Pinfen Yang

Flagellar radial spoke protein 2 is a calmodulin binding protein required for motility in Chlamydomonas reinhardtii.

ABSTRACT Genetic and morphological studies have revealed that the radial spokes regulate ciliary and flagellar bending. Functional and biochemical analysis and the discovery of calmodulin in the radial spokes suggest that the regulatory mechanism involves control of axonemal protein phosphorylation and calcium binding to spoke proteins. To identify potential regulatory proteins in the radial spoke, in-gel kinase assays were performed on isolated axonemes and radial spoke fractions. The results indicated that radial spoke protein 2 (RSP2) can bind ATP and transfer phosphate in vitro. RSP2 was cloned and mapped to the PF24 locus, a gene required for motility. Sequencing revealed that pf24 contains a point mutation converting the first ATG to ATA, resulting in only trace amounts of RSP2 and confirming the RSP2 mapping. Surprisingly, the sequence does not include signature domains for conventional kinases, indicating that RSP2 may not perform as a protein kinase in vivo. However, the predicted RSP2 protein sequence contains Ca2+-dependent calmodulin binding motifs and a GAF domain, a domain found in diverse signaling proteins for binding small ligands including cyclic nucleotides. As predicted from the sequence, recombinant RSP2 binds calmodulin in a calcium-dependent manner. We postulate that RSP2 is a regulatory subunit of the radial spoke involved in localization of calmodulin for control of motility.

IC97 Is a Novel Intermediate Chain of I1 Dynein That Interacts with Tubulin and Regulates Interdoublet Sliding

Our goal is to understand the assembly and regulation of flagellar dyneins, particularly the Chlamydomonas inner arm dynein called I1 dynein. Here, we focus on the uncharacterized I1-dynein IC IC97. The IC97 gene encodes a novel IC without notable structural domains. IC97 shares homology with the murine lung adenoma susceptibility 1 (Las1) protein--a candidate tumor suppressor gene implicated in lung tumorigenesis. Multiple, independent biochemical assays determined that IC97 interacts with both alpha- and beta-tubulin subunits within the axoneme. I1-dynein assembly mutants suggest that IC97 interacts with both the IC138 and IC140 subunits within the I1-dynein motor complex and that IC97 is part of a regulatory complex that contains IC138. Microtubule sliding assays, using axonemes containing I1 dynein but devoid of IC97, show reduced microtubule sliding velocities that are not rescued by kinase inhibitors, revealing a critical role for IC97 in I1-dynein function and control of dynein-driven motility.

Sequential assembly of flagellar radial spokes.

The unicellular alga Chlamydomonas can assemble two 10 μm flagella in 1 h from proteins synthesized in the cell body. Targeting and transporting these proteins to the flagella are simplified by preassembly of macromolecular complexes in the cell body. Radial spokes are flagellar complexes that are partially assembled in the cell body before entering the flagella. On the axoneme, radial spokes are “T” shaped structures with a head of five proteins and a stalk of 18 proteins that sediment together at 20S. In the cell body, radial spokes are partially assembled; about half of the radial spoke proteins (RSPs) form a 12S complex. In mutants lacking a single RSP, smaller spoke subassemblies were identified. When extracts from two such mutants were mixed in vitro the 12S complex was assembled from several smaller complexes demonstrating that portions of the stepwise assembly of radial spoke assembly can be carried out in vitro to elucidate the order of spoke assembly in the cell body.

Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 in Chlamydomonas flagella

The microtubule plus-end tracking protein EB1 moves into flagella and accumulates at the tip independently of intraflagellar transport. EB1 dwell for seconds at the tip, indicating stable EB1-binding sites. Simulations show that diffusion to capture is an alternative mechanism to accumulate proteins in cilia.

The versatile molecular complex component LC8 promotes several distinct steps of flagellar assembly

At the tip of flagella, an array of LC8 dimers binds to the spoke protein RSP3 in radial spoke precursors, triggering phosphorylation, stalk base formation, and axoneme targeting.

A flagellar A-kinase anchoring protein with two amphipathic helices forms a structural scaffold in the radial spoke complex

Amphipathic helices in the A-kinase anchoring protein RSP3 bind to spoke proteins involved in the assembly and modulation of the flagellar radial spoke complex, expanding the repertoire of these versatile helical protein motifs.

