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Typical elution profiles from each chromatography step (A and B) Elution profile of recombinant human a1B/b3-tubulin from two 1 mL HisTrap columns (A) and two 1 mL StrepTrap columns (B). (C) The chromatogram of the flow-through from HiTrap SP/HisTrap HP columns. (D and E) Elution profile of recombinant human a1B/b3-tubulin (D; peak volume, 81.1 mL) or bovine brain tubulin (E; peak volume, 80.4 mL) from size-exclusion chromatography. *, non-specific instrument singal. V 0 , void volume. 1CV, one column volume.
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α/β-tubulin heterodimers, which can harbor diverse isotypes and post-translational modifications, polymerize into microtubules that are fundamental to many cellular processes. Due to long-standing challenges in generating recombinant tubulin, however, it has been difficult to examine the properties of specific tubulin isotypes. Here, we provide a p...
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Context 1
... the second column and elute the first column until baseline, then attach the second column and continue the elution until baseline. See Figure 5A for a typical elution profile we observe from the two 1 mL HisTrap HP columns. ...
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... the second column and elute the first column until baseline, then attach the second column and continue the elution until baseline. See Figure 5B for the elution profile from two 1 mL StrepTrap HP columns. ...
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... and collect another 10 mL of strep elution buffer through the columns to maximize protein recovery. Figure 5C shows the chromatogram of the flow-through from HiTrap SP/HisTrap HP columns of purification with an above average yield of tubulin ($4 mg from 1.2 L cell culture). The absorbance of the flow-through at 280 nm is slightly higher than the background absorbance of the strep elution buffer. ...
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... quick way to do this is to compare the size exclusion trace from Step 69 with a sample of mammalian brain tubulin diluted in size exclusion buffer and run under identical flow rates. In both cases, tubulin should elute as a single peak, and the peaks from both runs should appear almost identical in shape and retention volume position ( Figures 5D and 5E). ...
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... Recombinant human tubulins (tubulin isotypes, α1A /βIII and α1A-Y /βIII in pFAST TM -Dual vector) were expressed and purified using a modified protocol from previous studies 56,70 . Briefly, 1 ng of plasmid DNA was transformed into 50 μl of DH10Bac competent cells, positive colonies were verified by PCR and subcultured for bacmid DNA, and the recombinant tubulins were expressed in Sf9 cells using the same methods described for KIF13B proteins. ...
Microtubules are major components of the eukaryotic cytoskeleton. Posttranslational modifications (PTMs) of tubulin regulates interactions with microtubule-associated proteins (MAPs). One unique PTM is the cyclical removal and re-addition of the C-terminal tyrosine of α-tubulin and MAPs containing CAP-Gly domains specifically recognize tyrosinated microtubules. KIF13B, a long-distance transport kinesin, contains a conserved CAP-Gly domain, but the role of the CAP-Gly domain in KIF13B’s motility along microtubules remains unknown. To address this, we investigate the interaction between KIF13B’s CAP-Gly domain, and tyrosinated microtubules. We find that KIF13B’s CAP-Gly domain influences the initial motor-microtubule interaction, as well as processive motility along microtubules. The effect of the CAP-Gly domain is enhanced when the motor domain is in the ADP state, suggesting an interplay between the N-terminal motor domain and C-terminal CAP-Gly domain. These results reveal that specialized kinesin tail domains play active roles in the initiation and continuation of motor movement.
... Further, while no ALS-related residues appear on the surface that mediates interactions with motor proteins, disruptions to protofilament assembly could alter the binding or movement of microtubule motors. Testing these ideas directly will require specific systems that use only the TUBA4A isotype (Davis et al., 1993;Sackett et al., 2010;Ti et al., 2020). However, experiments from COS7 cells overexpressing TUBA4A (W407X) suggest that both microtubule tracks and polymerization are compromised ( Fig. 3B) (Smith et al., 2014). ...
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by more than sixty genes identified through classic linkage analysis and new sequencing methods. Yet no clear mechanism of onset, cure, or effective treatment is known. Popular discourse classifies the proteins encoded from ALS-related genes into four disrupted processes: proteostasis, mitochondrial function and ROS, nucleic acid regulation, and cytoskeletal dynamics. Surprisingly, the mechanisms detailing the contribution of the neuronal cytoskeletal in ALS are the least explored, despite involvement in these cell processes. Eight genes directly regulate properties of cytoskeleton function and are essential for the health and survival of motor neurons, including: TUBA4A, SPAST, KIF5A, DCTN1, NF, PRPH, ALS2, and PFN1. Here we review the properties and studies exploring the contribution of each of these genes to ALS.
