Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation
- Department of Cell and Developmental Biology, University of Michigan, United States
- Department of Biophysics, University of Michigan, United States
- Department of Biomedical Engineering, University of Michigan, United States
- Department of Biological Chemistry, University of Michigan, United States
- Department of Molecular, Cellular and Developmental Biology, University of Michigan, United States
- Graduate School of Science and Technology, Kumamoto University, Japan
- International Research Organization for Advanced Science and Technology, Kumamoto University, Japan
Figures
Figure 1 with 3 supplements
Three probes that recognize the Y-αCTT.
(A, B) Schematic of tubulin protein and its conformational states within the microtubule lattice. (A) Schematic of tubulin heterodimer with the unstructured C-terminal tails (CTTs) protruding from the body of α- and β-tubulin. (B) Tubulin adds to the end of a microtubule in a GTP-bound and expanded state, resulting in a stabilizing GTP cap. In the microtubule lattice, β-tubulin undergoes GTP hydrolysis, resulting in a GDP lattice and compaction of α-tubulin. (C) Schematic of three sensors (YL1/2, A1aY1, and 4xCAPGly) generated to detect the accessibility of the Y-αCTT along the microtubule lattice. (D, E) Generation and validation of the YL1/2Fab probe. (D) Schematic of antibody proteins. YL1/2 IgG: typical mammalian IgG molecule containing two heavy (H) and two light (L) chains. Light chains are comprised of one variable (VL, light orange) and one constant (CL, dark orange) region. Heavy chains are comprised of one variable (VH, light gray) and three constant (CH1–3, dark gray) regions. Red dots indicate complementarity determining regions (CDRs) and purple lines indicate disulfide bonds. rMAb-EGFP: recombinant monoclonal antibody (rMAb) with EGFP (green star) fused to the C-terminus of the light chain. YL1/2Fab-EGFP: Fragment antibody binding (Fab) produced from rMAb-EGFP by papain cleavage. (E) GST-tagged αCTT sequences were probed by western blotting with (left) commercial YL1/2 monoclonal antibody, (middle) rMAb-YL1/2-EGFP, or (right) YL1/2Fab-EGFP. Each blot was also probed for GST protein as a loading control. Y: full-length and tyrosinated αCTT sequence; ΔY: detyrosinated αCTT sequence (lacking the C-terminal tyrosine); ΔC2: αCTT sequence lacking the C-terminal two amino acids. (F) Schematic of synthetic protein A1aY1 tagged at its N-terminus with sTag-RFP (red star). (G) Schematic of the domain organization of (top) full-length CLIP-170 and (bottom) the 4xCAPGly probe tagged at its C-terminus with mEGFP (green star). (H, I) Representative images of (H) YL1/2Fab-EGFP or (I) 4xCAPGly-mEGFP proteins binding to tyrosinated (Y–MT) or detyrosinated (ΔY-MT) microtubules. HeLa tubulin was polymerized into microtubules and Taxol-stabilized. The microtubules were used directly (Y-MTs) or detyrosinated by VASH/SVBP-containing lysate before adding the (H) YL1/2Fab-EGFP or (I) 4xCAPGly-mEGFP probes. Scale bars: 5 µm.
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Figure 1—source data 1
TIFF files of original western blots.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig1-data1-v1.zip
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Figure 1—source data 2
PDF file containing original gels for panel E indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig1-data2-v1.zip
Figure 1—figure supplement 1
Recombinant YL1/2 antibody and purified probes.
(A) Experimentally determined amino acid sequence of YL1/2 protein. The deduced amino acid sequences of YL1/2 IgG heavy and light chains are shown. The red text indicates the complementarity determining regions (CDRs) involved in antigen recognition. Asterisks demarcate every 10 aa. (B) Coomassie-stained SDS-PAGE gel of purified proteins. (C) Validation of 4xCAPGly specificity for the Y-αCTT. GST-tagged αCTT sequences were probed by far-western blotting with purified 4xCAPGly-mEGFP protein and with an antibody against GST protein as a loading control. Y: full-length and tyrosinated αCTT sequence; ΔY: detyrosinated αCTT sequence (lacking the C-terminal tyrosine); ΔC2: αCTT sequence lacking the C-terminal two amino acids.
