Multivalency of NDC80 in the outer kinetochore is essential to track shortening microtubules and generate forces
- Delft University of Technology, Netherlands
- Max Planck Institute of Molecular Physiology, Germany
Figures
Figure 1 with 3 supplements
CENP-T-mediated oligomerization of NDC80 produces particles that follow microtubule disassembly.
(a) Schematic representation of Alexa-488-labeled, phosphorylated CENP-T:MIS12 and TMR-labeled NDC80 in a single-molecule TIRF setup with dynamic microtubules. (b) Monomeric NDC80 at 200 pM does not …
Figure 1—figure supplement 1
Reconstitution of NDC80 and MIS12 on CENP-TAlexa-488 phosphorylated by CDK1:Cyclin-B.
(a) CENP-T2-373 labelled with Alexa-488 was separated from the excess of GGGGC-Alexa488 peptide by size-exclusion chromatography. (b) SDS-PAGE analysis to demonstrate the phosphorylation of CENP-T …
Figure 1—figure supplement 2
NDC80 promotes tip-tracking of reconstituted TN and TMN.
(a) TMN and TN (both at 0.4 nM) follow shortening microtubules in a single-molecule TIRF experiment. (b) TM does not bind to microtubules and does not follow microtubule shortening in a …
Figure 1—figure supplement 3
Tip-tracking by NDC80 oligomerized on CENP-T:MIS12.
(a) Two example kymographs as in Figure 1D. (b) Example kymograph of co-localizing NDC80TMR and CENP-TAlexa488:MIS12 on the microtubule lattice and at the shortening microtubule end in a flow …
Figure 2 with 3 supplements
Incremental addition of NDC80 results in hyperstable microtubule binding.
(a) NDC80SPY-SORT was fluorescently labelled and covalently bound to TS assemblies. The cartoon shows the formation of T1S3[NDC80]3 assemblies. Size-exclusion chromatography and SDS-PAGE analysis …
Figure 2—figure supplement 1
A reconstituted system to precisely control NDC80 stoichiometry (Part I).
(a) TySx variants were separated by ion-exchange chromatography based on the pI difference of T (5.1) and S (4.5). Collected assemblies were analyzed by SDS-PAGE as tetramers (not-boiled) or in a …
Figure 2—figure supplement 2
A reconstituted system to precisely control NDC80 stoichiometry (Part II).
Samples before (t0) and after (t18) the reaction were analysed by SDS-PAGE (samples not-boiled) to monitor coupling of SPC24SPY to TySx tetramers and fluorescent labelling of SPC25SORT. …
Figure 2—figure supplement 3
Characterization of oligomerized NDC80 on taxol-stabilized microtubules.
(a) Kymographs of mono-, di-, tri-, and tetravalent NDC80 complexes binding to taxol stabilized microtubules. Scale bar 5 µm. (b) Brightness distribution of TS-NDC80 assemblies on taxol-stabilized …
Figure 3 with 2 supplements
Trivalent TS-NDC80 efficiently tracks depolymerizing microtubules and transports cargo.
(a) Schematic representation of the experimental setup. (b) Kymographs showing NDC80 (green) assembled on T2S2, T1S3, or T0S4 tracking a depolymerizing microtubule (red). An example of a T1S3[NDC80]3…
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Figure 3—source data 1
Tip-tracking events for differently coated beads.
Accompanying Figure 3I.
- https://doi.org/10.7554/eLife.36764.014
Figure 3—figure supplement 1
Characterization of oligomerized NDC80 on dynamic microtubules.
(a) Brightness distribution of TS-NDC80 assemblies on dynamic microtubules. b) Distance travelled by TS-NDC80 modules moving with the tips of the shortening microtubules. (c–d) Presence of 25–50 mM …
Figure 3—figure supplement 2
Negative-stain EM of microtubules and nanogold particles coated with T1S3[NDC80]3.
Boxed areas in the upper micrograph are shown below at a higher magnification. The orange line marks the micrograph shown in main Figure 2E. Scale bars 100 nm.
Figure 4
TS-NDC80 modules stall and rescue microtubule depolymerization.
