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 4xCAP-Gly) 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.

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 3 experiments; Taxol=59 cells across 7 experiments. (E-H) Live-cell imaging of 4xCAPGly probe. (E) Representative images of the 4xCAPGly-mEGFP HeLa stable cell line before and 15 minutes 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 3 experiments; Taxol=17 cells across 5 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).

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.

MAPs that expand the MT lattice increase detyrosination of the Y-aCTT.

(A) Representative images of HeLa cells transiently expressing the indicated mEGFP-tagged MAPs and then fixed and stained with antibodies against detyrosinated microtubules (ΔY-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.

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 (ΔY-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.

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).

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 sidechains 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.

Recombinant YL1/2 antibody and purified probes.

(A) Experimentally-determined amino acid sequence 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.

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.

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.

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.

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.

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 wash out 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).

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.