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Cilia Reconstitution

Using Chlamydomonas reinhardtii as a model organism, we wished to investigate how cilia movement turns into propulstion, how cilia coordinate with each other hydrodynamically, and in general, develop techniques to manipulate cilia. This work was done by Andrew Fung and Hsiang-wei Lu during the Bioengineering Bootcamp of September, 2007.

The bulk of the three days devoted to the project involved obtaining a high percentage of reactivated cilia after purification, and the attachment of polystyrene microspheres to the cilia as handles to use with optical tweezers.

Experimental Methods

The protocol for cilia purification was developed by a cohort of scientists in the mid-70's by Borisy, Gibbons, Mitchell, and Witman, among many others. We opted for a relatively simple method, that of using dibucaine plus shear forces to strip the densely-grown Chlamydomonas (grown in TAP medium) of their cilia, then stripping the cilia of their membranes to facilitate bead attachment.

In order to get beads to attach to the cilia, we chose two pathways: 1) nonspecifically absorbing anti-alpha-tubulin antibodies onto the surface of the 1 micron diameter spheres, and 2) incubating antibody with protein-A coated spheres (protein A being a streptococcus protein that binds the Fc region of antibodies).


Our method for cilia purification worked well, but there was only a small percentage of reactivated cilia. If we watched long enough, it was apparent that some were undergoing non-Brownian motion, but they were incredibly slow, and the percentage was low. Below is a movie of cilia undergoing Brownian motion, and when ATP is added, we can see one cilia executing stereotypical motion.

Brownian motion of free cilia

Directional movement of free cilia

Stripping off the membrane worked well. We stained the cilia with FM464 dye before and after demembranation. See Figure below. It's an HSV image of a fluorescence image overlaying a brighfield image in HSV color space. One can see that there is a brighter fluorescence signal in the non-demembranated image (left, in blue). The long structures are cilia.

For some reason, the bead chemistry did not work that well.

Interactions between a green (532 nm) optical trap and the cilia resulted in a curious observation: that the cilia will orient along its long axis into the trap. Thus provides an alternate way to handle the cilia than through bead attachment.


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