Post-Translational Modifications of Microtubules Dorota Wloga and Jacek Gaertig

Erratum Post-translational modifications of microtubules Dorota Wloga and Jacek Gaertig

There was an error published in J. Cell Sci. 123, 3447-3455.

An incorrect version of Fig. 2 was published. The correct version, showing the severing of microtubules, is given below, together with its legend.

Key ODA Unmodified MT Kinesin-1 IDA Modified MT

CLIP-170 Katanin, spastin Kinesin-13

Fig. 2. Tubulin PTMs regulate diverse activities by affecting the activity of microtubule effectors. Tubulin modifications regulate diverse activities including motor protein movement, microtubule severing, microtubule-end polymerization and activity of +TIPs. See text for further details.

We apologise for this mistake. Author Correction Post-translational modifications of microtubules Dorota Wloga and Jacek Gaertig

There were errors published in J. Cell Sci. 123, 3447-3455.

In Fig. 1, the arrows showing enzymatic reactions for MEC-17, HDAC6 and SIRT2 were pointing in the wrong direction. In addition, the C-terminal amino acid on the tail of -tubulin is Y (and not T). The correct figure is shown below.

Ac CCP5 HDAC6 MEC-17 G ? CCP6 E SIRT2 TTLL10 G E TTLL6,9 Ac α E -tubulin E G E G G E Met TY Elongation G E β-tubulin G E E

G E E G E G G TTLL3 Initiation TTLL1 CCP1 E E G E E E E EE YT Met Δ2 TTL

After publication of this Commentary, Shida and colleagues reported independent identification of MEC-17 homologs in diverse organisms as -tubulin acetyltransferases (Shida et al., 2010). Contrary to what we stated in our article and in the referenced paper (Akella et al., 2010), the newest version of the assembled genome of Chlamydomonas reinhardtii contains a sequence encoding an apparent homolog of the MEC-17 -tubulin acetyltransferase (locus ID Cre07.g345150).

References Akella, A. J., Wloga, D., Kim, J., Starostina, N. G., Lyons-Abbott, S., Morrissette, N. S., Dougan, S. T., Kipreos, E. T. and Gaertig, J. (2010). MEC-17 is an alpha-tubulin acetyltransferase. Nature 467, 218-222. Shida, T., Cueva, J. G., Xu, Z., Goodman, M. B. and Nachury, M. V. (2010). The major -tubulin K40 acetyltransferase TAT1 promotes rapid ciliogenesis and efficient mechanosensation. Proc. Natl. Acad. Sci. USA doi/10.1073/pnas.1013728107.

The authors apologise for these mistakes. Commentary 3447 Post-translational modifications of microtubules

Dorota Wloga1 and Jacek Gaertig2,* 1Department of Cell Biology, Nencki Institute of Experimental Biology, Polish Academy of Science, 02-093 Warsaw, Poland 2Department of Cellular Biology, University of Georgia, Athens, GA 30602, USA *Author for correspondence ([email protected])

Summary Microtubules – polymers of tubulin – perform essential functions, including regulation of cell shape, intracellular transport and cell motility. How microtubules are adapted to perform multiple diverse functions is not well understood. Post-translational modifications of tubulin subunits diversify the outer and luminal surfaces of microtubules and provide a potential mechanism for their functional specialization. Recent identification of a number of tubulin-modifying and -demodifying enzymes has revealed key roles of tubulin modifications in the regulation of motors and factors that affect the organization and dynamics of microtubules. This article is part of a Minifocus on microtubule dynamics. For further reading, please see related articles: ‘Microtubule plus-end tracking proteins (+TIPs)’ by Anna Akhmanova and Michel O. Steinmetz (J. Cell Sci. 123, 3415-3419), ‘Kinesins at a glance’ by Sharyn A. Endow et al., (J. Cell Sci. 123, 3420-3424), ‘Tubulin depolymerization may be an ancient biological motor process’ by J. Richard McIntosh et al. (J. Cell Sci. 123, 3425-3434) and ‘Towards a quantitative understanding of mitotic spindle assembly and mechanics’ by Alex Mogilner and Erin Craig (J. Cell Sci. 123, 3435-3445).

Key words: Cilia, Modification, Tubulin

Introduction cells detected acetylation of K residues on multiple additional sites The - and -tubulin heterodimer – the building block of on - and -tubulin (Choudhary et al., 2009). However, K40 is the microtubules – undergoes multiple post-translational modifications main – if not the only – site of acetylation on tubulin in Tetrahymena (PTMs) (Table 1). The modified tubulin subunits are non-uniformly thermophila, on the basis of the observation that antibodies against distributed along microtubules. Analogous to the model of the acetylated K residues do not crossreact with the K40R point mutant ‘histone code’ on chromatin, diverse PTMs are proposed to form a of -tubulin that cannot be acetylated (Akella et al., 2010). biochemical ‘tubulin code’ that can be ‘read’ by factors that interact Acetylated of K40 residue (acetyl-K40) on -tubulin occurs on with microtubules (Verhey and Gaertig, 2007). In this Commentary, diverse microtubules including cytoplasmic, spindle, centriolar and