... Our systematic evaluation indicated that both the composition and position (N-or C-terminus) of the fused polypeptide are critical for the yield of functional human tubulin. To achieve optimal cleavage efficiency of the affinity tags, we incorporated a TEV-cleavable decahistidine tag with an Ala-Pro dipeptide linker to the N-terminus of human α1B-tubulin and a TEV-cleavable strep tag with a Gly-Gly-Ser-Gly-Gly pentapeptide linker to the C-terminus of human β2-and β3-tubulin (Ti et al., 2018;Ti et al., 2020). We note that the enzymatic digestion gets rid of the affinity tags but leaves residual 'scars' at the N-terminus of the α-tubulin (Gly-Ala-Pro) and the C-terminus of the β-tubulin (Glu-Asn-Leu-Tyr-Phe-Gln). We speculate that combining our approach with other protein engineering tools (e.g., protein ligation) will generate recombinant human tubulin with native sequence. ...
Microtubules are cytoskeletal filaments underlying the morphology and functions of all eukaryotic cells. In higher eukaryotes, the basic building blocks of these non-covalent polymers, ɑ- and β-tubulins, are encoded by expanded tubulin family genes (i.e., isotypes) at distinct loci in the genome. While ɑ/β-tubulin heterodimers have been isolated and examined for more than 50 years, how tubulin isotypes contribute to the microtubule organization and functions that support diverse cellular architectures remains a fundamental question. To address this knowledge gap, in vitro reconstitution of microtubules with purified ɑ/β-tubulin proteins has been employed for biochemical and biophysical characterization. These in vitro assays have provided mechanistic insights into the regulation of microtubule dynamics, stability, and interactions with other associated proteins. Here we survey the evolving strategies of generating purified ɑ/β-tubulin heterodimers and highlight the advances in tubulin protein biochemistry that shed light on the roles of tubulin isotypes in determining microtubule structures and properties.
... a1A only has two and eight different amino acids with a1B and a1C, respectively (Supplementary Figure S1A). To determine the polymerization properties of these a-tubulin isotypes, we expressed and purified a1A/b2A, a1B/b2A, and a1C/b2A tubulin heterodimers from insect cells using a protocol modified from the previously published methods (Minoura et al., 2013;Vemu et al., 2016;Ti et al., 2020). Homogeneous and single-isotype mouse a/b-tubulins were purified through a double-selection strategy using His-tagged a-tubulin isotypes (a1A, a1B, and a1C) and a Flag-tagged b-tubulin isotype (b2A) ( Figure 1A). ...
Microtubules consisting of α/β-tubulin dimers play critical roles in cells. More than seven genes encode α-tubulin in vertebrates. However, the property of microtubules composed of different α-tubulin isotypes is largely unknown. Here, we purified recombinant tubulin heterodimers of mouse α-tubulin isotypes including α1A and α1C with β-tubulin isotype β2A. In vitro microtubule reconstitution assay detected that α1C/β2A microtubules grew faster and underwent catastrophe less frequently than α1A/β2A microtubules. Generation of chimeric tail-swapped and point-mutation tubulins revealed that the carboxyl-terminal (C-terminal) tails of α-tubulin isotypes largely accounted for the differences in polymerization dynamics of α1A/β2A and α1C/β2A microtubules. Kinetics analysis showed that in comparison to α1A/β2A microtubules, α1C/β2A microtubules displayed higher on-rate, lower off-rate, and similar GTP hydrolysis rate at the plus-end, suggesting a contribution of higher plus-end affinity to faster growth and less frequent catastrophe of α1C/β2A microtubules. Furthermore, EB1 had a higher binding ability to α1C/β2A microtubules than to α1A/β2A ones, which could also be attributed to the difference in the C-terminal tails of these two α-tubulin isotypes. Thus, α-tubulin isotypes diversify microtubule properties, which, to a great extent, could be accounted by their C-terminal tails.
... Here, we took a biochemical reconstitution approach to define the minimal components required to methylate tubulin in vitro. Using purified tSETD2 and recombinant human tubulin (23)(24)(25) enabled precise control of tubulin isotype and PTMstate in vitro and allowed us to generate mutant versions of both proteins to probe site selectivity of SETD2 methylation. We demonstrate that tSETD2 is sufficient to methylate tubulin in vitro and has a higher activity towards tubulin dimers over microtubules. ...
... To determine if there are other methylation sites on tubulin, we utilized recombinant human tubulin. Recent advances in the expression and purification of recombinant single-isotype tubulin (23)(24)(25) allowed us to purify human αTub1B/βTub3 tubulin dimers (hereafter referred to as αβ-tubulin) from insect cells using the baculovirus expression system (Fig. 3A-B). This system allowed us to mutate the known methylation site K40 on α-tubulin by purifying αβ-tubulin dimers with a mutation of K40 to alanine [αβ-tubulin(αK40A)]. ...
