Dynein molecular motors are required for many essential motile cellular processes. In cilia/flagella, dyneins form the inner and outer arms that power these organelles;defects in these enzymes and their regulatory systems lead to a broad array of severe and diverse phenotypes in humans such as infertility, bronchiectasis and situs inversus. Dynein motors are highly complex macromolecular assemblies and the mechanisms by which they are assembled within the axoneme and how they function to generate specific waveforms in response to various signals are very unclear. In this application, we will address two key questions concerning dynein mechanics, regulation and assembly. The outer arm ? heavy chain (HC) appears to act as a key regulatory node which is the ultimate target of Ca2+, redox and mechanosensory signals.
The first aim will focus on Lis1 which is known to regulate cytoplasmic dynein motor activity. Lis1 is also present in cilia/flagella and intra-flagella levels of Lis1 are modulated in response to imposed alterations in flagella beat parameters. For example, Lis1 levels are highly elevated when flagella are placed under high viscous load which leads to an increase in the intrinsic flagella beat frequency. Thus, the cell has a previously unrecognized mechanism for monitoring motility that can control Lis1 entry into or retention within the flagellum and modify power output. In Chlamydomonas, most Lis1 is located in the basal body region and is present in several distinct cytoplasmic complexes. These will be purified to define their composition and genetic and biochemical approaches used to test their role in controlling the load-sensitive response.
The second aim, will address how outer arm dynein is assembled at specific sites within the axoneme. Although the docking complex and ODA5 are required for outer arm dynein assembly within the flagellum, it is now clear that they are not sufficient as two novel factors (CCDC103 and FBB18) are also necessary. CCDC103 is very tightly associated with the axonemal microtubules and has extraordinary biophysical properties that suggest it may self- assemble potentially acting as a "molecular ruler" to help pattern the fundamental 96-nm axonemal repeat. Cytoplasmic and flagella complexes containing these proteins will be analyzed to determine how they interact with both dynein and other axonemal components. This will allow the essential, and previously unsuspected, role these proteins play in the dynein assembly process to be defined.
Motile cilia/flagella are of central importance to human health and development;defects in these organelles lead to a broad array of disease and developmental phenotypes such as male/female infertility, situs inversus and severe bronchial problems. The beating of cilia is driven by dynein motors and this application will address the mechanisms by which dyneins are assembled and how they are controlled in response to varying regulatory inputs. This will yield fundamental insight into how these complex organelles generate motive force and fluid flow.
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