Novel LC8 Mutations Have Disparate Effects on the Assembly and Stability of Flagellar Complexes

LC8 functions as a dimer crucial for a variety of molecular motors and non-motor complexes. Emerging models, founded on structural studies, suggest that the LC8 dimer promotes the stability and refolding of dimeric target proteins in molecular complexes, and its interactions with selective target proteins, including dynein subunits, is regulated by LC8 phosphorylation, which is proposed to prevent LC8 dimerization. To test these hypotheses in vivo, we determine the impacts of two new LC8 mutations on the assembly and stability of defined LC8-containing complexes in Chlamydomonas flagella. The three types of dyneins and the radial spoke are disparately affected by dimeric LC8 with a C-terminal extension. The defects include the absence of specific subunits, complex instability, and reduced incorporation into the axonemal super complex. Surprisingly, a phosphomimetic LC8 mutation, which is largely monomeric in vitro, is still dimeric in vivo and does not significantly change flagellar generation and motility. The differential defects in these flagellar complexes support the structural model and indicate that modulation of target proteins by LC8 leads to the proper assembly of complexes and ultimately higher level complexes. Furthermore, the ability of flagellar complexes to incorporate the phosphomimetic LC8 protein and the modest defects observed in the phosphomimetic LC8 mutant suggest that LC8 phosphorylation is not an effective mechanism for regulating molecular complexes.

Chlamydomonas mutants display reversible deficiencies in flagellar beating and axonemal assembly.

Axonemal complexes in flagella are largely prepackaged in the cell body. As such, one mutation often results in the absence of the co‐assembled components and permanent motility deficiencies. For example, a Chlamydomonas mutant defective in RSP4 in the radial spoke (RS), which is critical for bend propagation, has paralyzed flagella that also lack the paralogue RSP6 and three additional RS proteins. Intriguingly, recent studies showed that several mutant strains contain a mixed population of swimmers and paralyzed cells despite their identical genetic background. Here we report a cause underlying these variations. Two new mutants lacking RSP6 swim processively and other components appear normally assembled in early log phase indicating that, unlike RSP4, this paralogue is dispensable. However, swimmers cannot maintain the typical helical trajectory and reactivated cell models tend to spin. Interestingly the motile fraction and the spokehead content dwindle during stationary phase. These results suggest that (1) intact RS is critical for maintaining the rhythm of oscillatory beating and thus the helical trajectory; (2) assembly of the axonemal complex with subtle defects is less efficient and the inefficiency is accentuated in compromised conditions, leading to reversible dyskinesia. Consistently, several organisms only possess one RSP4/6 gene. Gene duplication in Chlamydomonas enhances RS assembly to maintain optimal motility in various environments.

Chapter 7 – The Flagellar Radial Spokes

Publisher Summary The radial spokes are a major structural feature of 9 + 2 axonemes and are crucial for flagellar beating. The enormous variety of research tools that Chlamydomonas offers a model system, such as radial spoke defective mutants and in vitro functional assays, as well as the unique biochemical properties of spoke components, have all contributed to our understanding of the radial spokes. This chapter reviews the composition, assembly, and possible functions of this macromolecular complex. Chlamydomonas mutants that are defective in radial spoke assembly have played a central role in studies of spoke composition and function. The observation presented in the genetics, supports the hypothesis that the direct interaction between the spoke heads and central pair projections is crucial for the motility of 9 + 2 cilia and flagella. For decades, the axonemal dynein arms have been the target of biochemical and functional analysis due in large part to the ease with which they can be extracted and purified from isolated flagella. Extraction along with composition and functional domains are explained in the biochemical characterization. A variety of approaches including genetic, structural, and functional studies have provided significant insights into understanding the function of the radial spokes. These observations provide strong evidence that regulation of dynein-driven microtubule sliding involves a signal transduction pathway that includes the radial spokes. An enormous amount of evidence indicates that the radial spokes play an important role in modulating flagellar motility.

Radial Spokes-A Snapshot of the Motility Regulation, Assembly, and Evolution of Cilia and Flagella

Propulsive forces generated by cilia and flagella are used in events that are critical for the thriving of diverse eukaryotic organisms in their environments. Despite distinctive strokes and regulations, the majority of them adopt the 9+2 axoneme that is believed to exist in the last eukaryotic common ancestor. Only a few outliers have opted for a simpler format that forsakes the signature radial spokes and the central pair apparatus, although both are unnecessary for force generation or rhythmicity. Extensive evidence has shown that they operate as an integral system for motility control. Recent studies have made remarkable progress on the radial spoke. This review will trace how the new structural, compositional, and evolutional insights pose significant implications on flagella biology and, conversely, ciliopathy.