(a) The displacement of an optically trapped glass bead can be used to determine the force exerted by a shortening microtubule on a bead-bound TS-NDC80 oligomer. (b) Example of a trapped glass bead …
Figure 5
NDC80C oligomers stall microtubules through interaction with the shortening microtubule end.
(a) Experimental setup to compare forces generated by shortening microtubules (red box) with forces generating by a moving stage while a bead with T1S3[NDC80]3 is attached laterally to a dynamic …
Figure 6
Reconstitution of a dynamic kinetochore-microtubule attachment.
A graphical recapitulation of the kinetochore-microtubule interfaces reconstituted and characterised in this study and their occurrence in vivo.
Videos
Video 1
Single-molecule TIRF microscopy of TS-NDC80 modules on dynamic microtubules.
35 pM of T0S4[NDC80TMR]4 (green) in the presence of 8 µM tubulin labelled with HiLyte-642 (red) and in the absence of KCl. The two-channel images were acquired every 1.1 s (shown at 30 fps). Top …
Video 2
A disassembling microtubule tip pulls on a trapped bead.
A bead coated with 3% PLL-PEG-biotin and then saturated with T2S2[NDC80TMR]2 was attached to a microtubule with a trap (see also Figure 3B for still images and a complete QPD trace of this signal). …
Video 3
A microtubule is rescued five times at the bead attachment site.
A bead coated with 0.3% PLL-PEG-biotin and then saturated with T1S3[NDC80FAM]3 was attached to a microtubule with a trap. The microtubule experiences dynamic instability, but its shortening is five …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant DNA reagent | pLIB | Peters laboratory,Weissmann et al., 2016 | addgene plasmid 80610 | |
| Recombinant DNA reagent | pBIG1A | Peters laboratory,Weissmann et al., 2016 | addgene plasmid 80611 | |
| Recombinant DNA reagent | pLIB NDC80 | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pLIB NUF2 | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pLIB SPC25-HIS | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pLIB SPC24 | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pBIG1A with NDC80C (SPC25-HIS) | Musacchio laboratory,Huis In 't Veld et al., 2016 | combined by biGBac cloning (Weissmann et al., 2016) | |
| Recombinant DNA reagent | pLIB SPC25-SORT-HIS | Musacchio laboratory, this study | ||
| Recombinant DNA reagent | pLIB SPC24-SPY | Musacchio laboratory, this study | ||
| Recombinant DNA reagent | pBIG1A with NDC80C (SPC25-SORT-HIS) | Musacchio laboratory, this study | combined by biGBac cloning (Weissmann et al., 2016) | |
| Recombinant DNA reagent | pBIG1A with NDC80C (SPC25-SORT-HIS SPC24-SPY) | Musacchio laboratory, this study | combined by biGBac cloning (Weissmann et al., 2016) | |
| Recombinant DNA reagent | pGEX-6P GST-CENP-T2–373 SORT | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pBIG1A CDK1-GST:Cyclin-B1-HIS | Musacchio laboratory,Huis In 't Veld et al., 2016 | ||
| Recombinant DNA reagent | pET21a Core Traptavidin | Howarth laboratory,Chivers et al. (2010) | addgene plasmid 26054 | |
| Recombinant DNA reagent | pET21a Dead Strepatavidin SpyCatcher | Howarth laboratory,Fairhead et al. (2014) | addgene plasmid 59547 | |
| Peptide, recombinant protein | MIS12C (DSN1-d100-109) | Musacchio laboratory,Petrovic et al. (2016) | ||
| Peptide, recombinant protein | Sortase 5M and Sortase 7M | Ploegh laboratory, seeHirakawa et al. (2015) | addgene plasmids 51140 and 51141 | |
| Peptide, recombinant protein | GGGGC-Alexa488 | ThermoFisher | peptide for C-terminal sortase labeling | |
| Peptide, recombinant protein | GGGGK-TMR | GenScript | peptide for C-terminal sortase labeling | |
| Peptide, recombinant protein | GGGGK-FAM | GenScript | peptide for C-terminal sortase labeling | |
| Software, algorithm | Kymo.m | Dogterom laboratory, this study | Matlab script to trace fluorescent particles in kymographs |
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.36764.021
