The Study Of Fign Depletion On Rat Astrocytes Migration

Objective Microtubule polymetization and depolymerization are essential for many cellular events including mitosis,migration and membrane skeletal reorganization.Fidgetin(Fign)is a microtubule-dependent severing protein that can contribute to chromosome separation via regulating microtubule dynamics.Previous studies have found that Fign protein is still expressed in terminally post-mitotic neurons,where they participate in neuronal development by regulate microtubule movements and organization.However,it hangs in doubt about Fign's functional location,manner and the molecular mechanism,and even less is known whether Fign expresses in other cell types of the nervous system.In this study,we aim to address the effects and molecular mechanism of Fign depletion from cultured astrocytes of rat spinal cord on cell migration.Methods The total proteins were extracted from spinal cord tissues during different time points of rat postnatal development,and Western blotting was used to detect the protein level of Fign and Spastin,two microtubule severing proteins.The cultured astrocytes were obtained from the spinal cord tissues of new born rats(postnatal day1),then were purified and subcultured.The second generation astrocytes were transfected by Fign si RNA.After 3 days,astrocytes proliferation was evaluated by Ed U test,and astrocytes migration was evaluated by Transwell assay,wound-healing assay and live image methods.The q RT-PCR,Western Blotting and Immunofluorescence(IF)methods were used to investigate the Fign location,m RNA and protein level in astrocytes.We examined the microtubule pattern and post-translational modification of tubulin in astrocytes by si RNA/IF and Overexpression/IF.Results1.By Western Blotting test,we found the expression of Fign and Spastin in spinal cord gradually decreased following rat postnatal development.2.Compared to control siRNA,the cultured astrocytes migration was remarkably inhibited after Fign si RNA treatment for 3 days,while astrocytes proliferation was not changed significantly.3.Immunofluorescence results showed that the ratio of cells with curled microtubule ends at the cell cortical region was increased to0.26 ± 0.03 after Fign si RNA treatment,while in control si RNA treated astrocytes,the ratio was 0.96±0.03.4.The mass of tyrosinated-tubulin in cultured astrocytes was increased after Fign si RNA treatment,while the mass of tyrosinated-tubulin in cultured astrocytes was decreased after Fign gene overexpression.5.Previously,we have addressed Kinesin-12 depletion could significantly increase astrocytes migration.In this study,we found when knockdown Fign and Kinesin-12 expression,the inhibition of astrocytes migration was mitigated.ConclusionsThe expression of Fign and Spastin proteins decrease significantly following rat postnatal development.This result may interpret that microtubule stablility increase during rats mature.In the present study,in primary cultured astrocytes,Fign protein preferably severs labile tyrosinated-tubulin.Knocking down Fign expression decreased astrocytes migration notably,and increase the ratio of cells with curled microtubule ends at the cell cortical region remarkably.Loss of microtubule severing function may increase microtubule outgrowth,and result in microtubules tend to curl at cell cortical region,extending along the cell membrance.Previously,we have addressed Kinesin-12 depletion could significantly decrease mictotubule/microfilament dynamics since Kinesin-12/myosin IIB heteropolymer could link mictotubule andmicrofilament.Therefore,when knockdown Fign and Kinesin-12 expression,more microtubule could be perpendicular to the cell edge,the inhibition of astrocytes migration was particially mitigated.