Journal of Cell Science we will discuss recent advances in understanding the mechanism axonemal microtubules (Piperno and Fuller, 1985). The acetyl- and functions of tubulin PTMs. Recent data provide strong support K40 mark appears with a delay after microtubule assembly and is to a model that tubulin PTMs regulate microtubule effectors. seen as a marker of polymer age (Gundersen et al., 1984; Piperno et al., 1987; Schulze et al., 1987). Recently, MEC-17, a protein Conserved tubulin PTMs that occur on related to GCN5 histone acetyltransferases (Steczkiewicz et al., microtubules 2006) and required for the function of touch receptor neurons in Most PTMs appear on tubulin subunits after polymerization into Caenorhabditis elegans (Chalfie and Au, 1989; Zhang et al., 2002), microtubules. One exception is phosphorylation on serine residue was identified as an important -tubulin acetyltransferase (TAT) S172 of -tubulin by the Cdk1 kinase that occurs on the (Akella et al., 2010). MEC-17 deficiencies result in the loss of unpolymerized tubulin dimer and inhibits its incorporation into acetyl-K40 -tubulin in Tetrahymena, C. elegans, zebrafish assembling microtubules (Fourest-Lieuvin et al., 2006). Tubulin embryos and HeLa cells and – in vitro – recombinant mouse MEC- is also phosphorylated inside microtubules (Faruki et al., 2000; 17 exclusively acetylates K40 on -tubulin (Akella et al., 2010). Gard and Kirschner, 1985; Laurent et al., 2004; Ma and Sayeski, However, the genome of Chlamydomonas reinhardtii, an organism 2007; Matten et al., 1990; Wandosell et al., 1987), presumably on in which -tubulin acetylation was discovered and the TAT tyrosine (Y) or S residues that are different from S172 on the - activity characterized (Greer et al., 1985; L’Hernault and subunit, but the sites and significance of the post-assembly Rosenbaum, 1985; LeDizet and Piperno, 1987; Maruta et al., phosphorylation events are not known. Tubulin PTMs that occur 1986), lacks a MEC-17 homolog (Akella et al., 2010). Thus, on microtubules and have been well characterized include another ciliary TAT is likely to exist. In animal cells, several acetylation of lysine (K) residues, detyrosination, glycylation and acetyltransferases colocalize with acetylated microtubules and some glutamylation (see below). regulate the level of acetyl-K40 -tubulin, including N- acetyltransferase 1 (NAT) 1 (Ohkawa et al., 2008), NAT10 (Shen Acetylation of K40 on -tubulin et al., 2009) and elongator protein 3 (ELP3) (Creppe et al., 2009; Highly conserved acetylation of the -amino group of residue K40 Solinger et al., 2010), but it is not known whether any of these of -tubulin (L’Hernault and Rosenbaum, 1985; LeDizet and enzymes have direct activity on -tubulin. Deacetylation of acetyl- Piperno, 1987) is the only PTM that occurs on an amino acid K40 on -tubulin is catalyzed by HDAC6 (Hubbert et al., 2002; moiety that extends to the microtubule lumen (Nogales et al., Matsuyama et al., 2002) and sirtuin 2 (SIRT2) (North et al., 2003) 1998) (Fig. 1). A recent mass spectrometry study in mammalian deacetylases, enzymes that are related to histone deacetylases. 3448 Journal of Cell Science 123 (20)

Table 1. Known tubulin PTMs and tubulin-modifying and -demodifying enzymes PTM Subunit Residue(s) Forward enzyme Reverse enzyme References Lysine acetylation1 K40 MEC-172 HDAC63, 1(Choudhary et al., 2009; L’Hernault and K60, K112, K163, SIRT24 Rosenbaum, 1985; LeDizet and Piperno, 1987), K164, K311, K326, 2(Akella et al., 2010), 3(Hubbert et al., 2002; K370, K394, K401 Matsuyama et al., 2002), 4(North et al., 2003) K58 Arginylation , (Wong et al., 2007) 2 Penultimate E (Lafanechere and Job, 2000) Detyrosination5 C-terminal Y CCP16 TTL75(Argarana et al., 1978; Argarana et al., 1980; Hallak et al., 1977), 6(Kalinina et al., 2007), 7(Ersfeld et al., 1993) Glutamylation8 , Multiple E in CTT TTLL1,4,5,6,7,9, CCP510 8(Edde et al., 1990), 9(Ikegami et al., 2006; Janke et 11,139 al., 2005; Mukai et al., 2009; van Dijk et al., 2007; Wloga et al., 2008), 10(Kimura et al., 2010) Glycosylation , (Cicchillitti et al., 2008; Hino et al., 2003) Glycylation11 , Multiple E in CTT TTLL3,8,1012 11(Redeker et al., 1994), 12(Ikegami and Setou, 2009; Rogowski et al., 2009; Wloga et al., 2009) Lysine methylation (Iwabata et al., 2005) Glutamic acid , E434 -tubulin (Xiao et al., 2010) methylation Palmitoylation , C376 -tubulin (Caron, 1997; Ozols and Caron, 1997; Zambito and Wolff, 1997) Phosphorylation13 , S172 -tubulin Cdk114 13(Gard and Kirschner, 1985), 14(Fourest-Lieuvin et PSK15 al., 2006), 15(Mitsopoulos et al., 2003), 16(Faruki Syk16 et al., 2000) Sumoylation (Rosas-Acosta et al., 2005) Ubiquitylation17 Multiple Ks Parkin18 17(Huang et al., 2009), 18(Ren et al., 2003)

