Investigation Of Functionand Molecular Mechanism Of Fyn In Neuronal Migration

Embryonic development of the mammalian cerebral cortex requires the coordinatedmigration of postmitotic neurons from the internal ventricular zone to the marginal layers(Inside-Out) of the developing cortical plate. Neuronal migration is a complex processgoverned by numerous intrinsic and extrinsic factors. During brain development, newlygenerated postmitotic neurons have to migrate away from their birthplace to their finaldestinations and generate the synapse. Defects in neuronal migration are involved in manyneuronal disorders such as smooth brain, epilepsy and mental retardation. Fyn, a member ofthe Src tyrosine kinase family (SFK), plays an important role in the migration of corticalneurons and the development of cortical lamination, and has been involved in cell motilityand growth mediated by signalling pathways, such as reelin signal pathway. Fyn, a tyrosinekinase belongs Src family, is a non-receptor tyrosine kinase with molecular weight of about59KDa, the molecular structure of Fyn from N-to C-terminal is: SH3domain (Src homology3domain), SH2domains (Src homology2domain), the protein kinase domain (TyrKc). Srcfamily kinases (SFKs) are a family of histone proteins and transcriptional regulation factor,which achieves its functions by interacting with other members of the nuclear receptorsuperfamily. Fyn is highly expressed in the cerebral cortex during development. Mice deficitof Fyn show impaired neuronal migration and disrupted layer formation in cerebral cortex. Inaddition, cytoskeleton is constituted of actin microfilaments and microtubules. Adhesion andcytoskeletal rearrangement between the neurons and radial glial cells plays a critical role inthe neuronal migration process. These results proved that Fyn mediates neuronal migrationthrough signal transduction, Fyn knockout mouse shows abnormal neuronal morphology anddisorder structure of cortex, indicating that Fyn plays an important role in regulating neuronalmigration, but the mechanism remains unclear.Therefore, in this study, myristoylation, palmitoylation, methylation, inactivated SH3domain, inactivated SH2, kinase-deficient mutation, domain negative mutation, consisitantiveactivation of Fyn mutant plasmid and Fyn RNA-interference were employed to study itsfunction in neuronal migration. Cell transfection, molecular biological techniques,immunohistochemistry, in utero electroporation, time lapse and confocal microscopy were used to research and analyze the function of Fyn in neuronal migration, and these help us toelucidate the molecular mechanisms of neuronal migration and brain development. The mainfindings are as follows:1. Molecular mechanism of Fyn on pseudopodium movement and cell migration will berevealed by investigating effect of Fyn on cell morphology. Fyn gene was cloned and insertedinto pEGFP-N1by molecular cloning and recombination. After transfected, the CHO cellswere used for detecting the expression of Fyn mRNA and proteins by RT-PCR and westernblot, while some cells were stained with DAPI and taken time-lapse series. The resultsdemonstrated that the morphology of CHO cells transfected with the recombination vectorchanged remarkable and induced filopodia and lamellipodia formation. From this research wecan conclude that overexpression of mouse Fyn induces the formation of filopodia andlamellipodia in CHO cells and strengthens the activity of cell movement.2. As a main ancillary molecules involved in the regulation of proliferation,differentiation and migration of cortical neurons, Fyn has also been identified as a signalfactor in motility and growth involved with cytoskeleton. Our results showed that themorphology of the CHO cells transfected with the recombination vector changed remarkably.The numbers of stress fibers may be crunched to transformation for generating and supportingthe formation of filopodia and lamellipodia, but the changes of vinculin and tubulin areunremarkable. In addition, high concentrations of Fyn cause formation of lamellipodia as wellas filopodia. From this research we can draw a conclusion that overexpression of mouse Fyninduces the F-actin cytoskeleton rearrangement and provides a performance that both vinculinand tubulin are absent in the process of Fyn-mediated F-actin reorganization.3. The amino acids Y531of Fyn is of the C-terminal as a negative regulator of theactivity as well as a key residue. In this experiment, the Y531was changed from A to F(called consisitantive activation mutant, FynCA) while K299changed to A. The K299actsas an ATP-binding site (Fyn domain negative mutnat FynDN). The results showed that,overexpression of Fyn mutants cause significant morphological changes and arrestmigration. As an actin-related protein, Fyn plays an important role in cortical laminationand neuronal migration by promoting actin polymerization. Our studies confirm that Fynis an important factor to maintain cytoskeleton in neuronal migration.4. In the present study, we employed two Fyn mutants, Fyn259(Truncated Fyn, noprotein kinase domain) and Fyn390(FynD390A) to investigate the mechanism of Fyn inneuronal migration and cortical lamination. We found that the number of GFP-positiveneurons in cortical plate (CP) was dramatically increased in Fyn259and Fyn390group atpostnatal day (P)1due to increased total-FAK (T-FAK, Focal Adhesion Kinase). Our results also showed that over-expression of Fyn390but not Fyn259mutant led to remarkablemorphological change and both of them impaired migration of transfected neurons. Our workprovides in vivo evidence that tyrosine kinase domain of Fyn is indispensable for the radialmigration, and it is required for precise cooperation with focal adhesion in the process ofneuronal migration. The390thaspartic acid of Fyn in the kinase domain is a key site forcortical development and neuronal migration.