The long term goal is to determine the molecular mechanisms that regulate the molecular motor dynein and the control of ciliary / flagellar bending. Dyneins are ubiquitous molecular motors required for vital cell functions including directed cytoplasmic transport, function of the Golgi and mitotic apparatus and movement of cilia. Cilia are responsible for diverse and critical motile and sensory functions in the developing and adult human. Defective ciliary assembly or dynein function lead to abnormal Left-Right patterning, heart abnormalities, hydrocephaly, skeletal defects, """"""""primary ciliary dyskinesia"""""""" (a disease that manifests in failure in normal respiratory function, oviduct function and infertility in the male), blindness and polycystic kidney disease. Advances in understanding these and other diseases in the human are possible due to genetic and molecular advantages offered by Chlamydomonas. Chlamydomonas will continue as a key experimental system for gene discovery, functional analysis of dynein and the cilium and translational study of human biology and disease. The fundamental questions addressed by our studies include: How does dynein generate force? How is dynein activity regulated? How is dynein targeted and anchored to cellular cargoes? How is ciliary bending regulated? Diverse evidence indicates dynein is controlled by phosphorylation of intermediate chain subunits. The work proposed focuses on one axonemal dynein, inner arm dynein 11 and its regulatory intermediate chain IC138, which plays a central role in control of motility. Our focus is also on a """"""""solid-state"""""""" network of axonemal structures, including the radial spokes and proteins kinases (PKA) and phosphatases (PP1, PP2A) that regulate 11 dynein phosphorylation and control ciliary bending. The 11 dynein and Chlamydomonas offer a unique and exceptionally powerful system for study of dynein.
The specific aims are:[1] determine how phosphorylation of IC138 regulates 11 dynein activity, taking advantage of structural approaches and in vitro motility assays; [2] determine the regulatory domains in IC138 taking advantage of Chlamydomonas mutant strains, and define the role of the IC97 intermediate chain in 11 dynein; [3] define the axonemal machinery that anchors PKA in position for precise control of11 dynein, with particular focus on the axonemal A-kinase anchoring protein (AKAP) radial spoke protein 3 that localizes PKA in the axoneme, playing a role in mechano-chemical control of axonemal activities . We will also test the hypothesis that the dynein regulatory complex protein 1, DRC1, plays a role in organizing and anchoring kinases and phosphatases required for local control of dynein motor activity.
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