Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules - PubMed (original) (raw)
Long tethers provide high-force coupling of the Dam1 ring to shortening microtubules
Vladimir A Volkov et al. Proc Natl Acad Sci U S A. 2013.
Abstract
Microtubule kinetochore attachments are essential for accurate mitosis, but how these force-generating connections move chromosomes remains poorly understood. Processive motion at shortening microtubule ends can be reconstituted in vitro using microbeads conjugated to the budding yeast kinetochore protein Dam1, which forms microtubule-encircling rings. Here, we report that, when Dam1 is linked to a bead cargo by elongated protein tethers, the maximum force transmitted from a disassembling microtubule increases sixfold compared with a short tether. We interpret this significant improvement with a theory that considers the geometry and mechanics of the microtubule-ring-bead system. Our results show the importance of fibrillar links in tethering microtubule ends to cargo: fibrils enable the cargo to align coaxially with the microtubule, thereby increasing the stability of attachment and the mechanical work that it can do. The force-transducing characteristics of fibril-tethered Dam1 are similar to the analogous properties of purified yeast kinetochores, suggesting that a tethered Dam1 ring comprises the main force-bearing unit of the native attachment.
Keywords: anaphase; forced walk; laser tweezers; mathematical modeling; microtubule depolymerization.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Use of elongated protein fibrils to tether Dam1 to beads. (A) Schematic of an end-on kinetochore–MT connection with a Dam1 ring tethered to the kinetochore central hub. (B) Lateral attachment of a Dam1-coated bead to an MT in vitro (roughly to scale). Soluble Dam1 subunits are used to promote completion of the full Dam1 ring. (C) Chemical steps involved in Dam1 conjugation to the bead using the 100-nm-long CC tether or directly to the surface with a heterobifunctional cross-linker and anti-GFP IgG.
Fig. 2.
Motility and force transduction by CC-tethered beads. (A) Schematic of our motility assay. A Dam1 bead binds laterally to a segmented MT, which is attached to the coverslip at its minus end and carries a photodestructible stabilizing cap on its plus end. (B) Kymograph of a Dam1 bead moving with MT disassembly. (Scale bar: 5 μm.) (C) Motilities of beads coated with Dam1 by antibodies (control) or CC tethers are similar; error bars in all figures correspond to SEM unless stated otherwise. (D) Schematic of the stationary trap assay to measure force. (E) Examples of unprocessed quadrant photodiode signals recorded with either control or CC-tethered Dam1 beads (trap stiffness = 0.03 and 0.13 pN/nm, respectively). Amplitude of the force signal with CC tether is larger and the bead pauses longer before the detachment. (F) Quantification of force amplitude measured in a stationary trap. Whiskers show minimum to maximum; the box is 25–75%, and + shows the average.
Fig. 3.
Biomechanics of force transduction by the MT-encircling ring–bead system. (A) System configuration showing the initial position of an unloaded bead that is subsequently subjected to the trapping force Ftrap. (B) Enlarged view shows tilting of the ring under force acting through the bead; the image was generated with ANSYS software. (C) The slope of the calculated relationship between Ftrap and FMT at steady state reflects the lever arm factor. For lateral ring–bead attachment (black symbols), this factor is 12.5, which means that Ftrap applied at the bead’s center can equalize more than 10-fold larger force than the force that acts on the ring from the MT. For end-on attached bead (red symbols), the lever arm factor is one. (D) A drawing of the system configuration for the angle α = 0°, in which the tethered bead is aligned coaxially with the MT. (E) The lever arm factor decreases as the center of the bead cargo moves closer to the MT axis (note that angle-α from A decreases as the alignment of the bead improves).
Fig. 4.
Lateral to axial repositioning of the beads. (A) Illustration of a bead repositioning under the two oppositely directed forces that come from the depolymerizing MT and the trap. The force vectors are initially displaced but become aligned after the bead repositions. (B) Force and stage positions vs. time illustrate five experimental phases in one representative experiment. Phase I, laterally attached motionless bead. Phase II, bead is clamped and pulled to the MT plus end. After the stage settles and the clamp force is maintained constant, we trigger MT depolymerization. Phase III, the end of a shortening MT reaches the bead, and the stage moves to maintain constant force (in Right, the yellow arrowheads point to the MT minus end, and the arrows point to a bead moving in a stationary trap). (Scale bar: 3 μm.) Phase IV, we stop the stage and record the bead’s continued motion in the now stationary trap. Phase V, bead detaches from the MT tip and falls back to the trap’s center. (C) Example tracks of motions for CC-tethered beads (phase III). Tracks with increase in the speed are aligned to the center in the time that this change took place (time 0 for blue curves). (D) Average of centered two-phasic traces of the CC-tethered beads tracking the shortening MT end under an approximately constant trapping force of 2.6 ± 0.5 pN (right axis); dotted lines are SDs. (E) The percent of force signals measured in phase IV with amplitude that exceeded the value specified, normalized for different groups of beads. The CC-tethered beads that increased their speed during phase III (blue) showed larger force transients, on average, than the control beads (black, P < 0.05) or the CC-tethered beads that did not change their speed (green, P < 0.07). The maximum forces measured increased 5.8 and 2.6 times, respectively.
Fig. 5.
Quantitative analysis of Dam1 diffusion. (A) Scheme of the experiments with MTs attached to pedestals and a differential interference contrast image of such a coverslip with two immobilized MTs. (Scale bar: 5 μm.) (B) Example kymographs showing diffusion of GFP-Dam1 oligomers of various sizes. (Scale bar: 2 μm.) Contrast of these images was adjusted for clarity. (C) Mean squared displacements (MSDs) for groups of Dam1 oligomers with different number of subunits (details in
SI Materials and Methods
). (D) Diffusion coefficients plotted vs. the oligomer’s size on a semilog scale. Dotted lines show 95% confidence interval for the exponential fit.
Fig. 6.
Reconstructed Dam1 couplers perform like purified yeast kinetochores. (A) The duration of Dam1 bead attachment under maximal force is longest in CC-tethered beads with increased speed during phase III. (B) A force–velocity curve generated from the data for all CC-tethered beads’ motions from phases III and IV (
Fig. S2_C_
). Dotted lines show exponential fitting plotted on a semilog scale; the fitting for CC-tethered beads is for motions recorded with the load >5 pN. The theoretical curve is from ref. . (C) Comparison of the force–velocity curve for CC-tethered beads vs. measurements for the kinetochore particles (data from ref. 24).
References
- Cheeseman IM, Desai A. Molecular architecture of the kinetochore-microtubule interface. Nat Rev Mol Cell Biol. 2008;9(1):33–46. - PubMed
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