Structures of radial spokes and associated complexes important for ciliary motility

Abstract

In motile cilia, a mechanoregulatory network is responsible for converting the action of thousands of dynein motors bound to doublet microtubules into a single propulsive waveform. Here, we use two complementary cryo-EM strategies to determine structures of the major mechanoregulators that bind ciliary doublet microtubules in Chlamydomonas reinhardtii . We determine structures of isolated radial spoke RS1, and the microtubule-bound RS1, RS2, and the nexin-dynein regulatory complex. From these structures, we identify and build atomic models for 30 proteins including 23 radial-spoke subunits. We reveal how mechanoregulatory complexes dock to doublet microtubules with regular 96-nm periodicity and communicate with one another. Additionally, we observe a direct and dynamically coupled association between RS2 and the dynein motor IDA c , providing a molecular basis for the control of motor activity by mechanical signals. These structures advance our understanding of the role of mechanoregulation in defining the ciliary waveform.

LRRC23 is a conserved component of the radial spoke that is necessary for sperm motility and male fertility in mice

“…Immunoelectron microscopy uncovered that LRRC23 localized to the RS within spermatozoa (Fig 4B-4K). The LRRC23 homolog RSP15 in Chlamydomonas has previously been shown to interact with RSP3 and RSP22 [26]. Although we did not detect any interaction between LRRC23 and RSPH22, we did find that it was able to interact with RSPH3A/B (S5B-S5D Fig), strongly suggesting that LRRC23 is a RS component within sperm flagella.…”

Composition and function of ciliary inner‐dynein‐arm subunits studied in Chlamydomonas reinhardtii

“…LC8, also called DLL1 or FLA14, is a LC of IDA f/I1, ODAs and the IFT dynein (cytoplasmic dynein 2) (DiBella et al, 2001; Pazour, Wilkerson, & Witman, 1998; Figure 3; Tables 1 and 2). LC8 is also a component of ciliary radial spokes involved in the assembly of radial spoke structures (Gui et al, 2021; Gupta, Diener, Sivadas, Rosenbaum, & Yang, 2012; Yang, Diener, Rosenbaum, & Sale, 2001; Yang, Yang, Wirschell, & Davis, 2009). LC8 contains a “Dynein light chain type 1 (Dynein light)” domain in its structure, and LC8 sequence/structure has been studied in detail (Barbar, 2008; King & Patel‐King, 1995).…”

“…From the stoichiometric analysis, two p28 molecules were thought to interact with one actin monomer and bind to each one of the DHCs (IDAs DHC6/a, DHC9/c, DHC2/d, and most likely DHC11 and DHC12; Kamiya & Yagi, 2014; Yanagisawa & Kamiya, 2001). Recently, a p28 homodimer interacting with DHC9 and actin was identified in cilia (Gui et al, 2021). A mammalian orthologue of p28, DNALI1 (Table 3), is also a LC of axonemal dyneins (Rashid et al, 2006).…”

“…However, tests of these ideas require additional work, and the binding site(s) on microtubules of these LCs of the single‐headed IDAs, as well as of ICs/LCs of the two‐headed IDA f/I1 must both be biochemically and structurally analyzed. Predictably, high‐resolution/atomic structure(s) of IDA docking sites on doublet microtubules observed by the single‐particle cryo‐electron microscopy (Gui et al, 2021; Ma et al, 2019) will reveal IDA docking mechanisms.…”

“…Notable advances using Chlamydomonas include discovery of intraflagellar transport (IFT; Kozminski, Johnson, Forscher, & Rosenbaum, 1993), definition of the cytoplasmic “preassembly” of ciliary dyneins (Fowkes & Mitchell, 1998), identification of the molecular basis of the 96‐nm ciliary axonemal repeat (Oda, Yanagisawa, Kamiya, & Kikkawa, 2014), and a high‐resolution structure of the meshwork of inner proteins of doublet microtubules (Ma et al, 2019). Chlamydomonas mutants have also revealed the conserved components of the axonemal radial spokes (Gui et al, 2021; Pigino et al, 2011; Poghosyan et al, 2020), nexin‐dynein regulatory complex (N‐DRC; Bower et al, 2013; Heuser, Raytchev, Krell, Porter, & Nicastro, 2009), calmodulin spoke complex (CSC; Dymek, Heuser, Nicastro, & Smith, 2011; Dymek & Smith, 2007), and central pair complex (Loreng & Smith, 2017). In addition, Chlamydomonas has contributed to understanding of the basal bodies/centrioles (Dutcher & O'Toole, 2016) and the function of the Bardet–Biedl syndrome proteins in cilia (Lechtreck et al, 2013; Li et al, 2004; Liu & Lechtreck, 2018; Nachury et al, 2007).…”