Study Cytoskeletal Filaments in Real-time at the Nanoscale

Part of: Cytoskeletal Filaments

Force-extension, manipulation, and visualization of polymers and protein filaments

In this experiment, a vimentin filament is tethered between two optically trapped beads. We can obtain the force-distance curve and determine the mechanical properties of the microtubule by simultaneously stretching the filament and measuring the force and the extension.

The measured data in Figure 1 show the extension of a vimentin intermediate filament. The force-distance curve is measured while stretching and relaxing the intermediate filament at a slow speed, allowing for structural equilibrium. The retraction curve shows clear hysteresis due to the remodeling of the vimentin filament under high tension. Simultaneous confocal fluorescence imaging of the vimentin filament is used to resolve this intra-molecular remodeling.

Combining optical tweezers with simultaneous fluorescence measurements allows correlating the mechanical properties of the microtubule with local information.

1  Force-extension curve of vimentin filaments (left) and confocal fluorescence image showing the amount of tethered vimentin filaments (right).

Data courtesy of Prof. Dr. Sarah Köster at University of Göttingen.

Block et al. (2017) Physical Review Letter
Kurniawan et al. (2016) Biophysical Journal

Label-free observation of microtubules with high sensitivity

In this example, we anchored multiple microtubules to the surface through biotinylated GMPCPP-stabilized seeds. By using Interference Reflection Microscopy (IRM) to image the microtubules in a label-free manner, we observed the microtubules with high sensitivity and contrast (Figure 1).

Sections of the microtubules shown in figure 1 are not rigidly bound to the glass and can freely fluctuate in solution. Distinct patterns composed of alternating light and dark stripes are created by the IRM imaging method. This provides insights into the 3D orientation of the microtubules.

Next, using a C-Trap system equipped with Widefield and IRM we imaged fluorescently-labeled microtubules using both methods (Figure 2).

The new addition of the Surface Assay Toolkit brings fully correlated optical tweezers with IRM and TIRF or Widefield. Couple manipulation and measurements with live observation using different imaging methods and perform truly revolutionary experiments.

1 A typical field of view showing label-free microtubules on the surface. Imaged with an integration time of 40 ms.

Correlated Widefield IRM

2  Fluorescently-labeled microtubules imaged using IRM and Widefield. Imaged with an integration time of 20 ms and 100 ms for IRM and Widefield, respectively.

Sample courtesy Prof. Dr. Erwin Peterman at the VU University Amsterdam

Simmert et al. (2018) Optics Express
Mahamdeh et al. (2018) Journal of Microscopy

Label-free observation of microtubule dynamics

Using optical tweezers correlated with label-free microscopy you can study the dynamics of microtubule filaments to gain insights into the function and regulation of filament assembly. Dynamic microtubule assays can be easily reconstituted in vitro to visualize and measure intricate phenomena like protein binding, microtubule disassembly, severing, and cargo transport.

In the same experiment as the one previously described, we anchored multiple microtubules to the surface through biotinylated GMPCPP-stabilized seeds. This time, by using IRM to image the microtubules in a label-free manner, we observed microtubule dynamics over time (Figure 1).

Using this method it becomes possible to measure the force, speed, distance, conversion rate of filament growth and shrinkage, and to detect rare and transient events, which include rescue, catastrophe, and pause.

Sample courtesy Prof. Dr. Erwin Peterman at the VU University Amsterdam

1 Kymograph of a dynamic microtubule growing and shrinking over time with snapshot of microtubule shown at the left. The arrows indicate the stages where the microtubule is growing and shrinking.

Manipulation and visualization of microtubules

Microtubules can be manipulated to study their mechanical properties, dynamics, and interaction with several microtubule-binding proteins. Here a surface-tethered microtubule is attached to an optically trapped bead for the measurement of growth and shrinkage. In this assay, it is possible to apply force along the direction of the filament and measure precisely force and distance values with sub-pN and sub-nm resolution.

Figure 1 shows the force exerted by a microtubule upon bending (4-6 seconds) and followed by the disruption between the kinesin-coated bead and the filament (6-8 seconds). We can observe the disruption event between the kinesin-coated bead and the filament at a force of ~27 pN. Similar assays can be used to investigate the function and mechanism of various microtubule-associated proteins (MAPs), such as microtubule plus-end-tracking proteins (+TIPs) or (fluorescence) kinetochore particles.

Sample courtesy Prof. Dr. Erwin Peterman at the VU University Amsterdam

1 Force-time curve of microtubule bending. Data shown at 30 kHz (gray) and decimated to 50 Hz (red).

2 Snapshot of microtubule bending.

Visualization of filament-filament interactions

Here we use a quadruple trap configuration to trap beads and catch two fluorescently labeled microtubules in between. We then arrange the two microtubules in a crossed pattern and drag one microtubule across the other with a known force.

C-Trap Filament-Filament Interactions.png

Figure 1 shows the interaction between two fluorescently labeled microtubules, as one microtubule is dragged across the other with a known force. In the middle and left snapshot, we can observe that the friction force between the filaments causes one microtubule to adopt a curved conformational structure. When we stop the movement of the microspheres the microtubule slowly relaxes (right).

The surface properties of these filaments, the electrostatic and ionic forces and specific interactions, can generate friction when two filaments interact. This friction can be measured using a device such as the C-trap™ configured for quadruple optical trapping.

microtubule dragging quadruple optical tweezers fluorescence microscopy microfluidics

1 Two fluorescent microtubules held by two beads in a crossed pattern. One microtubule is dragged across the other with a known force.

Data courtesy of Dr. Zdeněk Lánský at BIOCEV Prague.

Block et al. (2017) Physical Review Letter
Kurniawan et al. (2016) Biophysical Journal

Solutions

C-Trap

Optical Tweezers and Fluorescence Microscopy

The C-Trap™ is the world’s first instrument that allows simultaneous manipulation and visualization of molecular interactions in real-time. It combines high-resolution optical tweezers, confocal microscopy or STED nanoscopy with an advanced microfluidics system in a truly integrated and correlated solution.

m-Trap

Optical Tweezers and Fluorescence Microscopy

The m-Trap™ is the first entry-level optical tweezers instrument specifically developed for high resolution single-molecule research. Ultra-high force resolution and stability, with incredible throughput, ease of use and modularity ‒ all at an unprecedented price level.

Key Product FeaturesC-Trap™m-Trap™
Mechanical characterization of filaments
Filament-filament interactions
Label-free visualization of microtubules
Fluorescence visualization of filaments in solution
Fluorescence visualization of filaments at the surface
Visualization of binding position and diffusion of proteins in solution
Visualization of binding position and diffusion of proteins at the surface
Rapid buffer exchange for fast experimental workflow

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