Revisiting the long-zero response: measurement of the flagellar growth and shortening rates in live, motile cells
Will Ludington1, Linda Shi2, Qingyuan Zhu3, Michael Berns2,3, and Wallace Marshall1
1) Depts. of Biochemistry and Biophysics, University of California, San Francisco
2) Dept. of Bioengineering, University of California, San Diego
3) Beckman Laser Institute, University of California, Irvine
We have developed new methods and tools to measure the instantaneous growth and shortening rates of Chlamydomonas flagella in live, motile cells by revisiting the classic Rosenbaum et al (1969) long-zero regeneration experiments. Our new methods allow us to choose the exact flagella that we perturb, simultaneously follow 15 cells in a single time course, and continuously change media during the course of an experiment. We first trap cells in a microfluidic device, trigger single flagellar deflagellation with an IR femtosecond laser, and then measure the ensuing length kinetics in a large number of cells. We additionally measure the rate of intraflagellar transport (total amount of protein per second) as a function of flagellar length in regenerating flagella. Both sets of experiments are then repeated in the presence of 10 micrograms/mL cycloheximide in order to shut off protein translation. Using a mathematical model of the flagellar system based on the Marshall et al (2001) balance point model, we then solve for the growth rate, shortening rate, and cytoplasmic pool of flagellar precursor. Our methods estimate a constant flagellar shortening rate of 0.5 microns per minute and indicate cytoplasmic pool kinetics that affirm qRT-PCR data. We are currently investigating how length-perturbing drugs and mutations affect growth rate, shortening rate, and cytoplasmic pool dynamics. Funding for this work was provided by the NIH, the Sandler Foundation, the Keck Foundation, an NSF predoctoral fellowship to WL, and the Beckman Laser Institute.
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