Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules
- University of Washington, United States
Figures
Figure 1 with 3 supplements
Ndc80 complex microtubule binding is auto-inhibited by the Spc24/Spc25 dimer.
(A) Cartoon of hypothesized model of auto-inhibition of the Ndc80 complex. Red arrow indicates hypothesized regions of intra-complex interactions. Black arrow indicates calponin homology domains on …
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Figure 1—source data 1
Residence times on microtubules measured for individual complexes of Ndc80, Broccoli, and Ndc80-MIND co-complexes, without and with the addition of Spc24/Spc25 dimer.
- https://doi.org/10.7554/eLife.44489.008
Figure 1—figure supplement 1
Purifications of Ndc80 complex constructs.
(A) (Left) Representative Superdex 200 16/60 elution profile of Broccoli-GFP, normalized to highest peak. Green bar shows SEC fractions collected. Dotted black line shows elution profile of gel …
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Figure 1—figure supplement 1—source data 1
Residences times on microtubules measured for Ndc80 complexes, without and with the addition of a large excess of Spc24/Spc25 dimer.
- https://doi.org/10.7554/eLife.44489.004
Figure 1—figure supplement 2
Representative kymographs of TIRF microscopy assays.
(A) Representative kymographs of each condition measured in Figure 1B,C. Brightness and contrast have been adjusted separately for each image for best visualization.
Figure 1—figure supplement 3
Spc24/Spc25 V159D dimer can inhibit the Ndc80-MIND co-complex.
(A) Coomassie blue-stained gel showing Spc24/Spc25 V159D. (B) Representative Superose 6 increase 3.2/300 elution profile of Spc24/Spc25, Spc24/Spc25 V159D, MIND, MIND +Spc24/Spc25 and MIND +Spc24/Spc…
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Figure 1—figure supplement 3—source data 1
Residence times on microtubules measured for individual Ndc80-MIND co-complexes, without and with the addition of mutant Spc24/Spc25 V159D dimer.
- https://doi.org/10.7554/eLife.44489.007
Figure 2 with 1 supplement
The Ndc80 complex can tightly bend.
(A) List of mutations made in the wild type Ndc80 complex to generate cysteine-light construct. (B) Model of the Ndc80 complex using the dwarf tetramer structure (DOI: 10.2210/pdb5TCS/pdb) plus …
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Figure 2—source data 1
Residence times on microtubules measured for individual, dual end-labeled Ndc80 complexes.
- https://doi.org/10.7554/eLife.44489.011
Figure 2—figure supplement 1
A functional Ndc80 complex can be sequentially end-labeled.
(A) Schematic of sequential end-labeling procotol for the Ndc80 complex used in bulk FRET assay. (B) (Left) Coomassie blue-stained gel showing end-labeled Ndc80 complex used in bulk FRET assay (SEC …
Figure 3 with 1 supplement
The Ndc80 complex fluctuates between tightly bent and more open conformations.
(A) (Left) Cartoon depicting method of tethering labeled control oligonucleotides to glass coverslip. (Left and middle) Cartoons show the distance in nanometers between FRET pair dyes for positive …
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Figure 3—source data 1
Analysis of threshold crossing from records of Ndc80 FRET versus time.
- https://doi.org/10.7554/eLife.44489.015
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Figure 3—source data 2
All individual FRET values measured for oligonucleotide controls and Ndc80 complexes.
- https://doi.org/10.7554/eLife.44489.016
Figure 3—figure supplement 1
Concurrent labeling of the Ndc80 complex is used for single molecule FRET assays.
(A) Schematic of concurrent end-labeling procotol for Ndc80 complex constructs used in single molecule FRET assays. (B) (Left) Coomassie blue-stained gel showing end-labeled Ndc80 complex used in …
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Figure 3—figure supplement 1—source data 1
Ratios of donor emission enhancement after photobleaching the acceptor dyes versus before photobleaching, measured for individual Ndc80 complexes and oligonucleotide control molecules.
- https://doi.org/10.7554/eLife.44489.014
Figure 4 with 2 supplements
The loop region of Ndc80 contributes to tight bending of the complex.
(A) (Top) Primary sequence of Ndc80 protein amino acids 460–520. The purple bar highlights the region of the loop deleted for the following experiment. (Bottom) Graph of probability of coiled-coil …
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Figure 4—source data 1
All individual FRET values and residence times on microtubules measured for individual mutant Ndc80 ∆490-510 loop-deletion complexes.
