Tuning the Biophysical Properties of Dynein 2 for Intraflagellar Transport

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DESCRIPTION (provided by applicant): Tuning of the Biophysical Parameters of Cytoplasmic Dynein 2 for Intraflagellar Transport Dyneins are large and complex molecular motors that transport cargo inside of living cells. There are two main classes of dyneins, the more well characterized dynein 1 family and the less understood dynein 2 family. While dynein 1 has a broad range of intracellular functions, dynein 2's role is restricted to transport within specialized cell extensions called cilia and flagella. The long-term objective of this proposal is to fully characterize the dynein 2 motor at the single molecule level and provide a genetic system for manipulating the motor in living organisms in order to determine the functional consequences of these single molecule properties. Because cilia are implicated in human diseases such as polycystic kidney disease and retinal degeneration, and dynein 2 is essential for normal functioning of these organelles, this proposal is directly relevant to human health. The proposal is divided into two core aims: the in vitro characterization of the dynein 2 motor properties at the single molecule level and the in vivo manipulation of these measured properties in the genetic model Caenorhabditis elegans (C. elegans). We will clone out a minimal dynein 2 motor domain from C. elegans and express the protein using the baculovirus system. We will characterize the enzymatic and motile properties of dynein 2 using a combination of biochemical and biophysical approaches. Parameters such as ATPase, stall force, processivity and velocity will be measured in vitro and mutations will be sought to modulate these single molecule properties. We will then reintroduce the mutated dynein 2 protein into a null background in C. elegans. This will allow us to determine how distinct biophysical properties relate to dynein 2's functions in vivo. Changes in single molecule parameters such as force production and processivity will be assayed for the first time in a living system to determine how critical those parameters are for normal motor function. PUBLIC HEALTH RELEVANCE: Ciliopathies are a broad spectrum of human diseases caused by malfunctions of cell protrusions called cilia. Cilia are built and maintained by specialized transport proteins whose functions are incompletely understood. This project will investigate, in detail, one of those transport proteins to determine how it works as a molecular motor and how its motor functions are related to the normal functioning of cilia. )