to wild-type myoblasts, whereas smaller differences were observed between plectin-deficient and wild-type keratinocytes, indicating that plectin might be more important for stabilizing cytoskeletal structures in myoblasts than in keratinocytes. Traction forces strongly correlated with the stiffness of plectin-deficient and wild-type myoblasts and keratinocytes. Contrary to that cell motility was comparable in plectin-deficient and wild-type myoblasts, but was significantly increased in plectin-deficient compared to wild-type keratinocytes. Thus, we postulate that the lack of plectin has divergent implications on biomechanical properties depending on the respective cell type.

Keywords: Cell motility; Cell stretching; Intermediate filaments (IF); Magnetic tweezer rheology; Mouse myoblasts and keratinocytes; Plectin; Traction force microscopy.

Figures

Fig. 1
Fig. 1
Cell stretcher. Schematic representation (A) and photographic image (B) of the cell stretcher device. Cells were plated on an elastomeric PDMS-membrane coated with the extracellular matrix–protein fibronectin. The cell stretcher was attached to an inverted microscope to observe cell behavior during uniaxial, cyclic stretch. The membrane was stretched up to 30% by a linear motor. (C) Statistical analyses of dead plectin+/+ and plectin−/− myoblasts and keratinocytes after cyclic stretch. A total of 40–60 cells were measured per cell type; bars represent mean±SE. p<0.05 from unpaired Student׳s t-test.
Fig. 2
Fig. 2
Magnetic tweezer device. Schematic representation (A) and photographic image (B) of the magnetic tweezer setup. A high magnetic field gradient was generated by a needle-shaped solenoid bound to a micromanipulator. The gradient force generated by the magnetic tweezer acted on fibronectin-coated paramagnetic beads. (C) Statistical analyses of cellular stiffness of plectin+/+ and plectin−/− myoblasts and keratinocytes. A total of 28–30 cells were measured per cell type; data represent mean±SE. p<0.05 and ⁎⁎p<0.01 from unpaired Student׳s t-test.
Fig. 3
Fig. 3
Analyses of bead displacement measurements in response to force ramps up to 80 nN. (A) Statistical analysis of cellular binding (adhesion) strength of plectin+/+ and plectin−/− myoblasts and keratinocytes, respectively. (B) Statistical analysis of cell fluidity, that is cellular dynamics (β-value). Data in (A) and (B) represent mean±SE; p<0.05 and ⁎⁎p<0.01 from unpaired Student׳s t-test. The number of cells analyzed was n=26–28 for myoblasts and n=54–56 for keratinocytes, respectively.
Fig. 4
Fig. 4
Traction force microscopy. (A) Schematic diagram of the setup. (B) Traction map with bright field image (inset) of a plectin+/+ and plectin−/− myoblast, respectively. Bars, 20 µm. (C) Statistical analysis of the strain energy normalized to the spreading area. Cells (n=15–21) were measured per cell type; the data represent mean±SE. p<0.05 from unpaired Student׳s t-test.
Fig. 5
Fig. 5
Analyses of cell motility. (A) Representative images from time lapse movies over 5 h. In red, the trajectories of individual myoblasts are displayed. Bars, 20 µm. (B) Statistical analysis of cell motility. The mean square displacement (MSD) was determined from trajectories of plectin−/− and plectin+/+ cells. For each cell line, the trajectories of more than 500 individual cells were evaluated; the data represent mean±SE. ⁎⁎p<0.01 from unpaired Student׳s t-test.

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References

    1. Wiche G., Winter L. Plectin isoforms as organizers of intermediate filament cytoarchitecture. Bioarchitecture. 2011;1:14–20. - PMC - PubMed
    1. Castañón M.J., Walko G., Winter L., Wiche G. Plectin-intermediate filament partnership in skin, skeletal muscle, and peripheral nerve. Histochem. Cell Biol. 2013;140:33–53. - PMC - PubMed
    1. Rezniczek G.A., Walko G., Wiche G. Plectin gene defects lead to various forms of epidermolysis bullosa simplex. Dermatol. Clin. 2010;28:33–41. - PubMed
    1. Winter L., Wiche G. The many faces of plectin and plectinopathies: pathology and mechanisms. Acta. Neuropathol. 2013;125:77–93. - PubMed
    1. Konieczny P., Fuchs P., Reipert S., Kunz W.S., Zeold A., Fischer I., Paulin D., Schröder R., Wiche G. Myofiber integrity depends on desmin network targeting to Z-disks and costameres via distinct plectin isoforms. J. Cell Biol. 2008;181:667–681. - PMC - PubMed

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