Cloning &in vivo role of flagellar protein Kinase A

University Of Wisconsin La Crosse, La Crosse WI

Investigators

Abstract

Numerous diseases are caused by defective cilia and/or flagella, including primary ciliary dyskinesia, polycystic kidney disease, lateralization defects, Bardet-Biedl syndrome, retinal degeneration, and male infertility. All of these diseases can be caused by a variety of molecular defects, some of which have recently been uncovered and some of which remain unknown. Progress in understanding cilia and flagella in health is largely due to the knowledge gained through basic science done in the model organism Chlamydomonas reinhardtii. The regulation of motility in cilia and flagella remains relatively poorly understood, both at a basic level and in the relationship to disease. This proposal is focused on determining the identity and the in vivo role of flagellar cAMP dependent protein kinase (PKA) in Chlamydomonas. Abundant evidence indicates that PKA regulates flagellar motility in both Chlamydomonas and humans. In Chlamydomonas, it has been shown that PKA slows the activity of dynein motors in vitro, but the in vivo role of PKA in flagella is not known. In addition, the PKA protein or gene has not been positively identified or characterized. In Aim 1, flagellar PKA proteins will be identified in an affinity-based approach using the same tools that were used to show PKA's function in vitro. This method allows the discoveries to be immediately related to previously established in vitro data. Identified PKA(s) will be partially sequence using tandem MS, and their full sequence located. A gene exists in the Chlamydomonas database that could possibly be PKA, but its predicted protein sequence does not clearly classify it as a PKA. Based on our preliminary data, there seems to be multiple PKAs in flagella. In Aim 2, cDNAs coding for flagellar PKA will be cloned and used to determine the number of different PKA transcripts that are made. In Aim 3, the in vivo role of flagellar PKA will be determined by disrupting its function, either by electroporatirig cells with biotin-PKI to inhibit kinase activity or by RNA-mediated interference (RNAi). Swimming velocity, phototaxis ability, and beat waveform parameters will then be measured. Together, this project will allow data about the in vitro control of dynein activity to be related to changes in flagellar beating in vivo. In addition the project will provide critical information about a key component of the pathway that regulates flagellar notility. An integral component of this project is the involvement of undergraduates in all aspects of the work.