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. 2019 Jan 15;30(2):228-241.
doi: 10.1091/mbc.E18-01-0047. Epub 2018 Nov 14.

The Roles of a Flagellar HSP40 Ensuring Rhythmic Beating

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Free PMC article

The Roles of a Flagellar HSP40 Ensuring Rhythmic Beating

Xiaoyan Zhu et al. Mol Biol Cell. .
Free PMC article

Abstract

HSP40s are regarded as cochaperones, perpetually shuttling client polypeptides to HSP70s for refolding. However, many HSP40s that are central for disparate processes diverge from this paradigm. To elucidate the noncanonical mechanisms, we investigated HSP40 in the radial spoke (RS) complex in flagella. Disruption of the gene by the MRC1 transposon in Chlamydomonas resulted in jerky flagella. Traditional electron microscopy, cryo-electron tomography, and sub-tomogram analysis revealed RSs of various altered morphologies that, unexpectedly, differed between the two RS species. This indicates that HSP40 locks the RS into a functionally rigid conformation, facilitating its interactions with the adjacent central pair apparatus for transducing locally varied mechanical feedback, which permits rhythmic beating. Missing HSP40, like missing RSs, could be restored in a tip-to-base direction when HSP40 mutants fused with a HSP40 donor cell. However, without concomitant de novo RS assembly, the repair was exceedingly slow, suggesting HSP40/RS-coupled intraflagellar trafficking and assembly. Biochemical analysis and modeling uncovered spoke HSP40's cochaperone traits. On the basis of our data, we propose that HSP40 accompanies its client RS precursor when traveling to the flagellar tip. Upon arrival, both refold in concert to assemble into the mature configuration. HSP40's roles in chaperoning and structural maintenance shed new light on its versatility and flagellar biology.

