Interaction Of CRMPs With Microtubules And Microfilaments In The Growthcone Of Hippocampal Neurons

Objective:To investigate the interaction of CRMPs in hippocampus neurons of rats andmicrotubules、microfilaments in the growth cone.Materials and methods:Using SPF17day-pregnant SD rats, gradient sucrose centrifugation to extractgrowth cone,transmission electron microscopy and scanning electron microscopyto observe the morphology of the isolate, immunofluorescence and immunoblott-ing to detect the express of GAP-43protein,using multiple immunofluorescencetechnique to analysis the spatial distribution of CRMPs respectively with microtu-bules and microfilaments in hippocampal tissue and in neurons，observe theinfluence of the CRMP5interference and over expression on microtubules andmicrofilaments.Results:（1）Gradient concentration sucrose centrifugation isolated the brain tissue into threelayers, the upper layer with low density, concentration located between thesupernatant and0.75sucrose, the middle layer with higher density, located between0.75sucrose and1.0sucrose, the lower layer with the highest density, locatedbetween1.0sucrose and2.66sucrose. The results obtained by scanning electronmicroscopy showed that the isolate were white, different sized, irregular shapedparticles. The particles in the upper layer were larger than these in the middle and lowlayer. Observed by transmission electron microscopy, the results showed that theupper layer contained a number of shallow-coloured vacuoles, and the particle sizewas uniform. The middle and lower layer contained a number of deep-coloured denseparticles and irregular shaped cell debris, and the particle size was not uniform. Theimmunofluorescence and immunoblotting results confirmed that all the isolate have ahigh expression of GAP-43. The particles in the upper layer almost has no GFAPexpression, however, in the middle and lower layer, there were more GFAPexpression. （2）In the hippocampus, the co-localization of CRMPs with microtubulesand microfilaments both were the most found in the central part of CA1-CA3area, the more in the edge part of CA1-CA3area and CA4area, the least inDG area, their co-efficient has no significant difference statistically;co-localizationof CRMPs and microtubules were mainly located in the cytoplasm, trunk andbranch of the projection, magnification of the growth cone, co-located mainlylocated in C district, less distribution of microtubules in P and T district.Co-localization of CRMPs and microfilaments mainly located in the cytoplasm,trunk and branch of the projection and the growth cone, magnificationof the growth cone, co-localization located in the hole growth cone, C zone、T zone and P zone all have much microfilaments distributed；in neurons or inthe growth cone for object,CRMPs and microfilaments colocalization coefficientwere higher than those of microtubules, both coefficient differencehave signific-ance difference statistically; immunoprecipitation experiments confirmed thatCRMPs can both respectively interact with microtubule and filaments; whenCRMP5over expression in neurons, microtubules and actin expression increased,especially in P district.When CRMP5interference, microtubule andmicrofilamentexpression decreased, also especially in P district.Conclusions:（1）The gradient sucrose centrifugation can isolate the brain tissue accordingto the size of particles，the components of the top isolate are identified asgrowth cones;（2）CRMPs coexist with microtubules and microfilaments in the same site of thehippocampus;（3）CRMPs can be combined with microtubules and microfilaments in vivo,and can influence the expression and distribution of microtubules and microfila-ments, CRMPs may be mediated the interaction of cytoskeleton to regulate neurit-e growth.