Ultrafast Force-Clamp Spectroscopy of Microtubule-Binding Proteins

Abstract

Optical trapping has been instrumental for deciphering translocation mechanisms of the force-generating cytoskeletal proteins. However, studies of the dynamic interactions between microtubules (MTs) and MT-associated proteins (MAPs) with no motor activity are lagging. Investigating the motility of MAPs that can diffuse along MT walls is a particular challenge for optical-trapping assays because thermally driven motions rely on weak and highly transient interactions. Three-bead, ultrafast force-clamp (UFFC) spectroscopy has the potential to resolve static and diffusive translocations of different MAPs with sub-millisecond temporal resolution and sub-nanometer spatial precision. In this report, we present detailed procedures for implementing UFFC, including setup of the optical instrument and feedback control, immobilization and functionalization of pedestal beads, and preparation of MT dumbbells. Example results for strong static interactions were generated using the Kinesin-7 motor CENP-E in the presence of AMP-PNP. Time resolution for MAP-MT interactions in the UFFC assay is limited by the MT dumbbell relaxation time, which is significantly longer than reported for analogous experiments using actin filaments. UFFC, however, provides a unique opportunity for quantitative studies on MAPs that glide along MTs under a dragging force, as illustrated using the kinetochore-associated Ska complex.

Keywords: Bead functionalization; CENP-E kinesin; Kinetochore; Microtubule-associated proteins; Microtubule-dependent diffusion; Molecular friction; Ska complex; Ultrafast force-clamp spectroscopy.

Fig. 1

Optical-trapping configurations to study force-dependent interactions between filaments and proteins with no motor activity. (a) A stationary trap holds a MAP-coated bead, which slides on a coverslip-immobilized MT, generating molecular friction [17, 19]. (b) Three-bead assay employing actin dumbbell and pedestal coated with high-affinity actin-binding molecules [42, 43]. (c) UFFC assay uses the three-bead geometry in combination with a feedback regime for ultrafast force application to actin-binding and DNA-binding proteins [44]. (d) Example UFFC recording for control MT dumbbell at 4 pN force clamp. Position of one of the dumbbell beads is shown in blue. Trapping force pulling the dumbbell against viscous drag is shown in red

Fig. 2

Laser-tweezers instrument. (a) Photograph of our microscope accommodating the UFFC system. (b) The optical microscope system consists of the following components: DIC pathway including light emitting diode (LED), condenser, polarizers and Wollaston prisms (not shown); sample (S), x,y,z piezo-stage, objective (O), and EMCCD. Dual optical tweezers are inserted and extracted from the optical axis of the microscope through dichroic mirrors (D1 and D2) and comprise: Ytterbium laser (1064 nm), half-wave plates, polarizing beam-splitter cubes (PBS), AOD and piezo-mirror (PM). Two tracking beams are aligned with two trapping beams, and the third tracking beam is projected through a pedestal bead. Dichroic mirrors D5 and D6, and three QPDs distribute beams and acquire information about dumbbell and pedestal beads. Tracking beam 4 (830 nm) coupled with QPD1 monitors position of bead 1 in TRAP1. Tracking beam 1 (780 nm) coupled with QPD2 monitors position of bead 2 in TRAP2. Tracking beam 5 (905 nm) coupled with QPD3 monitors position of pedestal bead and provides feedback #2 for stage stabilization. Signals from QPD1 and QPD2 are processed with an FPGA and sent to the AOD controller to implement UFFC feedback #1

Fig. 3

Operational principles of the UFFC feedback loops. (a–c) Schematics for different regimes to exert control of the trap(s) position in response to changes in the position of dumbbell bead(s), as determined with QPD(s). With all regimes, the MT dumbbell stretched with force F₀ is oscillated under constant force F, but the regimes differ in the noise level for signals collected from different beads (see text for more details). More narrow and darker cones depict trapping beams, whereas tracking beams are depicted with wide cones and light color

Fig. 4

Data transfer layout for feedback loops. (a) Our instrument is controlled with two feedback loops, in which data are transferred in the directions depicted with arrows. Blue arrows represent the force-clamp feedback loop, red arrows represent the stage stabilization feedback loop. AI analog input, AO analog output, DI digital input, DO digital output. (b) Blue curve shows the step-like signal generated by the FPGA to instruct the AOD to sweep the trapping beam across a coverslip-immobilized pedestal. The resultant QPD response signal (red curve) reveals instrument delay

Fig. 5

Immobilization of pedestals. (a) Schematic of the microscope chamber and application of flow by gravity. (b) Bright-field images of streptavidin-coated beads 1.87 μm diameter. Proper heat application results in partial bead melting, which firmly immobilizes beads on the coverslip while preserving the spherical shape of bead surface that was not in direct contact with the heated glass. Excessive heating leads to visible loss of spherical bead shape, resulting in immobilized but unusable pedestals