... An active truncated SETD2 construct (1418-2564) with a FLAG affinity tag (tSETD2-FLAG) in the pInducer vector for mammalian expression was generated by the Walker Lab. Single isoform αTub1B/βTub3 plasmid cDNA encoding Homo sapiens α-tubulin 1B (αTub1B, NP_006073.2) and β-tubulin 3 (βTub3, NM_178012.4) in pFastBac Dual vector (ThermoFisher 10712024) was obtained from the Kapoor lab for insect cell expression (25). Point mutations and domain deletions were generated using QuickChange sitedirected mutagenesis with Q5 Polymerase (NEB). ...
Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. It was recently shown that methylation of tubulin by the histone methyltransferase SETD2 occurs on mitotic spindle microtubules during cell division, with its absence resulting in mitotic defects. However, the catalytic mechanism of methyl addition to tubulin is unclear. We used a truncated version of human SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2 Rpb1 interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 had a higher activity towards tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrated that methylation was restricted to lysine 40 (K40) of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain (R1625C) allowed tSETD2 to bind to tubulin but not methylate it, whereas a mutation in the SRI domain (R2510H) caused loss of both tubulin binding and methylation. Further investigation of the role of the SRI domain in substrate binding and found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus the binding partner RNA Polymerase II for methylating histones in vivo, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Lastly, we found that substrate recognition also requires the negatively-charged C-terminal tail of α-tubulin. Together, this study provides a framework for understanding how SETD2 serves as a dual methyltransferase for both histone and tubulin methylation.
... Research Resource Identifier: Addg-ene_8827; Kapust et al., 2001). TEV was expressed in BL21-CodonPlus (DE3)-RIL and purified using Ni-nitrilotriacetic acid (NTA) and gel filtration following the methods described in Ti et al. (2020). 1 mg of TEV protease (stored in 40 mM Hepes, pH 7.5, 30% [wt/vol] glycerol, 150 mM KCl, 1 mM MgCl 2 , and 3 mM 2-mercaptoethanol) was diluted into 1 ml of gel filtration buffer and injected onto the NHStrap column, and proteolysis was allowed to proceed for 2 h at 4°C. ...
The formation of cellular microtubule networks is regulated by the γ-tubulin ring complex (γ-TuRC). This ∼2.3 MD assembly of >31 proteins includes γ-tubulin and GCP2-6, as well as MZT1 and an actin-like protein in a “lumenal bridge” (LB). The challenge of reconstituting the γ-TuRC has limited dissections of its assembly and function. Here, we report a biochemical reconstitution of the human γ-TuRC (γ-TuRC-GFP) as a ∼35 S complex that nucleates microtubules in vitro. In addition, we generate a subcomplex, γ-TuRCΔLB-GFP, which lacks MZT1 and actin. We show that γ-TuRCΔLB-GFP nucleates microtubules in a guanine nucleotide–dependent manner and with similar efficiency as the holocomplex. Electron microscopy reveals that γ-TuRC-GFP resembles the native γ-TuRC architecture, while γ-TuRCΔLB-GFP adopts a partial cone shape presenting only 8–10 γ-tubulin subunits and lacks a well-ordered lumenal bridge. Our results show that the γ-TuRC can be reconstituted using a limited set of proteins and suggest that the LB facilitates the self-assembly of regulatory interfaces around a microtubule-nucleating “core” in the holocomplex.
... Here, we took a biochemical reconstitution approach to define the minimal components required to methylate tubulin in vitro. By utilizing both purified tSETD2 and recombinant human tubulin (21)(22)(23) we had precise control of tubulin isotype and PTM-state in vitro and allowed us to generate mutant versions to probe site selectivity of SETD2 methylation. We demonstrate that tSETD2 is sufficient to methylate tubulin in vitro and has a higher activity towards tubulin dimers over microtubules. ...
... Single isoform αTub1B/βTub3 plasmid cDNA encoding Homo sapiens α-tubulin 1B (NP_006073.2), β-tubulin 3 (NM_178012.4) in pFastBac Dual vector (ThermoFisher 10712024) was obtained from the Kapoor lab for insect cell expression (23). Point mutations and domain deletions were generated using QuickChange site-directed mutagenesis with Q5 Polymerase (NEB). ...
... αTub1B/βTub3 tubulin. Purification was as previously described (23,64). Briefly, the Bac-to-Bac system (Life Technologies) was used to generate recombinant baculovirus in SF9 cells. ...
