Tight bending of the Ndc80 complex provides intrinsic regulation of its binding to microtubules

  1. Emily Anne Scarborough
  2. Trisha N Davis  Is a corresponding author
  3. Charles L Asbury  Is a corresponding author
  1. University of Washington, United States
5 figures, 1 table and 1 additional file

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 …

https://doi.org/10.7554/eLife.44489.002
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 …

https://doi.org/10.7554/eLife.44489.003
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.

https://doi.org/10.7554/eLife.44489.005
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…

https://doi.org/10.7554/eLife.44489.006
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 …

https://doi.org/10.7554/eLife.44489.009
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 …

https://doi.org/10.7554/eLife.44489.010
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 …

https://doi.org/10.7554/eLife.44489.012
Figure 3—source data 1

Analysis of threshold crossing from records of Ndc80 FRET versus time.

https://doi.org/10.7554/eLife.44489.015
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 …

https://doi.org/10.7554/eLife.44489.013
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 …

https://doi.org/10.7554/eLife.44489.017
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 …

https://doi.org/10.7554/eLife.44489.018
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) …

https://doi.org/10.7554/eLife.44489.019
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 …

https://doi.org/10.7554/eLife.44489.022
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 …

https://doi.org/10.7554/eLife.44489.023
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
DesignationSource
or reference
IdentifiersAdditional
information
Strain, E. coliRosetta (DE3) competent cellsMillipore SigmaCat#70954
Biological sampleBovine brain tubulinlab purificationProtocol adapted from Castoldi and Popov, 2003.
Sequence-based reagentFluorescently-labeled oligonucleotidesIDT
Sequence-based
reagent
Fluorescently-labeled, biotintylated oligonucleotidesIDT
AntibodyPenta-HIS biotin conjugate, monoclonal mouseQiagenCat#34440Diluted to 20 nM
Chemical compoundAlexa Fluor 488 succinimidyl esterThermo FisherCat#A20000
Chemical compoundAlexa Fluor 647 maleimideThermo FisherCat#A20347
Chemical compoundAlexa Fluor 568 succinimidyl esterThermo FisherCat#A20003
Chemical compoundCy3 maleimideGE Healthcare Life SciencesCat#PA23031
Chemical compoundCy5 maleimideGE Healthcare Life SciencesCat#PA25031
Chemical compoundTroloxMillipore SigmaCat#238813
Chemical compoundGlucose oxidaseMillipore SigmaCat#345386
Chemical compoundCatalaseMillipore SigmaCat#219261
Chemical compoundPOPCAvanti Polar LipidsCat#8504571-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
Chemical compoundBio-cap-PE/BioPEAvanti Polar LipidsCat#8702731,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl)
sodium salt
Chemical compoundBiotinylated bovine serum albumin (BSA)Vector LaboratoriesCat#B-2007
Chemical compoundAvidin DNVector LaboratoriesCat#A-3100
Chemical compoundTCEPThermo FisherCat#T2556
Chemical compound, drugPaclitaxel/TaxolMillipore SigmaCat#T7402
SoftwareLabviewNational Instruments
SoftwareIgor ProWavemetrics

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