Calcium-responsive Transactivator (CREST) Promotes Neurite Growth By Increasing Microtubule Stability And Dynamic Assembly

The calcium-responsive transactivator(CREST)is a nuclear protein highly expressed in post-mitotic neurons during neurodevelopment.It affects the development of neuronal dendrites by regulating calcium-dependent gene transcription,and involved in maintaining the morphology of mature neurons.CREST-knockout mice showed significant dyskinesia,abnormal dendritic growth and reduced branching.Previous studies in our laboratory found that the expression of CREST increased during the differentiation of mouse neuroblastoma cells(N2a cells)induced by serum-free medium,and distributed in the cytoplasm in a granular manner.Overexpression of CREST can promote neurite growth and neuronal differentiation by activating the transcription of genes related to neuronal differentiation.Neuronal differentiation is based on the growth and branching of neurites.The assembly and extension of microtubules in neurites is the skeleton support of neurite growth.The defects of microtubule development will cause abnormal morphology and function of nervous system.The formation and growth of neurites depend on the synergistic regulation of the stability and dynamics of microtubule skeletons by various microtubule interacting proteins,they regulate the assembly,stability,depolymerization or rupture of microtubules by binding to microtubule skeletons.The granular distribution of cytoplasm in the process of neuronal differentiation suggests that CREST may have cytoplasmic function and regulate the growth of neurites through the direct influence of microtubule cytoskeleton.In this study,the interaction characteristics between CREST and ?-tubulin as well as their molecular regions of interaction were analyzed by immunofluorescence and immunoprecipitation experiments.Through the morphological observation of the microtubule skeleton,the detection of the stability of the microtubule and the dynamic observation of the plus end assembly of the microtubule,the molecular mechanism of CREST promoting the neurite growth by regulating the stability and dynamics of the microtubule was deeply analyzed.CREST interacts with ?-tubulin middle region through its MFDIn order to determine whether the granular distribution of CREST in cytoplasm is related to microtubules,the distribution of endogenous CREST and ?-tubulin in N2 a cells was detected by immunofluorescence double labeling.The results showed that in undifferentiated N2 a cells,CREST was mainly distributed in the nucleus,?-tubulin was dispersed in the cytoplasm,there were fewer assembled(fibrous)microtubules,and only a small number of CREST particles were co-located with fibrous microtubules in the cytoplasm.In serum-free differentiated N2 a cells,a large number of ?-tubulin were assembled into fibrous microtubules,and the immunoreactivity of CREST in cytoplasm was significantly enhanced,showing a good co-localization and distribution relationship between granular and microtubule skeleton.Immunoprecipitation analysis showed that endogenous CREST and ?-tubulin interacted in the cerebral cortex of mice one week after birth and in serum-free differentiated N2 a cells.In order to further clarify the molecular region of the interaction between CREST and ?-tubulin,we used GFP and mCherry fluorescent protein as tags to construct the full-length of pEGFP-CREST and mCherry-tubulin and their respective structural deletion mutants,respectively.The patients were divided into groups for immunoprecipitation test,The results showed that CREST binds to the intermediate segment of ?-tubulin(132-227aa)through its multifunctional domain MFD(246-317aa).CREST increases the stability of microtubulesIn order to determine whether the binding of CREST to microtubules has an effect on the stability of microtubules,we first detected the assembly and fibrosis of microtubules during neurite growth of N2 a cells by immunofluorescence.The results showed that ?-tubulin aggregated into fibrous microtubule bundles in N2 a cells induced by serum-free culture or overexpressed CREST,while the formation of fibrous microtubule bundles is rare in cytoplasm in N2 a cells which were not differentiated or transfected with control plasmids.The acetylation and detyrosine modification levels of ?-tubulin(labeling of stable microtubules)were detected by Western blot.The results showed that serum-free differentiation or overexpression of CREST could increase the acetylation and detyrosine modification of ?-tubulin in N2 a cells.However,the above two modification levels of ?-tubulin were significantly decreased in CREST silenced cells.These results suggest that CREST increases the stability of microtubules by interacting with ?-tubulin during neurite growth.CREST is a new microtubule-associated protein(MAP).CREST increases dynamic assembly of microtubulesThe growth of neurites depend on the close coordination of microtubule stability and dynamics.A completely stable microtubule is not conducive to protuberance growth,and the dynamic assembly of the plus end of the stable microtubule skeleton is the key to neurites elongation.In order to dynamically observe the effect of CREST on the dynamics of microtubule end,we constructed a GFP-EB1 plasmid which can trace the dynamics of microtubule end.After transfected into N2 a cells,the number and movement speed of GFP-EB1 fluorescence points were tracked by laser confocal microscope.The growth of microtubules in neurites was recorded and analyzed.the results showed that in N2 a cells without serum-induced differentiation or overexpression of CREST,the number of GFP-EB1 fluorescent spots was significantly increased and the movement speed was significantly accelerated,indicating that the number of growing microtubules as well as the growth rate of microtubules was increased.Overexpress CREST in serum-free differentiated N2 a cells,the number of growing microtubules and the growth rate of microtubules in the neurites were both increased.However,the silencing of CREST in serum-free differentiated N2 a cells resulted in a significant decrease in the number of growing microtubules in the neurite and a significant decrease in the rate of terminal growth.These results suggest that CREST can promote the dynamic assembly of the microtubule.Conclusion: CREST is a new microtubule associated protein,which is located in the cytoplasm in the process of neuronal differentiation and interacts with the intermediate segment of ?-tubulin through its MFD domain.CREST increases the stability of microtubules by combining with ?-tubulin,and promotes the dynamic assembly at the plus end of microtubules in neurites,thus promoting the growth of neurites.