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Figure 1—figure supplement 1—source data 1
PDF file containing original gels for panels B and C indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
TIFF files of original western blots.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig1-figsupp1-data2-v1.zip
Figure 1—figure supplement 2
The A1aY1 probe must be imaged in live cells.
COS-7 cells transiently expressing sTagRFP-A1aY1 and mEGFP were (A) imaged live, (B) fixed and mounted, or (C) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.
Figure 1—figure supplement 3
The 4xCAPGly probe must be imaged in live cells.
COS-7 cells transiently expressing 4xCAPGly-mSc3 and mEGFP were (A) imaged live, (B) fixed and mounted, or (C) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.
Figure 2 with 1 supplement
Y-αCTT probes bind to the microtubule lattice after Taxol treatment.
(A–D) Live-cell imaging of A1aY1 probe. (A) Representative images of the sTagRFP-A1aY1 HeLa stable cell line before and 15 min after treatment with (top) 0.3% DMSO control or (bottom) 10 µM Taxol. Insets (boxes) show magnified views of A1aY1 probe. Scale bars: 10 µm in whole-cell views and 2 µm in magnified views. (B, C) Quantification of probe binding to microtubules. Paired data plots display the amount of A1aY1 probe bound to microtubules in individual cells before and after treatment with (B) DMSO or (C) Taxol. (D) Mean difference plot showing the fold change in A1aY1 probe binding in DMSO- vs Taxol-treated cells. DMSO = 29 cells across three experiments; Taxol = 59 cells across seven experiments. (E–H) Live-cell imaging of 4xCAPGly probe. (E) Representative images of the 4xCAPGly-mEGFP HeLa stable cell line before and 15 min after treatment with (top) 0.3% DMSO control or (bottom) 10 µM Taxol. Insets (boxes) show magnified views of CAPGly probe. Scale bars: 10 µm in whole-cell views and 2 µm in magnified views. (F, G) Quantification of probe binding to microtubules. Paired data plots display the amount of 4xCAPGly probe bound to microtubules in individual cells before and after treatment with (F) DMSO or (G) Taxol. (H) Mean difference plot showing the fold change in 4xCAPGly probe binding in DMSO- or Taxol-treated cells. DMSO = 28 cells across three experiments; Taxol = 17 cells across five experiments. Error bars in (D) and (H) indicate SD. ***: p<0.0002; ****: p<0.0001; ns: not significant Student’s t test (B, C, F, G: two-tailed; paired), (D, H: unpaired).
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Figure 2—source data 1
Excel file with fluorescence intensity measurements.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig2-data1-v1.xlsx
Figure 2—figure supplement 1
Controls for probe binding in response to Taxol-mediated lattice expansion.
(A, B) Y-αCTT probes bind to the microtubule lattice after Taxol-induced expansion. Representative images of (A) sTagRFP-A1aY1 or (B) 4xCAPGly-mEGFP probes transiently expressed in (top) HeLa or (bottom) COS-7 cells and imaged live before or 15 min after addition of 10 µM Taxol. Scale bars: 10 µm. (C) EB3-EGFP is rapidly evicted from microtubules after Taxol addition. HeLa cells stably expressing EB3-EGFP were imaged live before (0 min) and at the indicated time points after addition of 2 µM Taxol. Scale bar: 10 µm.
Figure 3
MAPs that expand the microtubule lattice increase Y-αCTT probe binding.
(A, B) Representative live-cell images of (A) A1aY1 or (B) 4xCAPGly HeLa stable cell lines transiently expressing the indicated mEGFP-tagged tubulin or MAP constructs. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. (C, D) Quantification of (C) sTag-RFP-A1aY1 or (D) CAPGly-mSc3 probe colocalization with mEGFP-tagged tubulin or MAP constructs. The threshold overlap score (TOS) was measured on a per-cell basis where 1.0 indicates perfect colocalization, –1.0 indicates perfect anti-colocalization, and values near 0 indicate no relationship. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. *: p<0.1; **: p<0.001; ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with TubA1A as the control). Number of cells analyzed (n) in (C): TubA1A=69, Tau = 57, MAP2=66, Kif5Crigor = 70, CAMSAP2=50, CAMSAP3=56, and MAP7=55 and in (D): TubA1A=62, Tau = 61, MAP2=61, Kif5C rigor = 76, CAMSAP2=62, CAMSAP3=71, and MAP7=57. (E) Schematic model depicting how expander and compactor MAPs regulate microtubule lattice conformation, influencing Y-αCTT accessibility.
