Mitchell Lab Research

Mitchell Lab Research Interests:

Cartoon of Chlamydomonas reinhardtii Isolated axoneme reactivated at low calcium Our work is directed toward understanding the mechanisms that regulate the microtubule-based motor dynein during motility of eukaryotic cilia and flagella. The approach focuses on mutations in a model organism, Chlamydomonas reinhardtii, that alter dynein activity by disrupting assembly of dynein motors themselves or regulatory complexes such as the central pair apparatus that modulate dynein activity. Based on many previous studies, projections from the two central pair microtubules interact with radial spokes, which in turn transmit regulatory signals to doublet microtubule-associated dyneins. A cascade of protein kinases has been implicated in this signaling process, but the mechanisms remain poorly understood.

Axoneme cross section All known dynein ATPase motors in cilia and flagella are attached to the A-tubule surface of each outer doublet (the cargo) and translocate along the B-tubule surface of an adjacent doublet (the track) to power flagellar bending. Much of our present understanding of these motors derives from studies of the many mutations that affect flagellar structure and function in Chlamydomonas (summarized in the figure to the right). Although many of these mutations completely block flagellar motility, others modify motility in ways that reveal the functional roles of their gene products. There are multiple unique dynein complexes that attach along the inner margin (inner row dyneins) or outer margin (outer row dyneins) of each A-tubule. We are studying outer row dyneins through isolation of outer row dynein assembly (oda) mutations and characterization of their gene products.

Dyneins: Cloning ODA Loci

Isolated axoneme reactivated at high calcium

One major thrust of current work in the lab is to clone and characterize loci that do not apparently encode dynein enzyme subunits, but that are essential for normal outer row dynein assembly and function. These include oda7, oda8, oda16, and pf13. The oda7 mutation is similar phenotypically to many other oda mutations, resulting in absence of outer row dyneins and reduced flagellar beat frequency. Unlike other oda mutations, oda7 appears to specifically reduce the unassembled cytoplasmic pool of one catalytic dynein heavy chain (Fowkes and Mitchell, 1998). Starting with a molecular marker that maps near oda7, we walked to the locus in a BAC library. Current work in the lab suggests that the oda7 gene product is a protein that interacts with both outer row and I1 inner row dyneins. The oda8 locus has also been recently cloned, and work is in progress to characterize its gene product. Mutations at PF13 not only reduce the assembly of outer row dyneins, they also block flagellar motility. Our unpublished results indicate that this mutation also blocks cytoplasmic preassembly of outer row dynein, and disrupts the assembly of a structure in the inner dynein row as well as the outer row. An insertional allele at this locus has been used to clone the gene, and we are beginning a molecular analysis of the gene product. The oda16 mutation only partially disrupts outer row dynein assembly, and has no effect on cytoplasmic pre-assembly of outer row dynein complexes. Two insertional mutations at this locus have been used to clone the gene, and we recently determined that the gene product is a highly-conserved flagellar matrix protein (Ahmed and Mitchell, 2005). Our working hypothesis is that Oda16p is part of a complex important for transport and assembly of axonemal dyneins.

Kinesins and the Central Pair Microtubule Complex

Little is known about the mechanism through which interactions between radial spokes and the central pair complex regulate dynein activity. We are currently exploring three aspects of central pair structure and function. Although most mutations affecting central pair structure completely block motility, we recently described a Chlamydomonas mutation that allows altered motility. This mutation, cpc1, specifically prevents assembly of one row of central pair microtubule projections (Mitchell and Sale, 1999). Insertionally tagged alleles were used to clone and sequence the gene, which encodes a large protein with an embedded adenylate kinase domain (Zhang and Mitchell, 2004) . However, the reduction in motility in this mutant probably results from a reduction in intraflagellar ATP concentration due to the reduction in a glycolytic enzyme, enolase, that resides in the Cpc1 complex (Mitchell et al., 2005).

We are also analyzing the kinesin-like proteins that have been identified as central pair proteins. Dyneins were long thought to be the only motor proteins present in eukaryotic cilia and flagella, but kinesins have been discovered in association with intraflagellar transport particles and with the central pair microtubules. Although at least two central pair kinesins have been identified by using pan-specific anti-kinesin antibodies, only one of these, klp1, was cloned and localized to one central pair microtubule (Bernstein et al., J. Cell Biology 125:1313-1326, 1992). We are working to identify the role of klp1 in flagellar motility (Yokoyama et al., 2004) and to molecularly characterize additional kinesin-like proteins in the flagellar axoneme.

We are also continuing to explore the electron microscopic ultrastructure of the central pair complex (Mitchell 2003 a, b). This has led to the conclusion that the Chlamydomonas central pair both twists and rotates, and maintains a specific orientation in bent regions. By examining the orientation and shape of the central pair in both straight and bending axonemes, and in both motile flagella and flagella of non-motile (radial spoke-defective) mutants, we found that the orientation of the central pair is constant with respect to bends. As bends propagate, the central pair orientation also propagates, giving rise to the apparent rotation of the central pair complex (Mitchell and Nakatsugawa, 2004). These observations have supported speculations about the evolution of the central pair regulatory apparatus and of 9 + 2 organelles in general (Mitchell, 2004, 2005, 2006).