Figures

FIGURE 1:
FIGURE 1:
Characterization of a new HSP40 mutant, pf33. (A) Western blots show a specific HSP40 deficiency (asterisk) in pf33 axonemes. The other proteins located at three major areas of the RS appear normal. Tubulin bands in the Ponceau S–stained membrane (bottom panel) show the protein loads. The WT strain serves as a positive control. The spokeless strain pf14 is a negative control. (B) Electron micrograph of axoneme cross-sections. Nearly all 45 RSs in all 5 collected WT axoneme sections (top panel) appear to be typical Y-shaped complexes (arrows), rendering an ordered appearance of axonemes. In contrast, the RSs in five images representing 12 focused pf33 axoneme sections appear disordered (bottom panel). The head/neck region of some RSs appears split or tilted (arrowhead). Only one seems normal (arrow). (C) A schematic depicting the HSP40 gene, the PCR genotyping strategy, and a partial MRC1 transposon inserted downstream of the stop codon in the reverse direction in pf33’s HSP40 gene. The primer pairs for PCR are depicted on arrows. The base pair number of each segment is indicated above or below. (D) DNA gels of PCR products from WT and pf33 cells (left) and restriction digest (right). PCR fragments of the N-terminal region from both strains are identical (e.g., the #2 fragment amplified by the primer pair 2S and 2AS). But fragments #3 and #4 from pf33 are 1.3 kb larger than those from the WT. The two fragments overlapped at the region around the stop codon. BglII and HindIII digests demonstrate specific PCR amplification. Sequencing of fragments #3 and #4 showed that the additional sequence is a partial 1.3-kb MRC1 fragment. The 3′ LTR is truncated.
FIGURE 2:
FIGURE 2:
Structural comparison of RSs from the WT (A, D) (EMD-2131; Bui et al., 2012), pf33 (B, E), and pf24 (C, F; Pigino et al., 2011) Chlamydomonas strain in cryo-electron tomograms. Density maps from averaged sub-tomograms of the 96-nm periodic unit are represented as transverse sections at the RS2 level (A–C) and longitudinal sections (D–F). The major differences among these three strains are at the neck (red arrow) and head regions. The density at these regions is weak and blurred for pf33 (B, E) as compared with WT (A, D); however, it is not completely lost, as in the headless strain pf24 (C, F). Scale bar: 24 nm.
FIGURE 3:
FIGURE 3:
Structural comparison between RS1 (A, C) and RS2 (B, D) of the WT (A, B) and pf33 (C, D) Chlamydomonas. (E) Schematic diagram illustrating the corresponding planes, crossing RS2 in the 96-nm periodic unit of C. reinhardtii flagella (EMDB 1941), of averaged slices shown in Figures 2 (plane 1) and 3 (plane 2). In the pf33 axoneme, the spokehead and one of the two neck branches in RS1 are blurred (C), whereas the other branch (red arrow) is completely missing compared with the WT reference (A). The head and neck regions in RS2 (D) also appear blurry compared with the RS2 in WT (B). However, the upper branch in RS2 is more intense than that in RS1 (C). For best visualization of RSs, densities represent an average of 15 slices along the z-axis (corresponding to 8.4 nm, which covers the entire thickness of the bifurcated spokeneck as well as the spokestalk), and rotation along the x-axis by +20° angle with respect to Figure 2, A–C. Scale bar: 24 nm.
FIGURE 4:
FIGURE 4:
pf33: classification of RS complexes in pf33 mutant axoneme. The 1D classification of subvolumes from the total average (pf33, top) results in two density maps (A, B). The subvolume representing class A, with ∼80% of all particles (N A = 541), has a distinct density at the interface between the spoke stalk and neck of RS1 (arrow in A). Particles in this class were further categorized into three subclasses, A1–A3 (N A1 = 102; N A2 = 131; N A3 = 193), each with a distinctive feature (red arrows). A1 has a weaker density at the spokehead periphery compared with the rest of the spokes. A2 has a slight change in the orientation of the RS2 neck region. A3, like class A, is prominent by the flexure of one branch in RS1 neck region (arrow in A3). The three subclasses from the particles forming class B (N B = 147) have variations distinct from those of class A (N B1 = 79; N B2 = 43; N B3 = 25). B1 is very similar to class B, with homogeneous density distribution in the RS complexes. In contrast, B2 has a slight distortion at the neck of RS2. In B3, the density of the spokehead region (dashed rectangle) appears very weak, indicative of further heterogeneity in this region. Control: classification of RS complexes in the axoneme of a motile pf14 transformant rescued by a full-length RSP3 transgene. With applied classification procedures, the data set representing the total, 96 nm–based average has diverged into two distinct classes. The majority of particles (∼60%) form class C1 (N C1 = 269) and have WT-like RS morphology with rather typical neck and head regions. The rest of particles are included in class C2 (N C2 = 174). The weak density of RS1 compared with RS2 suggests that RS1 might contain further structural variations that are not evident due to low signal-to-noise ratio of the particular class average. Scale bar: 24 nm.
FIGURE 5:
FIGURE 5:
Synchronized assembly of HSP40 and RSs. (A) Western blot of axonemes probed with anti-HSP40 antibody shows similar abundance of HSP40 from WT control cells and GFP-HSP40 from motile pf33 transformants. (B) Fluorescence microscopy of pf33 and GFP-HSP40 transformants. With the focus on GFP-HSP40–illuminated flagella, fluorescence distributes uniformly throughout the length of flagella in all cells from a motile transformant strain. Intense background fluorescence in the cell body of both strains is primarily derived from plastids. (C) Fluorescence images of live pf33::GFP-HSP40 cells taken periodically within 1 h following flagella excision. Fluorescence distributed throughout the regenerating flagella, regardless of the length. Dots show the tip of fluorescence. The experiment was performed three times. Cells from the same strain and same culture could vary in size. Scale bar: 10 μm.
FIGURE 6:
FIGURE 6:
Possible outcomes of dikaryon rescue experiments testing how HSP40 is transported and assembled. Immunofluorescence shows that RSs are restored to the RS-free flagella in the RSP3 mutant pf14 when a pf14 cell is fused to a WT donor cell. The same results are expected in the control group using motile pf14::RSP-GFP as donor cells. Illustrated are three possible outcomes for dikaryons of pf33 by the donor cell, pf33::GFP-HSP40. If HSP40 is delivered by IFT as RSs, the rescue pattern will be similar to the repair of pf14 flagella. If HSP40 cannot enter flagella, there will be no repair. On the other hand, if HSP40 diffuses into flagella, perhaps through the axonemal lumen, the fluorescence will appear in a base-to-tip direction.
