Role of osmotic pressure in the regulation of cytoskeleton during mitosis
Date of Issue2016
School of Biological Sciences
The dynamic actin cytoskeleton plays a pivotal role towards the regulation of various cellular functions, including cell motility and division. Adherent cells, which are usually flat during interphase assume a spherical shape at the onset of mitosis. This rounding primarily involves de-adhesion from the substrate and cortical retraction. The mitotic cell rounding is essential for the proper positioning and stabilization of the mitotic spindle and ensures correct chromosome segregation. The actin filaments, present at the cell periphery during interphase, undergo realignment and distribution to the spherical cortex during mitosis. The interaction of the cortical actin with the astral microtubules that radiate towards the cell cortex from the spindle poles generates tensile forces, thus favoring the rounded morphology. In addition to this inward contraction of the actomyosin network, an outward osmotic pressure also governs the mitotic cell rounding. Cells experience an increase in the hydrostatic pressure at the onset of mitosis. A rise in the intracellular osmotic pressure results in an increased cell volume and rounding pressure. A balance between the outward directed osmotic pressure and the inward actomyosin contraction is instrumental in driving the mitotic cell rounding. Earlier work from our lab has shown that an intact actin cytoskeleton is required for proper spindle assembly at the early stage of mitosis. Perturbation of the actin cytoskeleton resulted in a less-defined mitotic spindle with an aberrant increase in astral microtubules emanating from the spindle pole towards the cortex. Cells treated with inhibitors of Rho GTPase pathway to perturb the actin cytoskeleton showed centrosome defocusing and spindle misorientation, thereby suggesting that actin cytoskeleton plays an essential role in mitotic spindle assembly. Perturbation of the osmotic gradient also led to changes in rounding pressure and volume. Failure of the cells to round up is associated with defects in spindle assembly, pole splitting and a delay in progression. Since reduction of the osmotic pressure is associated with decrease in rounding pressure and volume and defects in cell rounding, we hypothesized that hypertonic stress could have adverse effects on the establishment of a bipolar spindle. Aim of this thesis was to investigate the effect of osmotic stress on cell cycle progression. More precisely, we examined the role of osmotic stress on the mitotic progression and metaphase spindle assembly. Our results show an arrest at the G2/M phase and a decreased expression of cyclin B1 and cyclinD1 protein levels under conditions of hypertonic stress. Further, we observed a delay in the mitotic progression of cells upon hypertonic stimulation. Besides possible defects in cytokinesis, cells displayed a prolonged metaphase arrest when subjected to hypertonic shock. Thus, our results imply the existence of a possible link between the osmotic pressure and cell cycle progression. We observed an arrest during metaphase, as shown by time-lapse microscopy and therefore we proceeded to study the effects of osmotic imbalance on metaphase spindle assembly. We observed defects in the astral microtubule arrangement and in misorientation of the mitotic spindle upon hypertonic challenge. The increase in the astralmicrotubule intensity was proportional to the duration of the hyperosmotic shock and the impaired astral microtubule arrangement was restored to normal when the cells were recovered in an isotonic medium, following a hypertonic shock. The ERM (Ezrin, Radixin, Moesin) family of proteins, known to cross-link the plasma membrane with the underlying actin cortex, are essential for mitotic cell rounding. Previous reports suggest the ERM proteins, upon phosphorylation and activation during mitosis redistribute towards the cell cortex, thereby contributing to cortical rigidity. Our studies have shown that hypertonic stress upregulates phospho-ERM protein levels with a concomitant increase in the cortical rigidity of mitotic cells. Hyperactivation of moesin also results in an aberrant increase in astral microtubule arrangement and spindle misorientation. Thus, our work suggests that hypertonicity-induced ERM phosphorylation might be responsible for the observed defects in metaphase assembly. In order to further elucidate the mechanistic details behind the observed phenotypic defects in the spindle assembly, we designed a model based on the fact that hypertonic stress results in a higher diffusion rate. The model predicts that a perturbation of the RanGTP gradient under hypertonic stress owing to the higher diffusion rate. As a result, the cortical recruitment of the proteins required for spindle assembly, namely Gα, LGN, NuMA and dynein/dynactin will be impaired. This atypical localization of the proteins is responsible for the spindle angle defects. Our experimental results corroborate the model as we observe a reduced cortical distribution of these proteins under conditions of hypertonic stress, thereby providing a possible explanation for the defects in mitotic spindle orientation. In conclusion, our study shows that osmotic balance is required for timely progression through the cell cycle and disruption of the osmotic gradient is responsible for delay in mitotic progression due to defects in the bipolar metaphase spindle assembly, characterized by an aberrant increase in astral microtubules and defects in spindle orientation. One possible reason behind these phenotypic defects might be the hyper activation of ERM proteins since moesin phosphorylation is implicated in the defects observed in mitotic spindle apparatus. Besides, we propose a second mechanism wherein the hypertonicity-induced aberrant cortical localization of several proteins associated with spindle orientation explains the observed impairment in astral microtubules and spindle assembly induced by hypertonic stress.
DRNTU::Science::Biological sciences::Molecular biology