ABSTRACT
Neuronal polarity is involved in multiple developmental stages, including cortical neuron migration, multipolar-to-bipolar transition, axon initiation, apical/basal dendrite differentiation, and spine formation. All of these processes are associated with the cytoskeleton and are regulated by precise timing and by controlling gene expression. The P-Rex1 (phosphatidylinositol-3,4,5-trisphosphate dependent Rac exchange factor 1) gene for example, is known to be important for cytoskeletal reorganization, cell motility, and migration. Deficiency of P-Rex1 protein leads to abnormal neuronal migration and synaptic plasticity, as well as autism-related behaviors. Nonetheless, the effects of P-Rex1 overexpression on neuronal development and higher brain functions remain unclear. In the present study, we explored the effect of P-Rex1 overexpression on cerebral development and psychosis-related behaviors in mice. In utero electroporation at embryonic day 14.5 was used to assess the influence of P-Rex1 overexpression on cell polarity and migration. Primary neuron culture was used to explore the effects of P-Rex1 overexpression on neuritogenesis and spine morphology. In addition, P-Rex1 overexpression in the medial prefrontal cortex (mPFC) of mice was used to assess psychosis-related behaviors. We found that P-Rex1 overexpression led to aberrant polarity and inhibited the multipolar-to-bipolar transition, leading to abnormal neuronal migration. In addition, P-Rex1 overexpression affected the early development of neurons, manifested as abnormal neurite initiation with cytoskeleton change, reduced the axon length and dendritic complexity, and caused excessive lamellipodia in primary neuronal culture. Moreover, P-Rex1 overexpression decreased the density of spines with increased height, width, and head area in vitro and in vivo. Behavioral tests showed that P-Rex1 overexpression in the mouse mPFC caused anxiety-like behaviors and a sensorimotor gating deficit. The appropriate P-Rex1 level plays a critical role in the developing cerebral cortex and excessive P-Rex1 might be related to psychosis-related behaviors.
ABSTRACT
Objective To investigate the role of cortactin in migration and invasion of U251 glioma cells and role of Rac1 activation in this process.Methods Human glioma U251 cells were cultured in vitro.The expressions and distributions of Rac1 and cortactin in U251 glioma cells were detected by immunofluorescence.U251 glioma cells assigned into 4 treatment groups:siRNA-cortactin group (transfected by siRNA specific cortactin),siRNA-NC group (transfected by negative control RNA sequence),siRNA-N group (transfected by empty vector) and siRNA-cortactin+Rac1 group (transfected by siRNA specific cortactin and Rac1 inhibitor).Forty-eighty h after grouping and each treatment,the protein expressions of cortactin and Rac1 in the 4 groups were detected by Western blotting;the migration and invasion of glioma cells were evaluated by wound-healing and Transwell-chamber invasion assays;the lamellipodia of glioma cells was observed by immunofluorescence.Results Cortactin and Rac1 were co-localized in the front ofglioma cells,where actin was polymerized and lamellipodia was formed.As compared with siRNA-NC group and siRNA-N group,siRNA-cortactin group and siRNA-cortactin+Rac1 group had significantly lower cortactin and Rac1 expressions (P＜0.05);siRNA-cortactin+Rac1 group had significantly lower cortactin and Rac1 expressions as compared with siRNA-cortactin group (P＜0.05).As compared with siRNA-NC group and siRNA-N group,siRNA-cortactin group and siRNA-cortactin+Rac1 group had significantly smaller healing areas and number of perforator cells (P＜0.05);siRNA-cortactin+Rac1 group had significantly smaller healing areas and number of perforator cells as compared with siRNA-cortactin group (P＜0.05).As compared with siRNA-NC group and siRNA-N group,siRNA-cortactin group and siRNA-cortactin+Rac1 group had decreased lamellipodia of glioma cells;siRNA-cortactin+Rac1 group had decreased lamellipodia of glioma cells as compared with siRNA-cortactin group.Conclusion Cortactin can promote the migration and invasion of glioma cells by regulating lamellipodia formation;combined inhibition of Rac 1 and cortactin may be an effective mean for treatment ofglioma.
ABSTRACT
Molecular mechanisms underlying the effects of Fyn on cell morphology, pseudopodium movement, and cell migration were investigated. The Fyn gene was subcloned into pEGFP-N1 to produce pEGFP-N1-Fyn. Chinese hamster ovary (CHO) cells were transfected with pEGFP-N1-Fyn. The expression of Fyn mRNA and proteins was monitored by reverse transcription-PCR and Western blotting. Additionally, transfected cells were stained with 4',6-diamidino-2-phenylindole and a series of time-lapse images was taken. Sequences of the recombinant plasmids pMD18-T-Fyn and pEGFP-N1-Fyn were confirmed by sequence identification using National Center for Biotechnology Information in USA, and Fyn expression was detected by RT-PCR and Western blotting. The morphology of CHO cells transfected with the recombinant vector was significantly altered. Fyn expression induced filopodia and lamellipodia formation. Based on these results, we concluded that overexpression of mouse Fyn induces the formation of filopodia and lamellipodia in CHO cells, and promotes cell movement.