C-terminal proteolytic events of -tubulin – detyrosination Polymodifications are catalyzed by TTL-like proteins (TTLLs) and 2 PTM that have homology with TTL (Ikegami and Setou, 2009; Janke et Tubulin detyrosination is a proteolytic removal of the C-terminal Y al., 2005; Rogowski et al., 2009; van Dijk et al., 2007; Wloga residue on -tubulin (Hallak et al., 1977). Detyrosination occurs et al., 2008; Wloga et al., 2009). A polymodification side chain is after incorporation of tubulin subunits into the microtubule lattice formed in two biochemically distinct steps: initiation and (Kumar and Flavin, 1981) on diverse microtubules (Geuens et al., elongation. These two steps are often, but not always, mediated by 1986; Gundersen and Bulinski, 1986; Gundersen et al., 1984; distinct TTLLs. First, a E or glycine (G) residue is attached to Johnson, 1998; Webster et al., 1987). Detyrosination is likely to be tubulin through an isopeptide bond with the -carboxyl group of a

Journal of Cell Science generated by the cytosolic carboxypeptidase 1 (CCP1, also known primary sequence glutamic acid, by a TTLL with chain-initiating as NNA1), a finding based on the observation that a mutation in activity, such as TTLL1, TTLL4 and TTLL7 glutamic acid ligases Ccp1 in mice greatly decreases the level of microtubule (E-ligases) (Janke et al., 2005; Mukai et al., 2009; Wloga et al., detyrosination (Kalinina et al., 2007). It remains to be determined 2008) and TTLL3 glycine ligase (G-ligase) (Rogowski et al., 2009; whether CCP1 has -tubulin detyrosination activity in vitro. Wloga et al., 2009). The side chain is extended by TTLLs with Detyrosinated tubulin heterodimers that are released from elongase activity such as TTLL6 and TTLL9 E-ligases (Suryavanshi microtubules can revert to an unmodified state by tyrosination (Barra et al., 2010; van Dijk et al., 2007; Wloga et al., 2008) and TTLL10 et al., 1973) catalyzed by tubulin tyrosine ligase (TTL) (Ersfeld et G-ligase (Ikegami and Setou, 2009; Rogowski et al., 2009). Some al., 1993). Detyrosinated -tubulin that remains in the microtubule TTLLs, such as mouse TTLL7 E-ligase (Mukai et al., 2009) or lattice may undergo a second proteolysis, 2 PTM, that removes the Drosophila melanogaster TTLL3 G-ligase (Rogowski et al., 2009) now terminal glutamic acid (E) residue. The 2 -tubulin that is have both side-chain-initiating and -elongating activities. In vivo, released from microtubules during depolymerization cannot revert the length of the side chain correlates with the microtubule type. to an unmodified state (Lafanechere and Job, 2000) and, thus, this For example, in ciliates, the side chains are longer in axonemes PTM can limit the amount of -tubulin that undergoes recycling. when compared with those present on microtubules in the cell body (Iftode et al., 2000; Wloga et al., 2008). The extent to which Tubulin polymodifications – glutamylation and glycylation either of the two tubulin subunits is modified is spatially regulated. Glutamylation (Edde et al., 1990) and glycylation (Redeker et al., For example, in superior cervical ganglion neurons, tubulin 1994) are two related PTMs that form as peptide side chains on the glutamylation is abundant on -tubulin in the somatodendritic C-terminal tail (CTT) domain of tubulin (Fig. 1). Because of their compartment, but -tubulin is glutamylated to a higher extent in polymeric nature, glycylation and glutamylation are referred to as the axon (Ikegami et al., 2006). Such patterns of tubulin ‘polymodifications’. The side chains branch off multiple glutamic polymodifications can be formed by selective targeting of G- and acids within the CTT of - and -tubulin. Tubulin glutamylation E-ligases to specific cellular locations such as neuronal projections is abundant on microtubules of axonemes, centrioles and basal or cilia (Ikegami et al., 2006; Suryavanshi et al., 2010; van Dijk et bodies, and on some cytoplasmic and spindle microtubules al., 2007; Wloga et al., 2009). The side-chain elongation occurs (Bobinnec et al., 1998b; Bre et al., 1994; Wolff et al., 1992). The gradually after microtubule assembly. Consequently, microtubules related PTM, tubulin glycylation, is restricted to ciliated cell types, in the newly forming organelles, such as the nascent basal bodies and is enriched on axonemes and basal bodies (Bre et al., 1996). and axonemes, have short side chains that lengthen as the organelle Post-translational modifications of microtubules 3449

Ac CCP5 HDAC6 MEC-17 G ? CCP6 E SIRT2 TTLL10 G E TTLL6,9 Ac α-tubulin E E G E G G E Met T Elongation G E G E E β-tubulin