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Figure 3—source data 1
Excel file with fluorescence intensity measurements.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig3-data1-v1.xlsx
Figure 4
MAPs that expand the MT lattice increase detyrosination of the Y-αCTT.
(A) Representative images of HeLa cells transiently expressing the indicated mEGFP-tagged MAPs and then fixed and stained with antibodies against detyrosinated microtubules (∆Y-tubulin) and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing the corresponding MAPs. Scale bars: 20 µm.(B) Quantification of the intensity of detyrosination on MAP-bound microtubules. The fluorescence intensity of detyrosination was measured on MAP-decorated microtubules and normalized against the total microtubule intensity of MAP-decorated microtubules. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 67, MAP2=70, Kif5Crigor = 75, CAMSAP2=67, CAMSAP3=70, and MAP7=67. (C) Quantification of the colocalization of MAPs and detyrosinated microtubules. The threshold overlap score (TOS) was measured on a per-cell basis. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 70, MAP2=72, Kif5Crigor = 76, CAMSAP2=66, CAMSAP3=69, and MAP7=67.
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Figure 4—source data 1
Excel file with fluorescence intensity measurements.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig4-data1-v1.xlsx
Figure 5 with 1 supplement
GTP-like tubulin state increases Y-αCTT accessibility and detyrosination.
(A–D) Live-cell imaging of Y-αCTT probes. (A, C) Representative images of (A) sTagRFP-A1aY1 or (C) 4xCAPGly-mSc3 HeLa stable cell lines transiently expressing PA-tagged WT or E254A α-tubulin with an IRES-driven mEGFP protein as a reporter of transfected cells. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. (B, D) Quantification of (B) A1aY1 or (D) 4xCAPGly probe binding to microtubules. The density was measured as the ratio of the skeletonized probe-decorated microtubule length to the total cell area. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. **: p<0.01 (Mann-Whitney U test). Number of cells analyzed (n) in (B): WT = 33, E254A=29 and in (D): WT = 42, E254A=50. (E,F) Detyrosinated microtubules. (E) Representative images of HeLa cells transiently expressing PA-tagged WT or E254A α-tubulin (TubA1A) and then fixed and stained with antibodies against the PA tag, detyrosinated microtubules (∆Y-tubulin), and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing α-tubulin. Scale bars: 20 µm. (F) Quantification of the intensity of detyrosination in cells expressing PA-tagged WT or E254A α-tubulin. The fluorescence intensity of detyrosination was measured on a per-cell basis and normalized against the total microtubule intensity. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with the untransfected sample (untr.) as the control). Number of cells analyzed (n): untransfected = 105, WT = 84, E254A=94.
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Figure 5—source data 1
Excel file with fluorescence intensity measurements.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig5-data1-v1.xlsx
Figure 5—figure supplement 1
Controls for nucleotide-mediated lattice expansion.
(A,B) Western blot of HeLa cells transiently expressing internal PA-tagged α-tubulin TubA1A. (A) sTagRFP-A1aY1 stable HeLa cells or (B) 4xCAPGly-mSc3 stable HeLa cells were untransfected (untr.) or transfected with plasmids for expressing PA-tagged WT or E254A α-tubulin (TubA1A). Whole cell lysates were prepared and analyzed by immunoblotting with the antibodies indicated on the left side of the blots. Size markers in kD are indicated on the right side of the blots. Asterisks denote upshifted PA-tagged tubulin bands.
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Figure 5—figure supplement 1—source data 1
PDF file containing original gels for panels A and B indicating the relevant bands.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig5-figsupp1-data1-v1.zip
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Figure 5—figure supplement 1—source data 2
TIFF files of original western blots.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig5-figsupp1-data2-v1.zip
Figure 6 with 1 supplement
MAPs but not nucleotide state promote Y-αCTT exposure in vitro.
