The Mechanism Of Dendritic Targeting Of MKLP1 In Primary Cultured Rat Hippocampal Neurons

Typical vertebrate neurons extend a single axon and several dendrites.Dendrites are short stout tapering processes that are equipped with ribosomes and Golgi elements, whereas axons are long thin processes of uniform diameter that are deficient in these organelles. It has been hypothesized that the unique morphological and compositional features of axons and dendrites result from their distinct patterns of microtubule polarity orientation. The microtubules within axons are uniformly oriented with their plus ends distal to the cell body, whereas microtubules within dendrites are nonuniformly oriented. These differences were believed to contribute to establishing the unique features that distinguish dendrites from the axon. For example, ribosomes and Golgi elements are thought to move toward minus ends of microtubules, and hence would have an appropriate track to move into the dendrites but not the axon.The kinesin-related motor protein MKLP1 was initially thought to be expressed only in mitotic cells, where it presumably transports oppositely oriented microtubules relative to one another in the spindle mid-zone. Later on it has been realized that that MKLP1 is also present in neurons. In hippocampal neurons the MKLP1 has properties to establish the non-uniform polarity orientation of microtubule arrays. Immunofluorescence analyses indicated that MKLP1 is absent from axons but is enriched in developing dendrites. MKLP1 transports microtubules from the cell body into the developing dendrite with their minus ends leading, thereby establishing the nonuniform microtubule polarity pattern of the dendrite. It has been also revealed that MKLP1 is expressed within various neuronal populations of the brain including those in the cerebral cortex, hippocampus, olfactory bulb and cerebellum. Dorsal root ganglion neurons, which generate axons but not dendrites, express significantly lower levels of mRNA for MKLP1 than hippocampal or sympathetic neurons.Before the development of dendrites, MKLP1 is present only within the cell body of the neuron and is diffuse in appearance. The protein does not enter immature processes or developing axons to any appreciable degree at any stage of development,indicating that it is not freely diffusible. As dendrites develop, MKLP1 begins to appear within the developing dendrites. Depletion of MKLP1 from cultured neurons causes a rapid redistribution of microtubules within dendrites such that minus-end-distal microtubules are chased back to the cell body while plus-end-distal microtubules are redistributed forward. The dendrite grows significantly longer and thinner, loses its taper, and acquires a progressively more axon-like organelle composition. These results suggest that MKLP1 are necessary for maintaining the minus-end-distal microtubules in the dendrite so acquire dendrite's property.This study consists of two parts.In the first place, we looked at the distribution of MKLP1 mRNA and protein in neurons. Using in situ hybridization, we found that MKLP1 mRNA was restricted to cell body in developed neuron. Observing green fluorescence, we observed that eGFP tagged MKLP1 was distributed to the dendrtite and soma of the neuron, closely resembling the distribution of MAP2. It is concluded that it is the MKLP1 protein, not its mRNA, which was distributed specifically to the dendrite.Secondly, we addressed the issue whether there is amino acids domains within the MKLP1 molecule which is responsible for its dendritic distribution.In NCBI Conserved Domain Search putative conserved domains have been detected in MKLP1. The amino-termianl half of the MKLP1 are conserved kinesin motor donnains ,central region between amnioacids 485 and 655 may assemble into a coiled-coil as MKLPl's stalk, amnioacids 656 and 856 is MKLPl's tail .A consensus phosphorylation site for p34cdc2 kinase (residues 13-20), ATP-binding site (residues 113-117),and two kinesin concerved domains the SSRSH domain (resioues 296-300) and the LAGSE domain (residues 334-340).It is plausible that one or or more of these functio...