Use the latter only if methanol is incompatible with the subsequent immunofluorescence protocol. Perform immunofluorescence protocol (Section 1, M4) using antibodies against – and -tubulin and/or against other centromeric markers (such as CENP-A or CREST) if visualization of the kMT interface is required (Fig. phosphorylation of serine 172 (S172) by Cdk1 in mitosis inhibits polymerization due to the close proximity to the exchangeable nucleotide-binding site. Thus, this phosphorylation seems to be fundamental for MT remodelling during mitosis (Fourest-Lieuvin et al., 2006). Tubulin palmitoylation consists of the covalent binding of a fatty acid group to a cysteine residue and has been reported to occur primarily at cysteine 376 (C376) of Rabbit polyclonal to ZNF473 -tubulin in function is usually unknown (Jaffrey, Erdjument-Bromage, Ferris, Tempst, & Snyder, 2001). Tubulin polyamination consists of the irreversible covalent binding of a polyamine to various glutamine residues on – and -tubulin by a transglutaminase (Mehta, Fok, & Mangala, 2006). This is the only PTM described to date that adds positive charges to the tubulin subunits. Studies using rat brain extracts revealed that polyamination ZK-756326 dihydrochloride is required for MT stability in neurons (Song et al., 2013). Ubiquitination involves the formation of an amide linkage between -amine of a lysine target and the C-terminus of ubiquitin (Hershko & Ciechanover, 1998). Tubulin is multiubiquitinated by several ubiquitin ligases (Xu, Paige, & Jaffrey, 2010). More recently, it was shown that loss of the ubiquitin E3 ligase activity of MGRN1 causes spindle misorientation and decreased -tubulin polymerization, suggesting a role for MGRN1 in regulation of MT stability. The same work proposed a further role in mitotic spindle orientation (Srivastava & Chakrabarti, 2014). Sumoylation is another regulatory system, similar to ubiquitination, in which a SUMO protein is added to lysine residues. – and -tubulins have been identified as candidates for sumoylation in global sumoylation screens, however the biological function of this modification to MTs is yet to be discovered (Rosas-Acosta, Russell, Deyrieux, Russell, & Wilson, 2005; Wohlschlegel, Johnson, Reed, & Yates, 2004). Tubulin glycosylation consists of the reversible enzymatic addition of O-linked -N-acetylglucosamine (O-GlcNAc) to serine/threonine ZK-756326 dihydrochloride residues in the tubulin sequence (Love & Hanover, 2005). It has been reported that O-GlcNAcylation inhibits dimerization and that O-GlcNAcylated tubulin does not incorporate into MTs (Ji et al., 2011). Methylation was the last tubulin PTM to be identified. -tubulin is also methylated at K40 by a dual-function histone and microtubule methyltransferase called SET-domain-containing 2 (SETD2). The same study reported that methylation varies between different MT populations. Moreover, acute loss of SETD2 function caused mitotic and/or cytokinesis defects (Park et al., 2016). How is the tubulin code read? The myriad of tubulin PTMs display a patterned distribution among the many MT subpopulations (Yu, Garnham, & Roll-Mecak, 2015). In mitosis, detyrosination also distributes stereotypically among the MT subpopulations that compose the mitotic spindle. Several studies have consolidated the hypothesis that these epigenetic marks affect the activity of molecular effectors working on MTs. It has been reported that detyrosination regulates kinesin-1 and kinesin-2 processivity and decreases the depolymerizing activity of kinesin-13 (Dunn et al., 2008; Peris et al., 2009; Sirajuddin, Rice, & Vale, 2014). Furthermore, polyglutamilation enhances kinesin-1 and kinesin-2 motility, whereas kinesin-13 and dynein are insensitive ZK-756326 dihydrochloride to this modification (Kaul, Soppina, & Verhey, 2014; ZK-756326 dihydrochloride Konishi & Setou, 2009; Sirajuddin et al., 2014). Dynein is not directly affected by detyrosination but the initiation of its processive movement in complex with dynactin and BicD2 is affected by detyrosination, as well as the recruitment of MT plus-end tracking proteins such as CLIP170 (McKenney, Huynh, Vale, & Sirajuddin, 2016; Peris et al., 2006). The first demonstration of tubulin PTMs impacting on mitosis came from the discovery that CENP-E preferentially moves along detyrosinated MTs to guide chromosomes towards the spindle equator during chromosome congression (M. Barisic et al., 2015). At the entry into mitosis, cyclin-dependent kinase 1 (CDK1) is activated and triggers a cascade of phosphorylation events that ultimately regulate the activity of MAPs and motors (Cassimeris, 1999; Ramkumar, Jong, & Ori-McKenney, 2018). The functional shift of this complex machinery leads to the reconfiguration of the MT landscape in mitosis and thus requires specific methodologies to investigate the implications of tubulin PTMs during this process. Methods In this chapter, we provide an overview of the methods currently used in our laboratory to investigate tubulin PTMs and their roles in mitosis, focusing on detyrosination. We address 3 main topics: 1) analysis of the levels and distribution of detyrosination in perturbed and unperturbed cells; 2) study of the effect of.