G E G E G E TTLL3 Initiation TTLL1 CCP1 E G E G E EEE EE T TTL Met Δ2

Fig. 1. Tubulin PTM types and sites. Shown are the well-characterized post-translational modifications that act on tubulin and the enzymes that are responsible for these modifications. Most of the modifications occur at the C-terminal tail (CTT) domains of - and -tubulin. Ac, acetylated lysine; E, glutamic acid; G. glycine; Met, methylated glutamic acid; 2, proteolytic removal of the penultimate C-terminal amino acid.

matures (Iftode et al., 2000; Sharma et al., 2007). Thus, the length Bre et al., 1998; Khawaja et al., 1988; Piperno et al., 1987). In of polymodification side chains could have a role in distinguishing migrating mammalian cells, a subset of microtubules that orient between microtubular organelles that are either in the assembly or towards the leading edge are more stable and enriched in acetylation maintenance state. Tubulin glutamylation is important for assembly and detyrosination (Palazzo et al., 2004). The question arises and motility of cilia (see below). Although tubulin glycylation is a whether tubulin PTMs have a role in stabilizing microtubules or marker of ciliated cells, there are evolutionary lineages with cilia whether they simply accumulate on microtubules that are stabilized that lack this PTM (Fennell et al., 2008; Schneider et al., 1997). In by other mechanisms such as plus-end capping (Infante et al., vivo, there is competition between the two polymodifications, 2000). In mammalian cells, experimental stabilization of probably because the E- and G-ligases attach side chains to the microtubules with paclitaxel increases the levels of tubulin same glutamic acids in the CTTs (Rogowski et al., 2009; Wloga et acetylation and detyrosination (Gundersen et al., 1987; Hammond al., 2009). Thus, the primary role of tubulin glycylation might be et al., 2010; Piperno et al., 1987; Witte et al., 2008). Thus, it is to negatively regulate tubulin glutamylation. Organisms that lack possible that at least some PTMs accumulate on long-lived tubulin glycylation could use other means to inhibit tubulin microtubules as a consequence of increased time of exposure to glutamylation, including tubulin deglutamylation (see below) or modifying enzymes. However, recent studies indicate that some other competing PTMs. In Toxoplasma gondii, a potentially tubulin PTMs can have a stabilizing effect on microtubules.

Journal of Cell Science polymodifiable residue of -tubulin, E434, undergoes carboxyl The -tubulin detyrosination and tyrosination cycle is important methylation (Xiao et al., 2010), a PTM of unknown effect, that in vivo. Mice that lack TTL have increased levels of detyrosinated could also compete with tubulin glutamylation (Plessmann et al., -tubulin, defects in the differentiation of neurons and die within 2004). hours of birth (Erck et al., 2005). The Purkinje cell degeneration Biochemical studies have detected activities that shorten (pcd) mice that carry a mutation in the gene encoding CCP1 polymodification side chains: deglutamylation (Audebert et al., (Kalinina et al., 2007) have increased levels of tyrosinated - 1993) and deglycylation (Bre et al., 1998). A recent study identifies tubulin, display adult-onset degeneration of Purkinje and retinal the carboxypeptidase CCP5 as the tubulin deglutamylase (Kimura neurons, and have structurally defective sperm (Fernandez- et al., 2010). CCP5 has an amino acid sequence related to the Gonzalez et al., 2002; Handel and Dawson, 1981; Mullen et al., putative detyrosination enzyme CCP1 (Kalinina et al., 2007). A 1976; Wang and Morgan, 2007). It seems that the phenotypes mutation of CCP5 in C. elegans increases the level of tubulin observed in mice deficient in those enzymes involved in the glutamylation in cilia of sensory neurons, and purified mammalian detyrosination–tyrosination cycle result from impaired microtubule CCP5 has tubulin deglutamylase activity in vitro (Kimura et al., dynamics. In vitro, fibroblasts isolated from Ttl-null mice have a 2010). To our knowledge, it has not yet been reported how less-polarized shape and defective motility (Peris et al., 2006). deregulation of CCP5 affects microtubules. It can be speculated These fibroblasts also have excessively detyrosinated microtubules that, among the CCP5-related carboxypeptidases that lack an that are abnormally long and hyperstable, and this phenotype is assigned enzymatic activity (Rodriguez de la Vega et al., 2007), rescued by overexpression of kinesin-13, a microtubule-end there are tubulin deglycylases. depolymerizer (Peris et al., 2009). Consistently, microtubules enriched in detyrosinated tubulin show increased resistance to Tubulin PTMs affect the organization and kinesin-13-mediated end depolymerization in vitro (Peris et al., dynamics of microtubules 2009). In mice, the knockout phenotypes for genes encoding TTL The use of antibodies that specifically recognize post-translationally and kinesin superfamily protein 2A (KIF2A, a kinesin-13-family modified tubulins reveals differences in the level of PTMs among protein expressed in the nervous system) are similar (Homma et distinct microtubules, or even along the length of the same al., 2003; Peris et al., 2009). Thus, detyrosinated -tubulin at or microtubule. Microtubules with a slow turnover and, in particular, near the microtubule end might protect against depolymerization ‘superstable’ centriolar basal body and axonemal microtubules by kinesin-13. Tubulin detyrosination also affects the dynamics of have high levels of diverse tubulin PTMs (Bobinnec et al., 1998b; the plus ends of microtubules by inhibiting binding of the plus-end 3450 Journal of Cell Science 123 (20)

