In vivo analyses of radial spoke transport, assembly, repair and maintenance

Abstract

Radial spokes (RSs) are multiprotein complexes that regulate dynein activity. In the cell body, RS proteins (RSPs) are present in a 12S precursor, which enters the flagella and converts into the axoneme-bound 20S spokes consisting of a head and stalk. To study RS dynamics in vivo, we expressed fluorescent protein (FP)-tagged versions of the head protein RSP4 and the stalk protein RSP3 to rescue the corresponding Chlamydomonas mutants pf1, lacking spoke heads, and pf14, lacking RSs entirely. RSP3 and RSP4 mostly co-migrated by intraflagellar transport (IFT). The transport was elevated during flagellar assembly and IFT of RSP4-FP depended on RSP3. To study RS assembly independently of ciliogenesis, strains expressing FP-tagged RSPs were mated to untagged cells with, without, or with partial RSs. Tagged RSPs were incorporated in a spotted fashion along wild-type-derived flagella indicating an exchange of RSs. During the repair of pf1-derived axonemes, RSP4-FP is added onto the preexisting spoke stalks with little exchange of RSP3. Thus, RSP3 and RSP4 are transported together but appear to separate inside flagella during the repair of RSs. The 12S RS precursor encompassing both proteins could represent a transport form to ensure stoichiometric delivery of RSPs into flagella by IFT.

DIAPH1 regulates ciliogenesis and trafficking in primary cilia

“…According to the supply‐limitation model, cilia will grow until the cell body pool of ciliary precursors is exhausted. The availability of cargoes, therefore, regulates IFT cargo loading and cilia length 50 . Our results are also consistent with this model, where we show basal body targeted DIAPH1 caused elongation or the formation of bulbs at the tips of cilia, probably owing to their ability to traffic vesicles and cargo to the base of the cilia.…”

“…As well, our findings that neither actin polymerization nor microtubule stabilization mutants of DIAPH1 rescue ciliation suggests that both functions of DIAPH1 are important in this process. Other studies have shown that changes in either actin or microtubule networks can influence cilia length by changing the level of soluble tubulin 49 or regulating the trafficking of vesicles to the base of cilia 50 …”

The roles of a flagellar HSP40 ensuring rhythmic beating

“…Compared with other radial spoke proteins (RSPs), spoke HSP40 is unusual. Axonemal components are imported from the cell body and then assemble at the microtubule plus ends at the flagellar tip (Rosenbaum and Child, 1967; Johnson and Rosenbaum, 1992; Lechtreck et al ., 2018). In general, components in an axonemal complex are first packaged into a precursor complex in the cell body (Fowkes and Mitchell, 1998; Qin et al ., 2004) and then delivered by anterograde intraflagellar transport (IFT) trains to the tip for the final assembly (reviewed by Rosenbaum and Witman, 2002).…”

In vivo analysis of outer arm dynein transport reveals cargo-specific intraflagellar transport properties

“…, 1996; Viswanadha et al. , 2014), radial spokes (Johnson and Rosenbaum, 1992; Lechtreck et al. , 2018), dynein regulatory complexes (DRCs; Bower et al.…”

“…Incorporation of GFP-RSP4 and GFP-DRC4, which are both transported more processively and less retrogradely than IC2-NG, occurs preferably at the tip with little exchange along the length of flagella (Wren et al. , 2013; Lechtreck et al. , 2018).…”