FIGURE 7:
FIGURE 7:
Distinct efficiencies in the repair of missing RSs and HSP40 in dikaryon rescues. Cells were imaged by bright-field (top) and fluorescence (bottom) microscopy approximately every 15 min after cells of opposite mating types were mixed. Each image represents at least five dikaryons. (A) Dikaryons of pf14(−) by pf14(+)::RSP3-GFP. Fluorescence, already detectable at the tips of receiver pf14(+) flagella after 15 min (arrowheads), extended toward the base and became stronger as time progressed. The experiment was performed twice. (B) Delayed restoration of GFP-HSP40 to receiver pf33(+) flagella from pf33(−)::GFP-HSP40. Fluorescence was not detectable in receiver pf33 flagella until 120 min after mixing. Fluorescence also appeared at the flagellar tip (arrowheads) of six dikaryons among many observed at the 120-min time point from a total of three experiments, but the intensity was exceedingly dim. Pseudocolor was used to highlight the restoration. Flagella were shed soon afterward. The experiment was performed three times. (C) Efficient restoration of GFP-HSP40 along with de novo assembly of RSs in newly generated flagella of dikaryons. At 30 min after flagellar excision, pf33(+) with half-length regenerating flagella was mixed with pf33(−)::GFP-HSP40 with full-length flagella. GFP-HSP40 was restored to the distal part of growing HSP40-minus flagella of pf33(+) in all dikaryons at 60 min after mixing. Fluorescence intensity in regenerating receiver flagella varies. All dikaryon rescue experiments were performed in the presence of cycloheximide to inhibit protein synthesis. The experiment was performed once. (D) Comparison of fluorescence intensity in dikaryons’ flagella. Fluorescence intensity at the brightest region near the flagellar tip was measured. Averaged receiver vs. donor (R/D) intensity ratio and SD at the flagellar tip was 0.62 ± 0.19 (n = 13, from 15- to 60-min time points) for restored RSs with RSP3-GFP, 0.24 ± 0.08 (n = 8, from 120-min time points) for GFP-HSP40 restored to existing RSs, and 0.72 ± 0.3 (n = 9, from 60-min time points) for de novo assembly of GFP-HSP40 and flagellar assembly. Only flagella from the same dikaryons and in focus were used for comparison. HSP40 repair is significantly lower than RS/RSP3 repair (*, Student’s t test, p < 0.001) and lower than the de novo assembly of GFP-HSP40 into RSs in growing flagella (**, Student’s t test, p < 0.001). Asterisks indicate statistically significant differences.
FIGURE 8:
FIGURE 8:
Analysis of the interaction of HSP40 and NDK5. (A) Live fluorescence imaging of ndk5 transformant expressing NDK5ΔC-Neongreen. The fragment containing the conserved 1–201 a.a. rescued the ndk5 mutant. Fluorescence distributed evenly throughout flagella as expected of RSs. Thus, this region is sufficient for the assembly of HSP40. Scale bar: 5 μm. (B) Analytical ultracentrifugation of recombinant HSP40. c(S) distribution of HSP40 shows a single peak with an sw value of 5 S, which is consistent with a dimer formation. (C) Little copurification of His-HSP40 and NDK51–201 in vitro. His-tagged NDK51–201 (dot), HSP40, and TEV protease were expressed individually in bacteria and affinity purified with Ni-NTA (left panel). In the presence of His-TEV, His-NDK51–201 that had a TEV cleavage site was incubated overnight without or with HSP40. The mixture (Pre) was then subjected to Ni-NTA purification. Cleaved NDK51–201 (asterisk) was in the flowthrough (Post) regardless of the absence (middle panel) or presence (right panel) of His-HSP40 that, alone with His-TEV, was pulled down by Ni-NTA (in the eluate [Elu]). Samples were revealed by Coomassie blue–stained protein gels.
FIGURE 9:
FIGURE 9:
Structural modeling of the RS neck region. (A) The distal view of the second RS (RS2) in each axonemal repeat from human (left panel; Electron Microscopy Data Bank [EMDB] accession number: 5950; Lin et al., 2014) and Chlamydomonas (right panel; EMDB accession number: 1941; Pigino et al., 2011). The overlay (middle panel) highlights the bifurcated neck with a nodule (arrow) below each branch and an asymmetric protrusion (double arrowheads). The top view shows two head modules positioned in twofold rotational symmetry (arrowheads of opposite orientation in the bottom panel). (B) The crystal structure of a dimeric bacterial HSP40 (PDB accession number: 4J80), with the G/F region between J domains to the V-shaped C domain dimer. It also exhibits rotational symmetry when viewed after a 90o rotation. (C) Schematic depicting the proposed RS structure core composed of homodimers of RSP3 (red) and RSP2’s (green) and NDK5’s (blue) Dpy30 domains. (D) Top view of a space-filled model illustrating the predicted key structural components in the region below the bifurcated branches. P, proximal; D, distal. (E) Distal view (top panel) and longitudinal view (bottom panel) of ribbon models overlaying human RS2 (left panels) and Chlamydomonas RS2 (right panels). Each of the neck nodules (arrows) harbors a unit of an AH from RSP3 (red) that anchors a Dpy30 domain homodimer from RSP2 (green) or NDK5 (blue) (an equivalent structure from a Set1-like histone methyltransferase complex; PDB accession number: 4RIQ). The two units are clenched together by a J domain (magenta; PDB accession number: 2CTP) and the invisible flexible G/F-rich region that connects to the V-shaped C domain dimer (purple; PDB accession number: 3AGX). The NDK domain dimer of NDK5 (cyan, represented by NDK1; PDB accession number: 4ENO) is placed in the asymmetric protrusion (double arrowheads).
FIGURE 10:
FIGURE 10:
Schematic picture depicting coupled trafficking and refolding of HSP40 and its RS client. During trafficking, HSP40 dimer (magenta) tethers to the Г-shaped RS precursor (panel I). RSP2’s and NDK5’s Dpy30 domains (green and blue) associate with each other but only RSP2’s binds to RSP3’s AH at the base of one arm. Upon their arrival at the flagellar tip, the precursor is released from IFT trains. With the AH in the second branch becoming available to anchor NDK5’s Dpy30 domain dimer (blue), formalizing the Y-shaped, albeit flexible RS (panel II). This transformation induces the disordered G/F domains (wiggly lines) and J domains (pink) of HSP40 to refold around the Dpy30/AH module to stabilize mature RS (III). Refolding may occur near the spokehead if the V-shaped C-termini is positioned along the bifurcated branches. This process may reverse (gray arrows) when the RS is transported back to the cell body for recycling.

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