Charles J. Brokaw
Motors, Motility, and Behavior of Eukaryotic Flagella
My work seeks to understand the mechanisms of motility of eukaryotic flagella and cilia. Specifically, I want to understand how the activity of the motor enzymes (dyneins) that cause sliding between the flagellar microtubules is regulated in order to produce particular patterns of bending.
Experimental work is carried out at the Kerckhoff Marine Laboratory, operated by the Division of Biology in Corona del Mar, about 60 miles from the Pasadena campus. This laboratory has facilities for maintaining sea urchins as sources of spermatozoa on a year-round basis, as well as access to spermatozoa from other marine invertebrates such as the tunicate, Ciona. We mostly use spermatozoa that have been demembranated with detergent and then reactivated with MgATP. This in vitro system provides an excellent system for precise measurements of the parameters of flagellar movement in response to various experimental manipulations. The measurements are made using high-speed photographic recording and computerized image analysis.
Our experimental search for rules that govern the initiation and propagation of bends by flagella proceeds in parallel with computer simulation studies using programs that solve the partial differential equation for the balance of active and resistive bending moments on a flagellum. This "black box biophysics" type of approach is made possible by the many variant bending patterns that can be obtained by manipulating the in vitro conditions for motility of the flagella. Since bending is generated by sliding between microtubules, measuring this sliding is important. Recent work has used gold beads as markers on exposed outer doublet microtubules in order to make direct measurements of microtubule sliding.
A second theme is analysis of the signal transduction steps involved in activation of the motility of Ciona spermatozoa. The first step is triggered by transfer from a high potassium ion environment to a low potassium environment, and results in an increase in cAMP concentration within the intact spermatozoa. Subsequent steps are blocked by inhibitors of protein phosphorylation, and can be carried out in vitro after sperm demembranation, by incubating the demembranated spermatozoa in the presence of cAMP and ATP. The initial steps of in vitro activation are blocked by specific inhibitors of cAMP-dependent kinase, but the final steps are carried out by a kinase (or more than one) that is not sensitive to these inhibitors. The flagellar axoneme carries several different tightly-bound kinases, with as yet unknown function.
General properties of dynein and other motor enzymes are also being explored by computer simulations, using a stochastic method that follows the behavior of individual molecules.