(A–D) Nucleotide state does not determine probe binding to microtubules polymerized in vitro. (A,C) Representative images of (A) 4xCAPGly-mEGFP or (C) YL1/2Fab-GFP probe binding to a mixture containing both AlexaFluor-568 labeled GMPCPP-stabilized microtubules and AlexaFluor-647 labeled GDP microtubules. Scale bars: 5 µm. (B,D) Quantification of the fluorescence intensity of (B) 4xCAPGly-mEGFP or (D) YL1/2Fab-EGFP probe binding per length of microtubule. Data are presented as scatter plots with data from three independent experiments in different shades of gray. ns: not significant (two-tailed, Student’s t test). (E–G) Stepping KIF5C can increase YL1/2Fab probe binding. (E) Flowchart of the in vitro reconstitution assay examining the effect of KIF5C(1-560) stepping on YL1/2Fab binding. (F) Representative images of YL1/2Fab-GFP probe binding to GDP-MTs in the absence or presence of KIF5C(1-560) in different nucleotide states. Scale bar: 5 µm. (G) Quantification of the mean fluorescence intensity of YL1/2Fab-GFP probe along GDP-MTs under the conditions shown in (F). Each spot indicates the probe binding on an individual microtubule. Number of microtubules (n)=74–105 from three independent experiments. ns, not significant, ****p<0.0001 (two-tailed, t-test).
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Figure 6—source data 1
Excel file with fluorescence intensity measurements.
- https://cdn.elifesciences.org/articles/109308/elife-109308-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Controls for washout of strongly-bound (apo) KIF5C.
(A,B) Wash out of strongly-bound (apo) KIF5C. (A) Representative images of KIF5C(1-560)-Halo554 binding to GDP-MTs in ATP (stepping) or no nucleotide (apo) conditions. In the washout condition, strongly-bound (apo) KIF5C was released from the microtubules by washing the flow chamber with buffer containing 3 mM ATP and 300 mM KCl. Scale bar: 5 µm. (B) Quantification of the mean fluorescence intensity of KIF5C(1-560)-Halo554 along GDP-MTs under the conditions shown in (A). Each spot indicates KIF5C(1-560)-Halo554 fluorescence intensity on an individual microtubule. Number of microtubules (n)=24–46 from two independent experiments. ****p<0.0001 (two-tailed, t-test).
Figure 7 with 1 supplement
The nucleotide state alters Y-αCTT interactions with the microtubule body.
(A) Representative image from MD simulations identifying four distinct sites where the Y-αCTT interacts with the body of tubulin subunits in the microtubule: sites 1 (green) and 2 (cyan) are on the adjacent β-tubulin along a protofilament (i.e. next tubulin towards the microtubule minus end), whereas sites 3 (purple) and 4 (salmon) are cis-interactions with α-tubulin itself. The tubulin body is shown in cartoon and colored gray. The Y-αCTT is shown in stick and colored yellow with the aspartate and glutamate side chains in red: 438-DSVEGEGEEEGEEY-451. (B) Interaction rate of the glutamate residues in the Y-αCTT with the four tubulin body sites for microtubules in the (gold) GDP or (blue) GTP states. (C) The fraction of Y-αCTTs that are inaccessible as a function of time for microtubules in the (gold) GDP or (blue) GTP state where inaccessibility is defined as one or more salt bridges formed between glutamates E445-E450 and the interaction sites in the microtubule body.
Figure 7—figure supplement 1
The Y-αCTT primarily contacts four sites in the tubulin body within a GDP microtubule lattice.
Jaccard index plot indicating the frequency of two residues simultaneously forming salt bridges with the Y-αCTT based on molecular dynamics simulations of a GDP microtubule lattice. The scale represents the Jaccard index where an index of 0 indicates that the two residues are never interacting with the Y-αCTT at the same time and an index of 1 indicates that when one of the two residues is interacting with the Y-αCTT, the other is also interacting with the Y-αCTT.
Author response image 1
Videos
Video 1
Movie of Y-αCTT interacting with the tubulin body from molecular dynamics (MD) simulations.
