Microtubules (MTs) are important for cell migration and differentiation. A subset of microtubules that are selectively stabilized are found in cells and these MTs are highly polarized. Wounded NIH-3T3 cells orient these stable microtubules towards the wound edge, in the direction of migration.
The precise proteins that directly stabilize MTs are unclear but certain features are apparent. The MT-stabilization protein(s) function at the end of a stable microtubule and not along their length since the rest of the stabilized microtubule is fully able to grow and shrink when the tip of the microtubule is severed, for instance by calcium treatment. This gave rise to the model that there is a microtubule cap at the plus ends of stable microtubules in NIH-3T3 fibroblasts. Based on biochemical characterization, it was determined the cap was sensitive to ATP and this ATP sensitivity could be blocked by AMP-PNP and high concentrations of vanadate. Both of these inhibitors are weakly selective for kinesins so a kinesin was suggested to be involved. The homolog of the kinesin Kif4 in Xenopus, XKLP1, was shown to have the ability in vitro to cap MTs. I tested whether Kif4 functioned to stabilize MT in vivo. I found that Kif4 was both necessary and sufficient for microtubule stabilization in NIH-3T3 fibroblasts and that it partially localized to the ends of stable microtubules. This encouraged me to conduct further explorations. A pathway that regulates microtubule stabilization in response to lysophosphatidic acid (LPA) has been discovered in NIH-3T3 fibroblasts. LPA, acting through its receptor, activates Rho GTPase which activates the formin mDia to stabilize MTs mDia appears to act with the MT tip proteins EB1 and APC in the MT-stabilization pathway. I found that Kif4 interacted with EB1 and that knockdown of Kif4 blocked EB1 and mDia mediated microtubule stabilization, suggesting Kif4 is downstream of these two proteins, perhaps directly acting on the microtubule. I also explored the localization and dynamics of GFP-Kif4 with live cell imaging. I observed that GFP-Kif4 moved in a fashion consistent with its localization on MT plus ends and that GFP-Kif4 puncta localized to the cell periphery in a MT-dependent manner. These behaviors are consistent with the idea that Kif4 may use its motor activity to move to the ends of MTs to stabilize them.
In addition I found that another kinesin, Kif3a, played a role in the other major microtubule rearrangement during cell polarization: centrosome polarization. I showed that Kif3a, but not other members of the Kinesin-II complex, was essential for centrosome polarization. The centrosome polarization activity by Kif3a and the microtubule stabilizing activity of Kif4 reveal the potential diversity of function in kinesin family members.
|School Location:||United States -- New York|
|Source:||DAI-B 71/03, Dissertation Abstracts International|
|Keywords:||Centrosome polarization, Kinesin, Microtubules|
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