tracking proteins (+TIPs) that have a CAP-Gly domain: CLIP170, In Tetrahymena cells that lack the K40-specific TAT, MEC-17, CLIP115 and p150Glued (Peris et al., 2006). most if not all microtubules appear normal but cells are hyper- Tubulin glutamylation also strongly influences the dynamics of resistant to paclitaxel and hypersensitive to oryzalin, indicating microtubules. Overexpression of the TTLL6-type E-ligase (- that MEC-17 has microtubule-stabilizing activity (Akella et al., tubulin elongase) in Tetrahymena thermophila leads to the 2010). Although our earlier study failed to detect a tubulin accumulation of hyperglutamylated nocodazole-resistant phenotype in a K40R -tubulin mutant that is drug resistant cytoplasmic microtubules (Wloga et al., 2010). The extent to which (Gaertig et al., 1995), re-examination of the same mutant strain by tubulin is glutamylated might affect the binding affinity of neuronal using longer drug exposure showed a drug-resistance phenotype stabilizing microtubule-associated proteins (MAPs) such as MAP2 similar to that seen in the MEC-17-knockout cells (Akella et al., and Tau (Bonnet et al., 2001; Boucher et al., 1994). Consistent 2010). with this, depletion of the TTLL7 E-ligase in differentiating PC- A recent study opens up the possibility that K40 acetylation 12 cells by RNA interference (RNAi) inhibits the emergence of regulates the levels of total tubulin (Solinger et al., 2010). In C. MAP2-enriched neurites (Ikegami et al., 2006). elegans nematodes with a genetic background that increases the However, tubulin glutamylation can also have a microtubule- level of MEC-12 proteins acetylated on K40, the total levels of destabilizing effect through the regulation of microtubule-severing transgenic MEC-12 decrease, and the effect is reversed by a K40Q factors katanin (McNally and Vale, 1993) and spastin (Hazan et al., substitution in MEC-12 (Solinger et al., 2010). Solinger and 1999). In Tetrahymena, a mutation of multiple adjacent colleagues propose that the acetyl-K40 of -tubulin is targeted for polymodifiable glutamic acid residues on the CTT of -tubulin degradation through ubiquitylation, presumably following the (Thazhath et al., 2002) phenocopies a loss of katanin (Sharma et disassembly of acetylated microtubules (Solinger et al., 2010). The al., 2007) (see next section). In C. elegans, substitution of a acetyl-K40-mediated tubulin degradation could involve HDAC6, glutamic acid residue in the CTT of -tubulin that is likely to serve because this protein binds to acetyl-K40 -tubulin and interacts in glutamylation rescues the embryonic lethality caused by with proteins that mediate ubiquitylation (Boyault et al., 2007). overproduction of katanin (Lu et al., 2004). Overexpression of the Moreover, - but not -tubulin undergoes ubiquitylation in the TTLL11 E-ligase in HeLa cells causes fragmentation of disassembling axonemes of Chlamydomonas reinhardtii (Huang et microtubules that can be rescued by knockdown of spastin using al., 2009). However, in Chlamydomonas, ubiquitylation was found small interfering RNA (siRNA); moreover, hyperglutamylated on -tubulin whose K-40 residues were acetylated, indicating that microtubules have increased sensitivity to spastin-mediated severing deacetylation of K40 is not a prerequisite of ubiquitylation (Huang in vitro (Lacroix et al., 2010). Spastin (and probably katanin) are et al., 2009). It is, however, possible that the acetyl-K40 mark predicted to function as a ring complex of six subunits, with a facilitates ubiquitylation by promoting binding of HDAC6, a model centrally located pore that incorporates the tubulin CTT (Roll- that is consistent with the observation that HDAC6 preferentially Mecak and Vale, 2008). Consistently, antibodies that recognize a deacetylates unpolymerized tubulin (Matsuyama et al., 2002). terminal E-residue (Rudiger et al., 1999) reduce spastin-mediated microtubule severing activity in vitro (Roll-Mecak and Vale, 2008). Tubulin PTMs regulate the assembly of To reconcile the microtubule stabilizing and destabilizing activities microtubular organelles