Representative movie from MD simulations showing interactions of the Y-αCTT with the tubulin body over 240 ns. The tubulin body is shown in cartoon and colored gray. The αCTT is shown in stick and colored yellow with the glutamate side chains in red. Residues in site 1 (green), site 2 (cyan), and site 3 (magenta) appear as spheres when the αCTT is forming salt bridges with those residues.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-β-tubulin, mouse monoclonal, clone E7 | DSHB | Cat# E7; RRID:AB_528499 | 1:1000 IF |
| Antibody | Anti-PA tag, rat monoclonal, clone NZ-1 | FUJIFILM Wako Pure Chemicals | Cat# 016–25861 | 1:1000 WB, 1:500 IF |
| Antibody | Anti-detyrosinated α-tubulin, rabbit monoclonal, clone RM444 | RevMAb Biosciences | Cat# 31-1335-00 | 0.1 µg/ml WB, 0.5 µg/ml IF |
| Antibody | Anti-tyrosinated α-tubulin, rat monoclonal, clone YL1/2 | Bio-Rad | Cat# MCA77G | 1:1000 WB |
| Antibody | rMAb-YL1/2-EGFP recombinant protein | This study | 1:1000 WB; Verhey lab | |
| Antibody | Anti-α-tubulin, mouse monoclonal, clone DM1α | MilliporeSigma | Cat# 05–829 | 1:3000 WB |
| Antibody | Anti-GFP, chicken polyclonal | Aves labs | Cat# GFP-1010 | 1:1000 IF |
| Antibody | Anti-GST, mouse monoclonal | Nacalai USA | Cat# 04435–26 | 1:1000 WB |
| Antibody | Anti-GAPDH, mouse monoclonal, clone G-9 | Santa Cruz | Cat# sc-365062 | 1:2000 WB |
| Antibody | Anti-TagRFP, mouse monoclonal, clone 6A11f | Kerafast | Cat# EFH005 | 1:1000 WB |
| Antibody | Anti-RFP (mScarlet), rabbit polyclonal | Rockland Immunochemicals | Cat# 600-401-379 | 1:1000 WB |
| Antibody | Anti-mouse IgG Alexa Fluor 680 AffiniPure, donkey polyclonal | Jackson ImmunoResearch | Cat# 715-625-150 | 1:500 IF |
| Antibody | Anti-rabbit IgG Alexa Fluor 594, goat polyclonal | Thermo Fisher | Cat# A-11012 | 1:1000 IF |
| Antibody | Anti-rat IgG Alexa Fluor 488 | Thermo Fisher | Cat# A-11006 | 1:1000 IF |
| Antibody | Anti-chicken IgY Alexa Fluor 488 | Thermo Fisher | Cat# A-11039 | 1:1000 IF |
| Antibody | Anti-α-tubulin Alexa Fluor 647, mouse monoclonal, clone DM1α | Millipore Sigma | Cat# 05–829-AF647 | 1:500 IF |
| Antibody | Anti-rat IgG Alexa Fluor 680, goat | Thermo Fisher | Cat# A-21096 | 1:5,000 WB |
| Antibody | Anti-mouse IgG Alexa Fluor 700, goat | Thermo Fisher | Cat# A-21036 | 1:5,000 WB |
| Antibody | Anti-mouse IgG DyLight 800, goat | Thermo Fisher | Cat# SA5-10176 | 1:10,000 WB |
| Antibody | Anti-rat IgG DyLight 800, donkey | Thermo Fisher | Cat# SA5-10032 | 1:5,000 WB |
| Antibody | Anti-rabbit IgG IRDye 800CW, goat | LI-COR | Cat# 926–32211 | 1:10,000 WB |
| Peptide, recombinant protein | Biotinylated porcine brain tubulin | Cytoskeleton | Cat# T333P | |
| Peptide, recombinant protein | HiLyte647 porcine brain tubulin | Cytoskeleton | Cat# TL670M | |
| Peptide, recombinant protein | HiLyte488 porcine brain tubulin | Cytoskeleton | Cat# TL488M | |
| Peptide, recombinant protein | Bovine brain tubulin | Ohi lab | ||
| Peptide, recombinant protein | HeLa S3 tubulin | Ohi and Verhey labs (Thomas et al., 2025) | ||
| Peptide, recombinant protein | Alexa Fluor 568 HeLa S3 tubulin | Ohi and Verhey labs (Thomas et al., 2025) | ||
| Peptide, recombinant protein | Alexa Fluor 647 HeLa S3 tubulin | Ohi and Verhey labs (Thomas et al., 2025) | ||
| Chemical compound, drug | Taxol | Cytoskeleton | Cat# TXD01 | |
| Chemical compound, drug | puromycin | MilliporeSigma | Cat# P8833 | |
| Chemical compound, drug | doxycycline | Thermo Fisher | Cat# BP26531 | |
| Chemical compound, drug | Lipofectamine 2000 | Thermo Fisher | Cat# 11668019 | |
