Hoenger Lab
University of Colorado at Boulder
Department of Molecular, Cellular and Developmental Biology

Structural and Functional Investigations
into Cytoskeletal Assemblies by
Cryo Electron Microscopy
and 3D Image Analysis

 
Research:: Molecular Motors

Molecular motors are involved in many of the cellular processes associated with microtubules. In nature, molecular motors such as kinesin walk along a microtubule to deliver their cargo, other kinesins are the driving force in spindle movement for chromosome separation.

Microtubules in vivo are commonly 13 protofilaments. When polymerized in vitro, microtubules may have 13, 14, 15, or 16 protofilaments. The uncommon microtubules of 15 protofilament twist longitudinally forming a helix This unusual property is used to examine the structure and attachment location of motors to the microtubule surface.

In the laboratory this helix is saturated with motors using non-hydrolyzable AMP-PNP in this way each tubulin dimer has a motor permanently attached. This helical arrangements of attached motors shows the motors in every possible orientation. Lengths of helical repeats are averaged together to provide a visual representation of the motors and microtubules.

To characterize the structure and binding of molecular motors to microtubules we combine electron microscopy with 3-D image reconstruction methods. The samples are imbedded in ice then examined using cryo-electron microscopy, high magnification photographs reveal the structural data. These images are digitized and the data is analyzed in 3-D helical reconstruction computer programs such as Suprim or Phoelix.

Kinesin walks along a microtubule protofilament. The purple and green subunits represent the alpha and beta tubulin monomers, the building blocks of these protofilaments.

An electron micrograph of a micortubule that has been decorated with molecular motors.
A 3-d reconstruction of a 15 protofilament microtubule decorated with eg5, a molecular motor involved in centrosome separation
The process of 3d reconstruction begins with interpreting the photos taken on the electron microscope.
The original image goes through the object. By looking at the diffraction patterns (right side)
and Fourier filtering, one can reconstruct (left side) a 3-d molecule from a 2-d image.
 

University of Colorado, MCDB, 347 UCB, Boulder, CO 80309-0347