Fig. 6

SD method for testing stability of immobilized pedestals. (a–c) Representative QPD signals for pedestal bead coordinates and running SDs (100 ms window) along y-axis, which in our system aligns with the MT dumbbell. Loosely attached pedestals are obtained via nonspecific adsorption in the presence of 8 mg/mL BSA (see step 4 in Subheading 3.2.2). (d) Example distributions of the running SDs for three types of pedestals are depicted using the same color coding as in panels A–C. (e) Pedestal SDs. Each dot shows average SD for one pedestal immobilized using indicated procedures. Bars with errors correspond to means ± SEM, p-value determined by Mann–Whitney test. (f). Histogram plot of the SDs of adsorbed pedestals shows considerable variability in pedestals stability (data from nine chambers, examining total 87 pedestals). Gray area shows pedestals with SD < 5 nm, which was chosen as a cutoff to select firmly attached pedestals for UFFC experiments

Fig. 7

Testing stability of pedestal immobilization by applying oscillating force. Changes in the coordinate of the bead center and corresponding fast Fourier transformation for streptavidin-coated polystyrene beads (diameter 1.8 μm) subjected to an oscillating laser force with 200-nm amplitude at 10 Hz (blue vertical lines). (a) A freely floating bead shows a full range of motion and strong peak at 10 Hz. (b) Pedestal immobilized via adsorption shows no detectable motion or distinct frequency. (c) Pedestal attached loosely via adsorption in the presence of BSA can be tilted by oscillating laser trap, as seen from periodic changes in its position

Fig. 8

Coating of immobilized pedestals with GFP-tagged proteins. (a) Representative DIC and GFP fluorescence images of functionalized pedestals. Partially melted pedestals were coated with anti-GFP antibodies and 200 nM GFP-tagged protein. Adsorbed pedestals were coated using SNAP-GBP and 10 nM GFP-tagged protein. (b) Quantifications of bead brightness and (c) percent of pedestals showing interactions with MT dumbbells. Each dot in (b) corresponds to the average result from one experimental chamber, in which brightness was collected from >30 pedestals. Each dot in (c) corresponds to the result from one experimental chamber in which MT dumbbell interactions were examined for >10 pedestals. Bars with errors are means ± SEM

Fig. 9

Dumbbell formation and stretching. (a) Schematic of experimental chamber with spatially segregated floating MTs and dumbbell beads. (b) An overlay of image of a stretched MT dumbbell (rhodamine channel) and dumbbell beads (DIC). (c) Steps to pre-tense MT dumbbell. (d) Example signals from two QPDs monitoring dumbbell beads during stretching. Unprocessed signals are shown with light colors, averaged signals are shown with darker lines

Fig. 10

Typical results for control (free) MT dumbbell. (a) Changes in the coordinate of one of the dumbbell beads, and (b) corresponding velocity distributions for the indicated force clamped via the Leading Trap Feedback regime. “Up” and “down” directions correspond to the rising and descending segments of bead trajectories shown in (a) [53]. (c) Velocity of dumbbell motion as a function of force. Each dot represents average velocity for 12–16 experiments, each recording the oscillations of a free MT dumbbell for 30 s. Error bars show SD, data collected from eight chambers. Line is a linear fit constrained at the origin

Fig. 11

Determining proper z-position for MT dumbbell during UFFC assay. (a) Rationale for the procedure to find the surface of pedestal bead. (b) Typical recordings of the y-coordinates of two dumbbell beads during motion of the piezo-stage, which brings the dumbbell closer to the coverslip with immobilized 1.8 μm pedestal. Diffuse red bar marks the approximate location of the pedestal surface

Fig. 12

Static MT-binding interactions. (a) Example UFFC recording showing changes in the position of one of the dumbbell beads near the pedestal coated with GFP-tagged CENP-E kinesin in the buffer with 1 mM AMP-PNP. Gray area highlights one binding event, during which the dumbbell maintains its position under 4 pN force. (b) Example recordings with a brief CENP-E binding event and a typical GFP-binding event. (c) Enlarged onset of the binding event shown by the red box in panel B. Time zero corresponds to the change in force direction, which led to the upward segment of bead trajectory immediately prior to the binding event. A trajectory smoothed using local regression (LOESS; 2.5 ms window) was fitted with a combination of a linear function and a single-exponential function with minimal R²; signals in which the amplitude of one of the functions was <10% were discarded. The characteristic time for single-exponential fit describes dumbbell stopping upon molecular binding with strong affinity. (d) Characteristic times for the onset of binding events observed with CENP-E (n = 77) and GFP (n = 49) at 4 pN. Bars shown with median lines and 1.5 inter quartile ranges, p-value determined by Mann-Whitney test. (e) Top graph shows individual curves (gray) for the onset of binding events for CENP-E in the presence of AMP-PNP. These curves were aligned to match the start of exponential fittings. The average signal was fitted using linear (red) and exponential (blue) functions. The same binding events were examined to plot changes in the y-position of the pedestal bead along the force application axis (bottom graph). The absence of a shift in pedestal position upon CENP-E binding confirms the high stability of pedestal immobilization

Fig. 13

Friction-generating gliding of the full-length Ska complex along MT under force. (a) Example signal with a processive gliding event (gray box) recorded at 6 pN. Close-up views of segments at the start and end of gliding (red boxes) are shown below. (b, c) Velocity distributions for MT dumbbell motion plotted for different directions for Ska-coated pedestals that showed interactions (b) or not (c). Gliding events lead to appearance of peaks with low velocity (arrows). Position of free velocity peaks is slightly different owing to experimental variables, such as different MT dumbbells, experimental chambers and optical alignments