Journal of Cell Science of tubulin glutamylation observed in various contexts, we propose Centrioles, basal bodies and cilia contain exceptionally stable and that the outcomes of this PTM depend on the specific structure of extensively post-translationally modified microtubules. In most the glutamyl side chain (length and positions) and the availability organisms, the microtubules in these organelles are compound of microtubule effectors (either stabilizing MAPs or severing polymers with tubules that have fused walls (triplets in basal bodies factors). and centrioles, doublets in axonemes). Tubulin PTMs, and The conserved -tubulin acetylation at K40 has only a subtle specifically detyrosination and polymodifications, have crucial effect on microtubule dynamics. Tetrahymena mutants assemble roles in the assembly, maintenance and function of complex diverse microtubules from -tubulin that contains the K40R microtubule-based organelles. Male pcd mice are infertile and mutation and cannot be acetylated (Gaertig et al., 1995), and mice produce fewer and slowly moving sperm with variable axonemal with excessively highly acetylated -tubulin (after deletion of defects (Fernandez-Gonzalez et al., 2002; Handel and Dawson, HDAC6 deacetylase) appear almost normal except for subtle 1981). In Chlamydomonas, detyrosinated tubulin is primarily increases in bone mineral content and reduced immune responses present on the outer doublets and the PTM levels gradually increase (Zhang et al., 2008). However, in cultured mammalian cells, broad after axoneme assembly (Johnson, 1998), suggesting that the PTM inhibition of HDACs with trichostatin A increases the level of is not needed for axoneme assembly but can stabilize axonemal acetyl-K40 -tubulin and causes stabilization of microtubules microtubules. because of their increased resistance to nocodazole (Matsuyama et Tubulin acetylation is also linked to ciliogenesis in mammals. al., 2002). Fibroblasts isolated from Hdac6-null mice have In cultured human retinal pigment epithelial (RPE1) cells, loss of stabilized microtubules that show shorter depolymerization events BBIP10, a Bardet-Biedl syndrome 4 (BBS4)-interacting protein, and increased resistance to nocodazole (Tran et al., 2007). However, inhibits the assembly of primary cilia and, at the same time, the potential microtubule-stabilizing effect of acetyl-K40 in strongly decreases the levels of acetylated cytoplasmic microtubules mammalian cells must be subtle because other studies have failed (Loktev et al., 2008). Also in RPE1 cells, inhibition of HDAC6 to detect a change in the parameters of microtubule dynamics in with tubacin blocks resorption of primary cilia (Pugacheva et al., response to manipulations of HDAC6 levels (Haggarty et al., 2003; 2007), pointing to a role for -tubulin K40 deacetylation in the Palazzo et al., 2003). In one study, inhibition of HDAC6 with depolymerization of axonemal microtubules. However, it is not tubacin (Haggarty et al., 2003), reduced the rate of microtubule clear whether BBIP10 and HDAC6 affect cilia through the growth, but no such effect was observed after depletion of HDAC6 acetylation and/or deacetylation of K40 on -tubulin or by other when using RNAi (Zilberman et al., 2009). mechanisms. In particular, HDAC6 deacetylates or binds to several Post-translational modifications of microtubules 3451

non-tubulin proteins (for a review, see Valenzuela-Fernandez et al., that drive intraflagellar transport (IFT), a motility pathway required 2008). It has already been reported that, in C. elegans, HDAC6 for the assembly and maintenance of cilia (for a review, see affects cilia in the chemosensory neurons, despite the fact that this Pedersen and Rosenbaum, 2008). There is already evidence that cell type does not detectably express the only isotype of -tubulin tubulin PTMs, including glutamylation, affect diverse motor with K40, MEC-12 (Fukushige et al., 1999; Li et al., 2010). proteins (see next section). Moreover, in protist models, -tubulin acetylation is not needed Tubulin glycylation might also have a role in axoneme assembly. for ciliogenesis (Akella et al., 2010; Gaertig et al., 1995; Kozminski In Tetrahymena, a knockout of TTLL3, a chain-initiating G-ligase, et al., 1993). Thus, in mammals the acetylation and/or deacetylation causes shortening of axonemes (Wloga et al., 2009). The depletion regulators are important in the assembly and disassembly of cilia, of TTLL3 expression in zebrafish leads to shortening and complete but it is not clear whether these activities are mediated by the loss of axonemes in the olfactory epithelium and Kupffer’s vesicle acetylation and/or deacetylation of -tubulin. (Wloga et al., 2009). siRNA-induced depletion of TTLL3 in Tubulin glutamylation has an increasingly well-documented role Drosophila arrests the assembly of sperm axonemes at the in the assembly and functions of complex microtubular organelles. individualization stage (Rogowski et al., 2009). It is probable that, Injection of antibodies that recognize glutamylation into at least to some extent, tubulin glycylation influences the process mammalian cells induces disassembly of centrioles (Bobinnec et of axoneme assembly through competitive inhibition of tubulin al., 1998a). In Tetrahymena, loss of TTLL1 and TTLL9 E-ligases glutamylation. Indeed, overproduction of the TTLL6 E-ligase in causes defects in the docking of nascent basal bodies to the plasma Tetrahymena inhibits axoneme assembly (Wloga et al., 2010). membrane (Wloga et al., 2008). A morpholino-mediated depletion However, a complicating factor in the interpretation of these TTLL3 of the TTLL6 E-ligase in zebrafish is associated with shortening studies is that, on the basis of observations in Drosophila, several and loss of olfactory cilia (Pathak et al., 2007). In mice, mutations non-tubulin proteins are glycylated by TTLL3 (Rogowski et al., in either the TTLL1 E-ligase or associated protein PGs1 cause 2009). various defects in the sperm axoneme that are associated with male In mammals and most other organisms that have ciliated lineages, infertility (Campbell et al., 2002; Ikegami et al., 2010; Regnard et TTLL10 catalyzes the elongation of glycyl side chains that are al., 2003; Vogel et al., 2010). Curiously, the Ttll1-deficient mice initiated by TTLL3 (Rogowski et al., 2009). Unexpectedly, in assemble structurally normal cilia in the trachea, despite low levels humans (but not in other mammals), the tubulin glycyl side chains of tubulin glutamylation (but the motility of these cilia is strongly are not elongated, a finding established because of the lack in affected; see below) (Ikegami et al., 2010; Vogel et al., 2010). As reactivity of these side chains to an antibody specifically discussed earlier for cytoplasmic microtubules, during axoneme recognizing polyglycylation (Bre et al., 1996; Rogowski et al., assembly tubulin glutamylation can also act by regulating the 2009). Although humans have a TTLL10 gene (Rogowski et microtubule-severing factors, specifically katanin. In Tetrahymena, al., 2009), the protein is enzymatically inactive on tubulin, owing substitution of aspartate for three glutamic acid residues that serve to the two amino acid substitutions that are human specific as polymodification sites on -tubulin result in assembly of (Rogowski et al., 2009). Rogowski and colleagues propose that a paralyzed cilia that have excessively short doublets and lack central non-elongated glycyl side chain is sufficient for axoneme assembly, microtubules. The same Tetrahymena mutants undergo an arrest in probably because it fulfills the proposed main function of

