Department of Physics, CFisUC, Center of Physics of the University of Coimbra, University of Coimbra, Coimbra, Portugal.
AdriaBio Llc, Postojna, Slovenia.
PLoS One. 2021 Dec 28;16(12):e0261227. doi: 10.1371/journal.pone.0261227. eCollection 2021.
We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.
我们研究了角蛋白聚集的形成以及可能涉及的机制。为此，我们观察了在体外培养的角质形成细胞中不同比例的突变型和野生型角蛋白表达可能对角蛋白聚集形成的影响，以及角蛋白聚集形成如何影响细胞皮质处细胞的机械性能。为此，我们制备了表达率尽可能接近 25%、50%和 100%的 EGFP-K14 蛋白（WT 或 R125P 和 V270M 突变体）的克隆。我们的结果表明，只有在 EGFP-K14 R125P 突变体的 25%的情况下才会出现显著差异。具体来说，在这种情况下，我们观察到角蛋白聚集的最大积累和细胞刚度的显著降低。为了深入了解这一观察结果背后的可能机制，我们通过实施更复杂的反应网络来扩展我们之前的角蛋白动力学数学模型，该网络考虑了细胞中野生型和突变型角蛋白的共存。新模型由一组耦合的非线性常微分方程组成，使我们能够得出关于细胞中间丝和聚集体相对含量的结论，并表明野生型和突变型角蛋白之间不对称结合导致的聚集体形成可以解释在培养中生长的细胞获得的数据。