- https://doi.org/10.7554/eLife.44489.021
Figure 4—figure supplement 1
Model of the Ndc80 complex with all combinations of dye locations.
(A) Model of the Ndc80 complex using the dwarf tetramer structure (DOI: 10.2210/pdb5TCS/pdb) plus inserted coiled coil between the tetramerization domain and globular domain of each dimer. Break in …
Figure 4—figure supplement 2
The Ndc80 complex has preferred orientations for intra-complex interactions.
(A) (Top) Fluorescence traces for one representative example of each combination of dye pairs for end-labeled Ndc80 complexes before and after photobleaching acceptor (Alexa Fluor 647). (Bottom) …
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Figure 4—figure supplement 2—source data 1
All individual FRET values measured for individual Ndc80 complexes with four pairwise combinations of dye locations.
- https://doi.org/10.7554/eLife.44489.020
Figure 5 with 1 supplement
Tight bending of the Ndc80 complex is inhibited by binding to either microtubules or the MIND complex.
(A) Fluorescence image of two end-labeled Ndc80 complexes (Nuf2 S2C Spc25 S154C) on Alexa Fluor 488 labeled-microtubules. Colors are off-set vertically. (B) (Top) Fluorescence traces of one …
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Figure 5—source data 1
All individual FRET values measured for individual Ndc80 complexes bound to microtubules or bound to the MIND complex.
- https://doi.org/10.7554/eLife.44489.025
Figure 5—figure supplement 1
Reproducibility of Ndc80 complex single molecule FRET measurements.
(A) (Left) Histograms of corrected FRET values for Ndc80 complex Nuf2 S2C Spc25 S154C (n = 1395, 48 particles), Ndc80 complex Nuf2 S2C Spc25 S154C measured from slides with MIND-GFP, but not …
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Figure 5—figure supplement 1—source data 1
All individual FRET values measured for individual Ndc80 complexes during technical and biological replicate experiments.
- https://doi.org/10.7554/eLife.44489.024
Tables
Key resources table
| Reagent type or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, E. coli | Rosetta (DE3) competent cells | Millipore Sigma | Cat#70954 | |
| Biological sample | Bovine brain tubulin | lab purification | Protocol adapted from Castoldi and Popov, 2003. | |
| Sequence-based reagent | Fluorescently-labeled oligonucleotides | IDT | ||
| Sequence-based reagent | Fluorescently-labeled, biotintylated oligonucleotides | IDT | ||
| Antibody | Penta-HIS biotin conjugate, monoclonal mouse | Qiagen | Cat#34440 | Diluted to 20 nM |
| Chemical compound | Alexa Fluor 488 succinimidyl ester | Thermo Fisher | Cat#A20000 | |
| Chemical compound | Alexa Fluor 647 maleimide | Thermo Fisher | Cat#A20347 | |
| Chemical compound | Alexa Fluor 568 succinimidyl ester | Thermo Fisher | Cat#A20003 | |
| Chemical compound | Cy3 maleimide | GE Healthcare Life Sciences | Cat#PA23031 | |
| Chemical compound | Cy5 maleimide | GE Healthcare Life Sciences | Cat#PA25031 | |
| Chemical compound | Trolox | Millipore Sigma | Cat#238813 | |
| Chemical compound | Glucose oxidase | Millipore Sigma | Cat#345386 | |
| Chemical compound | Catalase | Millipore Sigma | Cat#219261 | |
| Chemical compound | POPC | Avanti Polar Lipids | Cat#850457 | 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine |
| Chemical compound | Bio-cap-PE/BioPE | Avanti Polar Lipids | Cat#870273 | 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) sodium salt |
| Chemical compound | Biotinylated bovine serum albumin (BSA) | Vector Laboratories | Cat#B-2007 | |
| Chemical compound | Avidin DN | Vector Laboratories | Cat#A-3100 | |
| Chemical compound | TCEP | Thermo Fisher | Cat#T2556 | |
| Chemical compound, drug | Paclitaxel/Taxol | Millipore Sigma | Cat#T7402 | |
| Software | Labview | National Instruments | ||
| Software | Igor Pro | Wavemetrics |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.44489.026