Journal of Cell Science cytokinesis associated with an overgrowth of longitudinal glycylation: competitive inhibition of glutamylation (Rogowski et microtubule bundles (that resemble the midbody microtubules in al., 2009). The elongation of glycyl side chains that occurs in most mammalian cells) (Thazhath et al., 2004; Thazhath et al., 2002). ciliated lineages could have additional functions, such as the Importantly, knockouts of genes encoding subunits of the katanin generation of specific patterns of sperm motility. There is already complex produce a set of defects in cytokinesis and axoneme strong evidence that elongation of glutamyl side chains regulates assembly that are strikingly similar to the phenotype observed in ciliary motility (see below). the -tubulin polymodification-site mutants (Sharma et al., 2007). Consistently, a mutation in the katanin regulatory subunit p80 Tubulin PTMs influence navigation of motor inhibits the assembly of central axonemal microtubules in proteins on microtubules Chlamydomonas reinhardtii (Dymek et al., 2004). Subunits of the Growing evidence indicates that tubulin PTMs provide structural katanin complex are present inside cilia and are enriched on doublet cues for motor proteins. In mammalian neurons, the motor protein microtubules (Dymek et al., 2004; Sharma et al., 2007). KIF5 (a member of the kinesin-1 protein family) preferentially enters Consistently, the axonemal doublets and, in particular, the B- the axon (Jacobson et al., 2006; Nakata and Hirokawa, 2003). The subfiber are enriched in tubulin polymodifications (Kann et al., axonal shaft is enriched in acetylated and detyrosinated microtubules, 1995; Kubo et al., 2010; Lechtreck and Geimer, 2000; Multigner as compared with the dendrites (for a review, see Fukushima et al., et al., 1996; Suryavanshi et al., 2010). In the context of axoneme 2009). In fibroblasts, kinesin-1 also associates preferentially with assembly, katanin most probably interacts with glutamylated and microtubules that are enriched in acetylation and detyrosination (Cai not with glycylated tubulin, because Tetrahymena Ttll3-null cells et al., 2009; Dunn et al., 2008). In vitro, the binding affinity of that lack almost all tubulin glycylation assemble shortened but kinesin-1 to microtubules increases with increased quantities of K40- structurally normal 9+2 axonemes (Wloga et al., 2009). It remains acetylated -tubulin (Dompierre et al., 2007; Konishi and Setou, to be determined whether the function of katanin inside cilia 2009; Reed et al., 2006). Stabilization of microtubules by paclitaxel involves destabilization of the microtubule lattice or a new activity. in differentiating neurons elevates the levels of tubulin PTMs, In addition to the effects on katanin, tubulin glutamylation could including -tubulin acetylation on K40 and detyrosination, and affect complex microtubular organelles through its effects on motor promotes the accumulation of kinesin-1 and other axonal markers in proteins that transport precursors required for the assembly and multiple neurites (Hammond et al., 2010; Witte and Bradke, 2008). maintenance of these organelles. For example, tubulin glutamylation However, the unequal distribution of the acetylated tubulin between could regulate motors proteins (e.g. kinesin-2 and DH1b dynein) the axon and dendrites might not be sufficient for the preferential 3452 Journal of Cell Science 123 (20)

