Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2014 Apr;16(4):335-44.
doi: 10.1038/ncb2920. Epub 2014 Mar 16.

Regulation of microtubule motors by tubulin isotypes and post-translational modifications

Minhajuddin Sirajuddin  1 , Luke M Rice  2 , Ronald D Vale  1
Affiliations

Regulation of microtubule motors by tubulin isotypes and post-translational modifications

Minhajuddin Sirajuddin et al. Nat Cell Biol. 2014 Apr.

Abstract

The 'tubulin-code' hypothesis proposes that different tubulin genes or post-translational modifications (PTMs), which mainly confer variation in the carboxy-terminal tail (CTT), result in unique interactions with microtubule-associated proteins for specific cellular functions. However, the inability to isolate distinct and homogeneous tubulin species has hindered biochemical testing of this hypothesis. Here, we have engineered 25 α/β-tubulin heterodimers with distinct CTTs and PTMs and tested their interactions with four different molecular motors using single-molecule assays. Our results show that tubulin isotypes and PTMs can govern motor velocity, processivity and microtubule depolymerization rates, with substantial changes conferred by even single amino acid variation. Revealing the importance and specificity of PTMs, we show that kinesin-1 motility on neuronal β-tubulin (TUBB3) is increased by polyglutamylation and that robust kinesin-2 motility requires detyrosination of α-tubulin. Our results also show that different molecular motors recognize distinctive tubulin 'signatures', which supports the premise of the tubulin-code hypothesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Recombinant tubulin for testing the role of CTTs in motor function
a. Structure and sequence of the α (light grey) and β (dark grey) tubulin cores (ending with helix 12; pdb 4FFB) and the C-terminal tails (CTTs; red), which are disordered and extend from the polymer. Chimeric tubulins were created with the yeast core fused to the CTTs of TUBA1A/TUBB2A human tubulin at the junction shown. b & d. Histograms of kinesin-1 motor velocity and run lengths on yeast MTs (red), yeast core with TUBA1A/TUBB2A CTTs (blue) and porcine brain microtubules (grey). c & e. Single molecule motility of human kinesin-1, -2 and yeast dynein motor on yeast WT, yeast core-TUBA1A/TUBB2A CTT chimeric and porcine brain microtubules (mean and s.e.m, n = 3 independent experiments, each containing >100 motor measurements, for statistics source data see Supplementary Table 1), for absolute values see Table 1; mean velocity and run length were determined as described in Methods.
Figure 2
Figure 2. Minimal CTT requirement for motor function
a. Illustration of recombinant tubulins with TUBA1A/TUBB2A CTTs (blue), TUBB2A CTT alone (green), TUBA1A CTT alone (red) or no (Δ) CTTs (grey). Helix 12 highlighted in yellow and CTTs in red. b. Velocity and processivity of human kinesin-1, -2 and dynein was compared as a ratio to TUBA1A/TUBB2A CTT chimeric microtubule (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values).
Figure 3
Figure 3. The effects of the α-tubulin C-terminal tyrosine on motor performance
a. Microtubules were prepared from tubulin with (TUBA1A/TUBB2A) or without (TUBA1A-ΔY/TUBB2A) the C-terminal tyrosine (Y) on α-tubulin. b. The fold velocity and processivity of de-tyrosinated (ΔY) over tyrosinated (Y) microtubules is shown (mean and s.e.m, n = 3 independent experiments; see Supplementary Table 1 for individual experiment values and Table 1 for absolute values). c. The effect of the C-terminal α-tubulin tyrosine on microtubule depolymerization by mammalian kinesin-13; the kymographs (time versus microtubule end position) show the decrease in the length of the microtubule polymer over time. Histograms of kinesin-13 microtubule depolymerization rates of tyrosinated (TUBA1A/TUBB2A) and de-tyrosinated (TUBA1A-ΔY/TUBB2A) microtubules. The mean and s.d. were 1.5 ± 0.5 (n = 130) and 0.5 ± 0.2 (n = 92) respectively; n represents the number of microtubule ends analyzed. d. Kinesin-13 depolymerization activity with no α-CTT (Δα), the complete TUBA1A-CTT with its genetically encoded C-terminal tyrosine (α+Y), or the TUBA1A-CTT lacking this tyrosine (αΔY); experiments were performed with a tubulin heterodimer lacking β-CTT (Δβ) or containing the TUBB2A-CTT. Similar results were obtained with the βIV-CTT from two independent experiments (for absolute values and sample number see Table 1).
