Increased phosphorylation of dynein IC IC138 correlates with decreases in flagellar microtubule sliding and phototaxis defects. To test the hypothesis that regulation of IC138 phosphorylation controls flagellar bending, we cloned the IC138 gene. IC138 encodes a novel protein with a calculated mass of 111 kDa and is predicted to form seven WD-repeats at the C terminus. IC138 maps near the BOP5 locus, and bop5-1 contains a point mutation resulting in a truncated IC138 lacking the C terminus, including the seventh WD-repeat. bop5-1 cells display wild-type flagellar beat frequency but swim slower than wild-type cells, suggesting that bop5-1 is altered in its ability to control flagellar waveform. Swimming speed is rescued in bop5-1 transformants containing the wild-type IC138, confirming that BOP5 encodes IC138. With the exception of the roadblock-related light chain, LC7b, all the other known components of the I1 complex, including the truncated IC138, are assembled in bop5-1 axonemes. Thus, the bop5-1 motility phenotype reveals a role for IC138 and LC7b in the control of flagellar bending. IC138 is hyperphosphorylated in paralyzed flagellar mutants lacking radial spoke and central pair components, further indicating a role for the radial spokes and central pair apparatus in control of IC138 phosphorylation and regulation of flagellar waveform. INTRODUCTIONOur goal is to determine the mechanisms that regulate ciliary and eukaryotic flagellar bending. Based on informative mutations in Chlamydomonas, and effective in vitro functional studies, a surprisingly complex array of different dynein motors is required for generation and control of normal ciliary and flagellar bending (Mitchell, 1994;Gibbons, 1995;Porter, 1996;Porter and Sale, 2000;DiBella and King, 2001;Kamiya, 2002). For example, the outer arm dyneins are homogeneous structures responsible for control of beat frequency and power required for movement (Satir et al., 1993;Brokaw, 1994;DiBella and King, 2001;Kamiya, 2002). The inner arm dyneins, however, are more complex, composed of at least seven different dynein subspecies precisely organized in a 96-nm repeat pattern along each doublet microtubule of the axoneme (Porter, 1996;Porter and Sale, 2000). Diverse data indicate the inner arm dyneins control the size and shape of the flagellar bend (Brokaw and Kamiya, 1987;Brokaw, 1994;Kamiya, 2002). The mechanism for control of flagellar waveform involves additional structures (e.g., radial spokes, central pair apparatus, and the dynein regulatory complex) and control of dynein phosphorylation (Porter and Sale, 2000;DiBella and King, 2001;Kamiya, 2002;Smith and Yang, 2004).The present study is focused on a single inner arm dynein, the I1 complex, also called the f-dynein (Goodenough et al., 1987;Piperno et al., 1990;Kagami and Kamiya, 1992;Porter et al., 1992). The I1 complex is a tripartite structure, or triad, located near the base of the S1 radial spoke, at the proximal end of the axonemal 96-nm repeat (Goodenough and Heuser, 1985;Piperno et al., 1990;Mastronar...

Among the major challenges in understanding ciliary and flagellar motility is to determine how the dynein motors are assembled and localized and how dyneindriven outer doublet microtubule sliding is controlled. Diverse studies, particularly in Chlamydomonas, have determined that the inner arm dynein I1 is targeted to a unique structural position and is critical for regulating the microtubule sliding required for normal ciliary/flagellar bending. As described in this review, I1 dynein offers additional opportunities to determine the principles of assembly and targeting of dyneins to cellular locations and for studying the mechanisms that regulate dynein activity and control of motility by phosphorylation. Cell Motil.

␥-Tubulin is a conserved essential protein required for assembly and function of the mitotic spindle in humans and yeast. For example, human ␥-tubulin can replace the ␥-tubulin gene in Schizosaccharomyces pombe. To understand the structural/functional domains of ␥-tubulin, we performed a systematic alanine-scanning mutagenesis of human ␥-tubulin (TUBG1) and studied phenotypes of each mutant allele in S. pombe. Our screen, both in the presence and absence of the endogenous S. pombe ␥-tubulin, resulted in 11 lethal mutations and 12 cold-sensitive mutations. Based on structural mapping onto a homology model of human ␥-tubulin generated by free energy minimization, all deleterious mutations are found in residues predicted to be located on the surface, some in positions to interact with ␣-and/or ␤-tubulins in the microtubule lattice. As expected, one class of tubg1 mutations has either an abnormal assembly or loss of the mitotic spindle. Surprisingly, a subset of mutants with abnormal spindles does not arrest in M phase but proceeds through anaphase followed by abnormal cytokinesis. These studies reveal that in addition to its previously appreciated role in spindle microtubule nucleation, ␥-tubulin is involved in the coordination of postmetaphase events, anaphase, and cytokinesis.

γ-Tubulin is essential for the nucleation and organization of mitotic microtubules in dividing cells. It is localized at the microtubule organizing centers and mitotic spindle fibres. The most well accepted hypothesis for the initiation of microtubule polymerization is that α/β-tubulin dimers add onto a γ-tubulin ring complex (γTuRC), in which adjacent γ-tubulin subunits bind to the underlying non-tubulin components of the γTuRC. This template thus determines the resulting microtubule lattice. In this study we use molecular modelling and molecular dynamics simulations, combined with computational MM-PBSA/MM-GBSA methods, to determine the extent of the lateral atomic interaction between two adjacent γ-tubulins within the γTuRC. To do this we simulated a γ-γ homodimer for 10 ns and calculated the ensemble average of binding free energies of -107.76 kcal/mol by the MM-PBSA method and of -87.12 kcal/mol by the MM-GBSA method. These highly favourable binding free energy values imply robust lateral interactions between adjacent γ-tubulin subunits in addition to their end-interactions longitudinally with other proteins of γTuRC. Although the functional reconstitution of γ-TuRC subunits and their stepwise in vitro assembly from purified components is not yet feasible, we nevertheless wanted to recognize hotspot amino acids responsible for key γ-γ interactions. Our free energy decomposition data from converting a compendium of amino acid residues identified an array of hotspot amino acids. A subset of such mutants can be expressed in vivo in living yeast. Because γTuRC is important for the growth of yeast, we could test whether this subset of the hotspot mutations support growth of yeast. Consistent with our model, γ-tubulin mutants that fall into our identified hotspot do not support yeast growth.

Dyneins are minus end directed microtubule motors that play a critical role in ciliary and flagellar movement. Ciliary dyneins, also known as axonemal dyneins, are characterized based on their location on the axoneme, either as outer dynein arms or inner dynein arms. The I1 dynein is the best-characterized subspecies of the inner dynein arms; however the interactions between many of the components of the I1 complex and the axoneme are not well defined. In an effort to elucidate the interactions in which the I1 components are involved, we performed zero-length crosslinking on axonemes and studied the crosslinked products formed by the I1 intermediate chains, IC138 and IC140. Our data indicate that IC138 and IC140 bind directly to microtubules. Mass-spectrometry analysis of the crosslinked product identified both α- and β-tubulin as the IC138 and IC140 binding partners. This was further confirmed by crosslinking experiments carried out on purified I1 fractions bound to Taxol-stabilized microtubules. Furthermore, the interaction between IC140 and tubulin is lost when IC138 is absent. Our studies support previous findings that intermediate chains play critical roles in the assembly, axonemal targeting and regulation of the I1 dynein complex.