Relative distribution of polyglutamylated tubulin and alpha tubulin within mitotic glial cells.

Introduction Mitosis is the visually dramatic process of eukaryotic cell division (Nigg, 2001). Microtubules (MTs) are the fibers that structure the mitotic spindle and integral components of cell division (Walczak & Heald, 2008). MTs are polymers of tubulin, a protein with a heterodimer tertiary structure consisting of one alpha-subunit and one-beta subunit (Kandel, 2013). Fascinatingly, MTs display dynamic and disparate properties within the mitotic spindle (De Forges, 2012). Post-translational modifications of tubulin have been found to influence properties of MTs that determine their function in the cell (Hammond, Cai & Verhey, 2008). Particularly, there is evidence to support that the poly-glutamylation, or addition of multiple glutamate residues to alpha and beta tubulin (van Dijk, 2008) is prevalent in neuronal cells, centrioles, axonemes, and the mitotic spindle and could influence the stability and/or structure of MT assemblies (Hammond, Cai & Verhey, 2008). Other studies have examined distribution tyrosination and detyronsination of alpha-tubulin during mitosis using immunofluorescence and found a relative abundance of post-translationally modified tubulin in the polar regions containing the centrosome (Gundersen & Bulinski 1986). Further, a study of the distribution of glutamylated tubulin isoforms along the sea urchin sperm axoneme found gradiated distribution that correlates with nonuniform properties along the axoneme (Huitorel et al., 2002). Past research also using immunofluorescence techniques concluded that centrioles are poly-glutamylated in all cell types tested (Kahn et al., 2003). Alas, the distribution of post-translational modifications of tubulin is suggested to play a role in the structure and function of the mitotic spindle and work in sea urchin axoneme specifically displays the role of poly-glutamylation in heterogeneous behavior of MTs. However, no studies could be found that integrate these findings and specifically examine distribution of poly-glutamylation within the mitotic spindle. Accordingly, this study examines the hypothesis that there is a higher relative abundance of poly-glutamylated tubulin than alpha tubulin near the poles of a mitotic Schwann cell. Glial cells are the most plentiful cell type in the nervous system and function to support neurons. Macroglia are categorized into three major subcategories, astrocytes, oligodendrocytes and Schwann cells (Kandel, 2013). Schwann cells function to myelinate axons of neurons and are found in the peripheral nervous system. Thus the glia in this study obtained from primary culture of 10 day-old domestic chicken (Gallus gallus) embryos are Schwann cells. Chick (Gallus gallus) embryos are used because dorsal root ganglia and sympathetic nerve chains that are fairly easy to obtain through dissection grow well in the culture method used. Research on the intricacies of the mitotic spindle is of the utmost significance because MTs are a known target for anticancer drugs (Kavallaris, 2001;Islam&Iskander, 2004;Dumontet&Jordan, 2010; Jordan&Wilson, 2004; Rowinsky&Calvo, 2006). A recent review discussed improvements in anti-tubulin agents that exhibit enhanced tumor specificity and reduced neurotoxicity (Loong &Yeo, 2014). Thus, knowledge gained in this study could help to improve anti-tubulin agents that cause apoptosis in cancer cells by attacking the mitotic spindle. In addition, this study could be useful for research on traumatic injury to the peripheral nervous system, particularly to the sciatic nerve (Yao, L. et al., 2014) or demyelinating diseases such as Charcot-Marie-Tooth type 1A disease (Nobbio, L. et al., 2004) and Guillain-Barre Syndrome (Rubin, 2014) because of the type of glial cells used in this study. Many traumatic injurys to the peripheral nervous system and demyelinating diseases impact the body through lack of cell division so this study could provide novel information for possibly promoting mitosis. In short, this study will first confirm the hypothesis that glial cells are mitotic. If said objective is accomplished, this study will use ratiometric analysis to test the hypothesis that there is a higher relative abundance of poly-glutymylated tubulin compared to alpha-tubulin at the poles of a mitotic Schwann cell.