Figure 4
Figure 4. Effects of polyglutamylation on motor performance
a. To study how polyglutamylation affects motor function, peptide of either 3 or 10 glutamates (3E or 10E) was crosslinked using maleimide chemistry to a single cysteine in the CTT (see Methods). A 10E peptide is shown in this illustration. b. The crosslinked reaction product is shown by SDS PAGE as reflected by an upward gel shift of the indicated α- and β-tubulin bands (estimated ~60% crosslinked product). In a control without the introduced cysteine in the CTT, this gel shift did not occur, indicating the crosslinking occurred at the correct position of the CTT (Supplementary Figure 6). c and d. The ratio of the motor velocity (red hatched), processivity (solid red) or microtubule depolymerization rate (solid blue) on uncrosslinked versus crosslinked microtubules is shown (mean and s.e.m; n = 3 independent experiments, see Supplementary Table 1). Absolute values are shown in Table 1.
Figure 5
Figure 5. Motility regulation by β-tubulin isotypes
a. Sequence alignment of helix 12 (yellow) and CTTs (red) of human β-tubulin isotypes. The basic residues in TUBB1 and TUBB3 CTTs are highlighted in blue. b. Kinesin-1 and dynein motors velocity and processivity values measured against various β-tubulin isotypes as indicated (in Δα-CTT background). Mean velocity and run length were determined as described in Fig 1. c. Run length histograms of kinesin-1 motors moving on TUBB3 (blue) and TUBB3ΔK (red) microtubules (mean run length of 0.6 and 1.35 μm respectively). Examples of raw kymographs (inset panel; scale, 2 sec and 1.5 μm).
Figure 6
Figure 6. Cross-regulation of isotype specificity by polyglutamylation
a. Illustration of TUBA1A-E452C/TUBB3 heterodimer crosslinking strategy with 10E. b. SDS-PAGE of crosslinked product of 10 and 3 glutamic acid peptides (10E and 3E) as indicated in Fig. 4. c. Kinesin-1 motility kymographs (inlet panel) and run length histograms of TUBB3/TUBA1A- E452C and +10E (red) microtubule (mean run lengths of 0.5 and 1.5 μm respectively). Scale bars and bin size are similar to 4c. c. The ratio of the motor velocity (red hatched), processivity (solid red) on uncrosslinked versus 10E and 3E crosslinked TUBA1A- E452C/TUBB3 microtubules is shown (mean and s.e.m; n = 3 independent experiments, see Supplementary Table 1). Absolute values are shown in Table 1.
Figure 7
Figure 7. Summary of CTT mediated effects on different motors
α/β-tubulin heterodimer in light and dark grey respectively with helix 12 colored in yellow and unstructured C-terminal tails (CTTs) in red. K (blue), Y (yellow) and the EEEx (red) represent the TUBB3 β-isotype, tyrosination in α-tubulin and polyglutamylation respectively. The specific inhibitory and enhancing effects of tubulin variations on different motors are as indicated.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Nogales E. Structural insight into microtubule function. Annu Rev Biophys Biomol Struct. 2001;30:397–420. - PubMed
    1. Amos LA, Schlieper D. Microtubules and maps. Adv Protein Chem. 2005;71:257–298. - PubMed
    1. Ludueña RF, Banerjee A. Role Microtubules Cell Biol Neurobiol Oncol. Humana Press; 2008. pp. 123–175. (MD, T. F.) at < http://link.springer.com/chapter/10.1007/978-1-59745-336-3_6>. - DOI
    1. Banerjee A, et al. A monoclonal antibody against the type II isotype of beta-tubulin. Preparation of isotypically altered tubulin. J Biol Chem. 1988;263:3029–3034. - PubMed
    1. Burgoyne RD, Cambray-Deakin MA, Lewis SA, Sarkar S, Cowan NJ. Differential distribution of beta-tubulin isotypes in cerebellum. EMBO J. 1988;7:2311–2319. - PMC - PubMed

Publication types

-