Effects Of Myosin II Absence On The Cytokinesis Of Adherent Mammalian Cells

Cytokinesis is a complicated life process involved biology and mechanics. Literature findings demonstrated a diversity of mechanisms by which animal cells carried out cytokinesis. Cytokinesis mode B was a cell cycle-coupled, adhesion-dependent cytokinesis that did not depend on myosin II. This cytokinesis mode existed universally in adherent eukaryotes and higher animal cells and might be a more primary cytokinesis mode than classic contractile ring-depended model. However, the mechanism of cytokinesis mode B was still not clear. Since not only adherent eukaryotes but also higher animal cells employed cytokinesis mode B, it must play an important role in cell division. Understanding the mechanism of cytokinesis mode B will not only provide insight into how the multiple modes of division worked in cooperative manner from a comprehensive view, but also provide theoretical evidence for revealing the mystery of cell proliferation and disease related cytokinesis failure (such as cancer).The previous studies of the mechanism of cytokinesis B were mainly focus on Dictyostelium cells. However, such investigation has not been done on the mammalian cells. In the present study, Micro-image dynamic collecting and analysis system, immunofluorescence method and cell mechanics techniques were used to explore the effects of myosin II absence on cytokinesis using NRK cells. Our findings provided the first direct evidences that cell material mechanical properties and cell motility orchestrated to dictate a well-controlled cytokinesis mode B of adherent mammalian cells. The main work and conclusions are as follows: 1. First, the cytokinesis process of normal NRK cells was observed and described in detail. The possible mechanism of blebbing behavior and the relation between the daughter cells adhesion pattern and the mother cell at interphase were discussed. Second, cell surface tension and vicroelastic properties of NRK cells at interphase were determined using micropipette aspiration technique and its relative theoretical model. The change of force and strrss in dividing furrow regions during cytokinesis were analyzed according to the morphology parameters,cell mechanical properties and myosin II motor work.2. The behavior of NRK cells absence of myosin II undergoing division was explored. The Micro-images of NRK cells under division were collected and analyzed after myosin II inhibitor was released at metaphase and anaphase respectively. The distribution of actin at anaphase and early cytokinesis were examined using immunofluorescence method. Our findings showed that:(1) Myosin II absence neither affected the lamellipodia motion of NRK cells at interphase nor assembly and recruitment of actin to the dividing furrow.(2) Inhibition of myosin II activity at metaphase resulted in failure of karyokinesis or delay into cytokinesis. The lamellipodia motion of daughter cells lost their polarity.(3) The results of intercellular bridge thinning morphology analysis showed that whether in the control or NRK cells absence of myosin II, the trajectories of intercellular bridge thinning behaved a combination of a linear decrease and an exponential decay, with the exponential decay preceding the linear decrease. The intercellular bridge trajectories of cells absence of myosin II was obvious differently from the controls with relative diameter decrease more quickly before Dx point, while the trajectories were identical after Dx point.3. The role of cell material properties on the cytokinesis absence of myosin II was in investigated using the existed intercellular bridge thinning mechanics model. The intercellular bridge thinning trajectories of the control and NRK cells absence of myosin II were analyzed respectively. Intercellular bridge thinning morphology parameters and cell mechanical properties from experiments were employed during analysis. The results showed that cell surface tension contributed greatly to the intercellular bridge thinning trajectories. It indicated that internal pressure difference between the intercellular and the two daughters could help NRK cells absence of myosin II to complete cytokinesis.A new intercellular bridge thinning dynamics model was established on the base of fluid mechanics principle. This model can allow the parameters of morphology or mechanics to vary temporally or spatially as intercellular bridge-thinning proceeded.4. The role of cell motility in myosin II absent cytokinesis was investigated using cyto-immunofluorescence method and actin flament polymerization inhibitor (cytohalasin D). The results showed that a mass of actin filament recruited to the leading lamellipodia of daughter cells from the cytoplasm in cells treated with myosin II inhibitor. The process of cytokinesis absence of myosin II was obviously held back by releasing cytohalasin D. The relative pole to pole diatance decreased in contrast with other groups. Our results provided the first evidence that cell motility was involved the cytokinesis absence of myosin II.Our results indicated that the force required in cytokinesis might obviously reduce because the whole cell surface tension decreased during cytokinesis of adherent NRK cells absence of myosin II. Traction force generated from daughter cells motility not only facilitated two daughter cells separate, but also led to the surface tension increase in intercellular bridge, which resulted in the internal pressure difference increase between the intercellular bridge and two daughter cells, the latter accelerated the cytoplasm flow from the intercellular bridge into two daughter cells. While the intercellular bridge thinning further facilitated internal pressure difference effect to help the cytokinesis of adherent NRK cells absence of myosin II.