accumulation of kinesin-1, because inhibition of HDAC6 activity induces excessive levels of K40-acetylated -tubulin in multiple neurites, but does not affect the biased entry of kinesin-1 into the presumptive axon in differentiating neurons (Hammond et al., 2010; Witte and Bradke, 2008). However, overexpression of a nonacetylatable K40A mutant -tubulin in cortical neurons inhibits the migration and branching of neurite projections (Creppe et al., 2009), and decreases the velocity of brain-derived neurotrophic factor (BDNF)-associated vesicles that move along microtubules (Dompierre et al., 2007). Loss of MEC-17 and its paralog W06B11.1 in C. elegans leads to a loss of acetyl-K40 MEC-12 -tubulin and strong functional deficiency of touch receptor neurons (Akella et al., 2010). Adult C. elegans nematodes that express only a K40R or K40Q mutant MEC-12 -tubulin have a reduced touch response, but the remaining level of touch response is higher when compared with animals that lack both TATs MEC-17 or W06B11.1 (Akella et al., 2010). One possible explanation is that MEC-17 acetylates a K residue on - or -tubulin distinct from K40 (Choudhary et al., 2009). An antibody against acetylated K failed to react with microtubules that contain K40R -tubulin in Tetrahymena (Akella et al., 2010). However, we cannot exclude the possibility that MEC- 17 acetylates a K residue(s) distinct from K40 on animal tubulin in vivo. Alternatively, in animals, MEC-17 might acetylate a non- Key tubulin protein that is important in neurons. In zebrafish, depletion ODA Unmodified MT of MEC-17 expression using morpholinos decreases the levels of Kinesin-1 IDA Modified MT acetyl-K40 -tubulin in a number of different neurons and is associated with developmental defects, including decreased head CLIP-170 Katanin, spastin Kinesin-13 and eye size, a curved body axis and reduced mobility (Akella et al., 2010). It remains to be determined to what extent the significance of Fig. 2. Tubulin PTMs regulate diverse activities by increasing the activity MEC-17 in vertebrates involves its function as an TAT. of microtubule effectors. Tubulin modifications regulate diverse activities Tubulin detyrosination has an important role in regulating motors including motor protein movement, microtubule severing, microtubule-end in the context of neural differentiation. Compared with dendrites, polymerization and activity of +TIPs. See text for further details. the axon is enriched in detyrosinated -tubulin (Konishi and Setou, 2009). In vitro, kinesin-1 binds more strongly to microtubules

Journal of Cell Science enriched in detyrosinated -tubulin (Konishi and Setou, 2009; by exerting transient forces on the B-tubule of an adjacent doublet Liao and Gundersen, 1998). In hippocampal neurons, depletion of (for a review, see Lindemann and Lesich, 2010). Tubulin TTL by using RNAi increases the levels of detyrosination in glutamylation was first implicated in ciliary motility based on the dendrites and leads to increased entry of kinesin-1 into dendrites observation that anti-glutamylated protein antibodies inhibit (Konishi and Setou, 2009). Konishi and Setou propose that the motility of demembranated cilia exposed to ATP (Gagnon et tyrosinated tubulin acts as a negative cue that excludes kinesin-1 al., 1996). Mutants deficient in either the TTLL9 E-ligase in from dendritic microtubules (Konishi and Setou, 2009). Chlamydomonas or TTLL6 E-ligase in Tetrahymena have greatly Interestingly, in vitro, kinesin-1 moves more slowly along reduced rates of cilia-dependent cell motility, but apparently normal microtubules that contain predominantly detyrosinated tubulin axonemes. In both protists, loss of the E-ligase activity reduces the (obtained by carboxypeptidase treatment), as compared with beat frequency and, in Tetrahymena, results in a grossly abnormal untreated microtubules (Dunn et al., 2008). waveform (Kubo et al., 2010; Suryavanshi et al., 2010). TTLL9 Tubulin glutamylation also influences motors in the nervous and TTLL6 homologs are associated with activities that elongate system. Neurons of mice that carry a mutation in PGs1, a subunit glutamyl side chains (Suryavanshi et al., 2010; van Dijk et al., of the -tubulin-specific E-ligase complex TTLL1, have reduced 2007; Wloga et al., 2008). Thus, in protists, non-elongated glutamyl levels of -tubulin glutamylation and kinesin-3 KIF1A, and these side chains might be sufficient for axoneme assembly, but their changes are associated with abnormal synaptic transmission elongation is needed for ciliary motility. In both protists, tubulin (Ikegami et al., 2007). Although the TTLL1-dependent tubulin glutamylation is present mainly on the outer doublet microtubules; glutamylation is not essential in mice, it could have a subtle role within the doublets, the PTM is mainly present on the B-subfiber in the brain – a suggestion based on the observation that male (Kubo et al., 2010; Suryavanshi et al., 2010), the surface to which Ttll1-null mice are less aggressive than male wild-type mice dynein-arm motor domains bind during the ATP-dependent cross- (Campbell et al., 2002). bridging cycle that underlies ciliary motility. Recent studies have uncovered a key function of tubulin There are two types of dynein arms: inner (IDA) and outer glutamylation in ciliary motility. The beating of cilia is driven by (ODA). ODAs strongly affect the beat frequency, whereas IDAs coordinated activity of axonemal dynein-arm motors. In a typical mainly affect the waveform (for a review, see Kamiya, 2002). 9+2 motile axoneme (Fig. 2), each of the nine doublets has a Remarkably, both protist studies link tubulin glutamylation complete A-subfiber and an incomplete B-subfiber. Dynein arms specifically to IDAs (Kubo et al., 2010; Suryavanshi et al., 2010). are stably attached to the A-subfiber and cause axoneme bending Chlamydomonas cells that lack TTLL9 and ODAs have paralyzed Post-translational modifications of microtubules 3453

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