Characterization Of NudCL And Twa1, Centrosome-associated Proteins In Lis1/Dynein Pathway

Cytoplasmic dynein is a major motor protein associated with microtubule in eukaryotic cells, and its major function is transporting cargos along microtubules. Dynein appears to be regulated by Lisl (Lissencephaly 1), a causative gene for classic lissencephaly, wich is required for neuronal migration, cell proliferation and survival. In the filamitous fungus A. nidulans, nudF(nuclear distribution gene F), the homology of Lisl is regulated by nudC(Nuclear distribution gene C), which mutation greatly reduces the protein level of NudF. Thus, NudC, Lisl and dynein form a conserved signaling pathway from fungi to human.However, in mammalian cells the Lisl/Dynein pathway is much more complicated. Both NudC and Lisl have their own paralogues. Together with dynein complex, the proteins in this pathway always form a complex and take part in cell cycle regulation and cell migration. Most of them are localized at centosome or along with the microtubule. As a microtubule organizing center (MTOC), centrosome is involved in all microtubule-dependent processes and contributes to bipolar spindle formation, spindle positioning, cytokinesis, cell organelles transporting and cell migration. Therefore, the importance of Lisl/Dynein pathway is widely recognized. However, many proteins in this pathway have not been found and the regulation among these proteins is still not very clear. In the last few years our lab has been engaged in this pathway to identify new members and their functions. Here we focus on two new members of Lisl/Dynein pathway:NudCL (NudC-Like protein) and Twal (Two hybrid associated protein No.1 with RanBPM, Lisl homologue). It is the frist time that both of these two proteins were found to be localized to centrosome. To further explore the Lisl/Dynein pathway, we investigated the functions of NudCL and Twal in mammalian cells as well as in Xenopus embryos.Part I:Roles of a centrosome associated protein NudCL in cytokinesis and cell proliferationOur previous work demonstrated that NudCL localizes to Golgi and it maintains a normal mitosis by keeping the Dynein intermediate chain (DIC) stable. In this work, we found that NudCL was associated with the centrosome in inerphase and spindle poles at metaphase. During cytokinesis, NudCL was accumulated at the midbody. Cell number counting and MTT assays showed that overexpression of NudCL significantly inhibited cell proliferation. Immunofluorescence and time-lapse microscopy technology showed a significant increase in multinucleated cells in cells overexpressing NudCL. These multinucleated cells are always caused by the failure of cytokinesis. Most of NudCL-overexpressing cells had an abnormal structure of microbubule bundles at the midbody or with a flemming body formation failure. Furthermore, overexpression of NudCL also induced the mitotic delay, formation of multipolar spindle or chromosomes lagging. Depleted endogenous NudCL also induced cytokinesis defects besides multiple mitotic defects. These data suggest that besides the functions in mitosis, NudCL also plays an important role in cytokinesis.Partâ…¡:Roles of a centrosome associated protein Twal in cell migration and embryonic developmentLisl has been reported to be required for neuronal migration, cell proliferation and survival. Depletion of Lisl in mice results to embryonic lethal. Mutant of Lisl induces type-1 Lissencephaly in human, and the patients always with smoth brain, mental retardation, and short life. However, no Lis1 homologue has been identified yet. Using gene and protein database, we found a Lisl homologue Twal, which has been reported to be localized to the nuclei and interact with RanBPM (Ran-binding protein M) and Muskelin. By bioinformatics analysis we found that this protein is highly conserved in evolution. Twal contains a Lis homologue domain (LisH domain) and a CTLH domain (C-terminal to LisH domain). Immunofluorescence data showed that besides the nuclear location, Twal also locates at centrosome and midbody. Moreover, Twa1 interacts with Lis1 and Dynein in vivo. Cell wound assay and transwell assay showed that mammalian cells with Twal RNAi suffered migration defects. Futher investigation revealed that compaired with control cells the distrubusion of Dynein in the leading edge was disappeared in Twal RNAi cells. Interestingly, overexpression of Twal-â–³LisH also leads to cell migration defects. RT-PCR and in situ hybridization analysis in Xenopus embryos showed that Twal highly expresses at gastrulation (st. 10-13) and organogenesis (st.19-30) stages and predominantly expresses in eyes and brain. Microinjection of Twal-ALisH in Xenopus embryos induces loss of head structures and sometimes results to embryonic lethal. These results indicated that Twa1 may be involved in cell migration and is important for embryonic development.Taking together, these two centrosome associated proteins, NudCL and Twal in Lis1/Dynein pathway play crucial roles in cells. NudCL is essential for cytokinese, while, Twal is required for embryonic development by regulating cell migration. The study of these two centrosomal proteins gives us an extensive and in-depth understanding of the Lis1/Dynein pathway and provides a new approach for the centrosome function investigation.