| Chemical compound, drug | Opti-MEM | Thermo Fisher | Cat# 31985070 | |
| Chemical compound, drug | Bovine serum albumin | MilliporeSigma | Cat# A9647 | |
| Chemical compound, drug | Casein | MilliporeSigma | Cat# C8654 | |
| Chemical compound, drug | Glycerol | Thermo Fisher | Cat# BP229-4 | |
| Chemical compound, | Glucose | MilliporeSigma | Cat# G7528 | |
| Chemical compound, | Glucose oxidase | MilliporeSigma | Cat# G7141-10KU | |
| Chemical compound, | Catalase | MilliporeSigma | Cat# C3515 | |
| Chemical compound, | GTP | MilliporeSigma | Cat# G8877 | |
| Chemical compound, | GMPCPP | Jena Bioscience | Cat# NU405S | |
| Chemical compound, | ADP | MilliporeSigma | Cat# A2754 | |
| Chemical compound, | Hexokinase | MilliporeSigma | Cat# H5000 | |
| Chemical compound, | Apyrase | MilliporeSigma | Cat# A6535 | |
| Chemical compound, | ATP | MilliporeSigma | Cat# A7699 | |
| Chemical compound, | Janelia Fluor X 554 (JFX554) Halo ligand | Janelia Farms | Cat# JFX554 | |
| Chemical compound, | BSA-biotin | MilliporeSigma | Cat# A8549 | |
| Chemical compound, | NeutrAvidin | Thermo Fisher | Cat# 31000 | |
| Chemical compound, | Fish skin gelatin (FSG) | MilliporeSigma | Cat# G7765 | |
| Chemical compound, | Lysozyme | MilliporeSigma | Cat# L6876 | |
| Chemical compound, | SIGMAFAST protease inhibitor cocktail | MilliporeSigma | Cat# S8830 | |
| Chemical compound, | Benzonase nuclease | MilliporeSigma | Cat# E1014 | |
| Chemical compound, | Biotin | MilliporeSigma | Cat# B4501 | |
| Strain, strain background (Escherichia coli) | DH5α | Thermo Fisher | Cat# 18258–012 | |
| Strain, strain background (Escherichia coli) | Rosetta2(DE3)pLysS | Novagen | Cat# 71403–3 | |
| Strain, strain background (Escherichia coli) | BL21-CodonPlus-RILC | Aligent Technologies | Cat# 230245 | |
| Strain, strain background (Escherichia coli) | DH10Bac | Thermo Fisher | Cat# 10361012 | |
| Cell line (Ceropithecus aethiops) | COS-7 cells, male kidney fibroblast | ATCC | RRID:CVCL_0224 | |
| Other | Dulbecco’s modified Eagle medium (DMEM) | Gibco, Thermo Fisher | Cat# 11960044 | |
| Other | Fetal Clone III | HyClone | Cat# SH3010903 | |
| Other | GlutaMAX (L-alanyl-L-glutamine dipeptide in 0.85% NaCl) | Gibco, Thermo Fisher | Cat# 35050061 | |
| Cell line (Homo sapiens) | HeLa Kyoto cells, female | Shuh Narumiya | RRID:CVCL_1922 | |
| Cell line (Homo sapiens) | Knock-in HeLa Kyoto cell lines expressing 4xCAPGly-mEGFP, 4xCAPGly-mSc3 or sTagRFP-A1aY1 | This study | Ohi lab | |
| Other | Dulbecco’s modified Eagle medium (DMEM) | Gibco, Thermo Fisher | Cat# 11965118 | |
| Other | Fetal Bovine Serum (FBS) | Cytiva | Cat# SH3007103T | |
| Chemical compound, drug | Penicillin-Streptomycin | Gibco, Thermo Fisher | Cat# 15140122 | |
| Cell line (Homo sapiens) | HeLa S3 cells, female | ATCC (CCL-2.2) | RRID:CVCL_0058 | |
| Other | Dulbecco’s modified Eagle medium (DMEM) | Gibco, Thermo Fisher | Cat# 11965092 | |
| Other | Leibovitz’s L-15 Medium | Gibco, Thermo Fisher | Cat# 21083027 | |
| Cell line (Spodoptera frugiperda) | Sf9 cells | Thermo Fisher | Cat# 11496015, RRID:CVCL_JX36 | |
| Other | sf900 II SFM medium | Thermo Fisher | Cat# 10902088 | |
| Chemical compound, drug | Antibiotic antimycotic | Gibco, Thermo Fisher | Cat# 15240062 | |
| Chemical compound, drug | Cellfectin II | Thermo Fisher | Cat# 10362100 | |
| Other | Grace’s insect cell culture medium | Gibco, Thermo Fisher | Cat# 11595–030 | |
| Other | Poly-D-lysine coated glass-bottom dishes | MatTek | Cat# P35GC-1.5–14 C | |