Post-translational modifications to tubulin are important for many microtubule-based functions inside cells. A recently identified tubulin modification, methylation, occurs on mitotic spindle microtubules during cell division, and is enzymatically added to tubulin by the histone methyltransferase SETD2. We used a truncated version of human SETD2 (tSETD2) containing the catalytic SET and C-terminal Set2 Rpb1 interacting (SRI) domains to investigate the biochemical mechanism of tubulin methylation. We found that recombinant tSETD2 has a higher activity towards tubulin dimers than polymerized microtubules. Using recombinant single-isotype tubulin, we demonstrate that methylation is restricted to lysine 40 (K40) of α-tubulin. We then introduced pathogenic mutations into tSETD2 to probe the recognition of histone and tubulin substrates. A mutation in the catalytic domain, R1625C, bound to tubulin but could not methylate it whereas a mutation in the SRI domain, R2510H, caused loss of both tubulin binding and methylation. We thus further probed a role for the SRI domain in substrate binding and found that mutations within this region had differential effects on the ability of tSETD2 to bind to tubulin versus RNA Polymerase II substrates, suggesting distinct mechanisms for tubulin and histone methylation by SETD2. Lastly, we found that substrate recognition also requires the negatively-charged C-terminal tail of α-tubulin. Together, this work provides a framework for understanding how SETD2 serves as a dual methyltransferase for histone and tubulin methylation.
Tubulin posttranslational modifications (PTMs) modulate the dynamic properties of microtubules and their interactions with other proteins. However, the effects of tubulin PTMs were often revealed indirectly through the deletion of modifying enzymes or the overexpression of tubulin mutants. In this study, we directly edited the endogenous tubulin loci to install PTM-mimicking or-disabling mutations and studied their effects on microtubule stability, neurite outgrowth, axonal regeneration, cargo transport, and sensory functions in the touch receptor neurons of Caenorhabditis elegans . We found that the status of β-tubulin S172 phosphorylation and K252 acetylation strongly affected microtubule dynamics, neurite growth, and regeneration, whereas α-tubulin K40 acetylation had little influence. Polyglutamylation and detyrosination in the tubulin C-terminal tail had more subtle effects on microtubule stability likely by modulating the interaction with kinesin-13. Overall, our study systematically assessed and compared several tubulin PTMs for their impacts on neuronal differentiation and regeneration and established an in vivo platform to test the function of tubulin PTMs in neurons.
Microtubule nucleation in cells is templated by the gamma-tubulin ring complex (gamma-TuRC), a 2.3 MDa multiprotein assembly concentrated at microtubule organizing centers (MTOCs). Current gamma-TuRC structures exhibit an open conformation that deviates from the geometry of alpha/beta-tubulin in the microtubule, potentially explaining their low in vitro microtubule-nucleating activity. Several proteins have been proposed to activate the gamma-TuRC, but the mechanisms underlying activation are not known. Here, we isolated the porcine gamma-TuRC using CDK5RAP2's centrosomin motif 1 (CM1) and determined its structure with cryo-electron microscopy. 3D heterogeneity analysis revealed an unexpected conformation of the gamma-TuRC, in which five protein modules containing MZT2, GCP2, and CDK5RAP2 decorate the outer face of the holocomplex. These decorations drive a long-range constriction of the gamma-tubulin ring, bringing the GCP2/GCP3-rich core of the complex in close agreement with the architecture of a microtubule. A purified CDK5RAP2 fragment stimulated the microtubule nucleating activity of the porcine gamma-TuRC as well as a reconstituted, CM1-free human complex in single molecule assays. Our results show that CDK5RAP2 activates the gamma-TuRC by promoting gamma-tubulin ring closure, providing a structural mechanism for the regulation of microtubule nucleation by CM1 motif proteins in mammals and revealing conformational transitions in gamma-tubulin that prime it for templating microtubule nucleation at MTOCs.
Tubulin isotypes are critical for the functions of cellular microtubules, which exhibit different stability and harbor various post-translational modifications. However, how tubulin isotypes determine the activities of regulators for microtubule stability and modifications remains unknown. Here, we show that human α4A-tubulin, a conserved genetically detyrosinated α-tubulin isotype, is a poor substrate for enzymatic tyrosination. To examine the stability of microtubules reconstituted with defined tubulin compositions, we develop a strategy to site-specifically label recombinant human tubulin for single-molecule TIRF microscopy-based in vitro assays. The incorporation of α4A-tubulin into the microtubule lattice stabilizes the polymers from passive and MCAK-stimulated depolymerization. Further characterization reveals that the compositions of α-tubulin isotypes and tyrosination/detyrosination states allow graded control for the microtubule binding and the depolymerization activities of MCAK. Together, our results uncover the tubulin isotype-dependent enzyme activity for an integrated regulation of α-tubulin tyrosination/detyrosination states and microtubule stability, two well-correlated features of cellular microtubules.
