The Mechanism Of Palladin-mediated Proper Spindle Orientation In Mitosis

Palladin is known as an actin-binding protein and molecular scaffold protein involved in the regulation of microfilament dynamics in interphase cells,and play a key role in cell motility,the interactions between cells and cells,or cells and extracellular matrix.However,its role in mitosis remains largely unknown.Our study showed that besides its cell cortical localization,palladin also located at microtubule based structures like mitotic spindles and centrosomes at metaphase in He La cells.Therefore,palladin was a tubuliin-associated protein and interacted with?-tubulin via its 3^rd Ig C domian.After excluding the possiblity that palladin knockdown-induced spindle misorientation caused by abnormal connection between chromosomes and microtubules,prolonged division time,and adhesion defects,we demonstrated palladin as a new regulator of spindle orientation,to sustain proper spindle orientation via stabilizing astral microtubules rather than keeping the integrity of F-actin structures.Further investigation showed that palladin knockdown-induced instability of astral microtubules was because of its defects in cell cortical anchoring,but not related to abnormal microtubules regrowth or defeted centrosomes nucleation.Proper spindle orientation contributes to well-oriented cell division.We found that palladin knockdown caused cell division misorientation in cultured He La cells.In line with this,radial glial progenitors displayed defected cell division orientation in the brain of E12.5 Palld-/-embryo compared with Palld+/+embryo.Palladin asscociated with AKT1 at metapahse in He La cells,and palladin knocdown induced the downregualtion of p AKT T308,p AKT S473,and GSK3?S9 as compared to scramble cells.Hence,palladin directly regulated AKT1activation,in turn inhibited GSK3?function at metaphase.AKT1-GSK3?maintains cell cortical anchoring of astral microtubules to stabilize them.Spindle misorientation and astral microtubule instability caused by palladin knockdown could be rescued by overexpression of constitutively activated AKT1(AKT1 CA)or dominant negative GSK3?(GSK3?DN),on the contray,scramble cells with enforced expression of dominant negative AKT1(AKT1 DN)or constitutively activated GSK3?(GSK3?CA)displayed similar phenotypes to Palladin knockdown cells.Therefore,palladin promoted astral microtubule cell cortical anchoring to stabilize it via AKT1-GSK3?pathway.In conclusion,palladin is critical for the maintenance of spindle orientation via AKT1-GSK3?pathway.