| Other | Prolong Gold | Thermo Fisher | Cat# P36930 | |
| Other | Prolong Diamond | Thermo Fisher | Cat# P36970 | |
| Other | HiLoad 16/600 Superdex 200 prep grade column | Cytiva | Cat# 28989335 | |
| Other | Strep-Tactin XT 4Flow resin | Iba Life Sciences | Cat# 2-5010-002 | |
| Commercial assay, kit | Fluorescent Protein Labeling Kit | Thermo Fisher | Cat# A10235 | |
| Commercial assay, kit | Fab Preparation Kit | Thermo Fisher | Cat# 44985 | |
| Commercial assay, kit | HiPure Plasmid DNA miniprep kit | Thermo Fisher | Cat# K20003 | |
| Recombinant DNA reagent | pN1-4xCAPGly-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pN1-4xCAPGly-mSc3 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pEM791-4xCAPGly-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pEM791-4xCAPGly-mS3 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pET15b-6×His-4xCAPGly-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pC1-superTagRFP-A1aY1 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pEM791-superTagRFP-A1aY1 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pET15b-6xHis-A1aY1-superTagRFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-internal PA-TubA1A-IRES-EGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-internal PA-TubA1A(E254A)-IRES-EGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-internal PA-TubA1A | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-internal PA-TubA1A(E254A) | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-mEGFP-TubA WT | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-mEGFP-TubA WT | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pPA-MAP2-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pPA-MAP7-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pN1-KIFC(1–560,G235A)-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pmEGFP-Tau | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pmEGFP-CAMSAP2 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pmEGFP-CAMSAP3 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-mEGFP-Tau | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS-MAP2-mEGFP | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS- mEGFP-CAMSAP2 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pCAGGS- mEGFP-CAMSAP3 | This study | Lab plasmid | Ohi and Verhey labs |
| Recombinant DNA reagent | pFastBac1- KIF5C(1-560)-Halo-2xstrepII | This study | Lab plasmid | Ohi and Verhey labs |
| Software, algorithm | Fiji/ImageJ | Schindelin et al., 2012 | https://fiji.sc/ | |
| Software, algorithm | Fiji Lpx_bilevelThin plugin | Higaki et al., 2010 | ||
| Software, algorithm | Fiji Ridge Detection plugin | Wagner and Hiner, 2017 | ||
| Software, algorithm | Fiji EzColocalization plugin | Stauffer et al., 2018 | ||
| Software, algorithm | AIVIA | DRVision | ||
| Software, algorithm | R version 4.3.1 | R Core Team | https://www.R-project.org/ | |
| Software, algorithm | Bio3D | Grant et al., 2006 | ||
| Software, algorithm | Prism version 10.4.1 | GraphPad Software | https://www.graphpad.com | |
| Software, algorithm | Adobe Illustrator version 29.0 | Adobe |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/109308/elife-109308-mdarchecklist1-v1.docx
-
Supplementary file 1
Sequences of oligonucleotide primers.
- https://cdn.elifesciences.org/articles/109308/elife-109308-supp1-v1.docx
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Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation
eLife 14:RP109308.
https://doi.org/10.7554/eLife.109308.3
