Molecular Motors Research Group

  
 

MICROSCOPY TECHNIQUES
Single molecule fluorescence imaging is a highly powerful technique to understand how proteins physically perform their function in a time course. To be able to observe single proteins, we specifically label them with organic fluorophores and shine a laser light to observe the emitted fluorescent light under a microscope. The emitted light is collected with a highly sensitive CCD camera for quantitative analysis.

Our fluorescence microscope is equipped with 488 nm, 532 nm and 633 nm laser lines for the excitation of wide variety of fluorescent probes. All three lasers are combined to follow the same optical path and focused to the back focal plane of the microscope objective (Nikon 100X, 1.49 NA). The beam is then shifted laterally along the focal plane that creates tilted and parallel laser beam emitted from the microscope objective. When the tilt is larger than the critical angle, transmitted light will be total internally reflected back from the glass/water boundary. Total Internal Reflection (TIR) of light creates an evanescent field which penetrates only 100 nm into the flow chamber.


TIRF is ideal to study surface immobilized cells and in vitro systems since it results in minimal fluorescent background and sensitively detects fluorescent photons. Compared to the scanning confocal setup, TIRF is a wide field illumination technique that readily allows simultaneous observation of many single molecules on the surface. Fluorescent photons are filtered and collected by an EMCCD camera (Ixon EM+, Andor Technology) which can detect 92% of incoming photons with a minimal electronic noise.


To specifically label the motors, we generated a cysteine-light dynein motor and conjugated it with malemide reactive probes. We also use genetic tags (e.g. HaloTag and SNAP Tag) to covalently link motors with TMR or biotin. To achieve high signal to noise ratio, we label biotinylated proteins with a streptavidin coated quantum dot that is ~20 times brighter and more stable than Cy3 and TMR dyes. Labeled motors were introduced into the flow chamber after immobilizing microtubules on the glass surface. In the presence of limited ATP, individual steps of motors can be discerned.

FRET
We use our single molecule TIRF scope to detect fast conformational changes and particle tracking experiments. Specifically, by labeling a single protein with a pair of fluorescent dyes whose spectrum overlaps to allow Fluorescent Resonance Energy Transfer (FRET). Closely spaced fluorophores can transfer their energy via dipole-dipole interaction to each other. Excitation of the donor molecule whose emission is in resonance with the acceptor’s absorption yields the emission of an acceptor. The energy transfer depends on the distance (R) between the donor and the acceptor molecule with R6 that makes FRET highly sensitive to measure changes in the distance. Labeling of two sites of a protein with a FRET pair yields information on conformational dynamics for the distances between 2-10 nm. We are using FRET to study conformational changes in moving dynein molecules.

Fluorescence Imaging with One Nanometer Accuracy
To track the movement of motor proteins with high precision in vitro, we use Fluorescence Imaging with One Nanometer Accuracy (FIONA). The image of a point-like fluorescent object is as wide as 250 nm in the visible region of the light because of the diffraction limit. The position of an object, however, can be localized very precisely by determining the center of its emission pattern. The precision depends on maximizing the photon detection per image and minimizing the noise factors. Yildiz et al. 2003 showed that millions of photons can be collected from a single molecule before it photobleaches. Organic dyes (Cy3, TMR, Cy5) were localized within ~100 msec, which enables to measure the step size of the motor proteins kinesin and cytoplasmic dynein.

A. The Airy pattern of a diffraction-limited-spot in two dimensions. B. Fluorescence images of several single Cy3-DNA molecules immobilized on a glass surface. The data was taken with TIRF scope in 0.5 sec. C. Expanded view of one PSF with 2-D elliptical Gaussian curve fit (solid lines).  The center of this PSF can be located to within 1.5 nm.

Kinesin motors are labeled with a streptavidin coated quantum dot and their stepwise motion was tracked under TIRF. Above graph represents the stepping pattern of wild-type kinesin (WT) and engineered kinesin mutants with elongated neck-linker. As WT head takes regular 16 nm steps towards microtubule plus end, neck-mutants take more variable and bidirectional steps along.

Optical Trapping Microscopy
Using a tightly focused laser beam (1064nm), we can apply picoNewton forces to single biological molecules whilst simultaneously measuring position to sub-nanometer accuracy and fluctuations at 20 kHz. Our optical trap is built around a commercial Nikon Ti-E microscope that stabilizes the z-height of our sample, eliminating long-term axial drift. The trap is built in an acoustically quiet and temperature-controlled room to minimize vibrational noise factors. It is fully automated and remotely controlled by a custom-written Labview software. The position of our trap in the sample image plane is controlled by acousto-optical deflectors (AOD - red box in the picture below) to generate a force-feedback mode. We use the trap to apply forces to individual dynein motors walking along axonemes, allowing us detailed insight into the stepping behavior and mechanochemistry of the motor. Additionally, we have two TIRF lines coupled into our scope (488nm and 633nm) so that we have the option to observe single fluorophores.

 
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