MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement

Abstract

Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained in vivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C. elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

Keywords: C. elegans; MAP9; axoneme; cilia; dynein; kinesin; microtubule; microtubule doublet; microtubule-associated protein; polyglutamylation.

Figure 1.. MAPH-9 localizes to microtubule doublets and preferentially binds polyglutamylated microtubules

(A) Endogenously tagged GFP::MAPH-9 (green), TBB-4::RFP, and RFP::SPD-5 (magenta) in indicated ciliated sensory neurons in adult worms. TBB-4/SPD-5 brightness scaled differently in PQR. Scale bars: Amphids and Phasmids, 5 μm; ADE, PDE, PQR, 1 μm. (B) Schematic depicting phasmid axoneme structure. Cross section in the doublet or singlet region with the A-tubule (magenta) and B-tubule (blue) indicated. (C) Localization of endogenous GFP::MAPH-9 (green) and XBX-1::RFP (magenta) in adult phasmid cilia. Scale bar, 1 μm. (D) Quantification of protein localization length (μm) from ciliary base marked by XBX-1. Dashed horizontal line is MTD length from EM in amphid cilia (~4.5 μm). XBX-1: 7.86±1.33 n=50; MAPH-9: 4.69±0.63 n=50; p<0.0001. (E) Averaged line profiles of MAPH-9 (green) and TBB-4 (black) fluorescence (a.u.) drawn perpendicular to the middle segment of the axoneme in 3D SIM imaging (yellow line on 1F): MAPH-9: 0.35±0.062 μm n=16; TBB-4: 0.30±0.036 μm n=13. p<0.05 (F) 3D structured illumination imaging of endogenous GFP::MAPH-9 (green) and TBB-4::RFP/RFP::SPD-5 (magenta) in adult phasmid cilia. Scale bar, 1 μm. (G) Schematic depicting the timeline of MTD development at centrioles (top, cross-sectional view) in amphid neurons during C. elegans embryonic development (bottom, side view) at indicated minutes post-fertilization (m.p.f.). (H) Localization of endogenous GFP::MAPH-9 (green) and RFP::SPD-5 (magenta) in amphid neurons through embryonic development. Scale bar, 1 μm. (I) Longitudinal view schematic depicting A-tubule (magenta), B-tubule (blue), and MAPH-9 (green) localization during amphid ciliogenesis in the embryo at indicated m.p.f. (J) Localization of endogenous GFP::MAPH-9 (green) and RFP::SPD-5/TBB-4::RFP (magenta) during ciliogenesis in amphids. Scale bar, 1 μm. (K) Total Internal Reflection Fluorescence microscopy images of: Top: microtubules assembled from tubulin from wild-type (WT) mice (white arrowheads) or Ttll1^−/− and Ttll7^−/− mice (orange arrowheads); Bottom: MAPH-9 localization. Scale bar, 5 μm. (L) Quantification of normalized fluorescence intensity of MAPH-9 localized to microtubules assembled from tubulin extracted from mice with indicated genotypes. WT: 1.0±0.31 n=105; Ttll1^−/−: 0.61±0.15 n=31 p<0.0001; Ttll7^−/−: 0.54±0.15 n=31 p<.0001; Ttll1^−/− Ttll7^−/−: 0.25±0.11 n=26 p<0.0001; Atat1^−/−: 0.89±.31 n=24 p=0.15. (M) Heat map localization of endogenous GFP::MAPH-9 in phasmid cilia in Control and ttll-4(tm3310), ttll-5(tm4059), ttll-9(tm3889), ttll-11(tm3360), tll-15(tm3871) polyglutamylation mutant (5xE mutant). Scale bar, 1 μm. (N) Quantification of fluorescence of α-polyglutamylated tubulin (GT335 antibody) staining. Control: 3.94±3.79 n=63; 5xE mutant: 0.28±0.19 n=25 p<0.0001. (O) Quantification of MAPH-9 fluorescence per unit length normalized to Control. Control:1±0.211 n=140; 5XE:0.83±0.19 n=68 p<0.0001. Values are mean±SD. p-values are calculated by Student’s t-test and represent a comparison to Control. Graphs present individual data points with horizontal bar representing the mean.

Figure 2.. MAPH-9 modulates motor speed and promotes cilia function

(A) Schematic of endogenous maph-9 locus with sgRNA cut sites and BFP (blue fluorescent protein) replacement indicated to make maph-9(0) mutant allele. (B) Top: Localization of endogenous TBB-4::RFP in Control and maph-9(0) mutant. Scale bar, 1 μm. Bottom: Quantification of TBB-4 fluorescence length (μm). Control: 7.15±0.76 n=76. maph-9(0):7.05±0.93 n=67. osm-3(0): 3.82±0.61 n=38. osm-3(0); maph-9(0):3.88±0.42 n=36. kap-1(0):6.73±0.78 n=66. kap-1(0); maph-9(0):5.51±1.34 n=106. *: p<0.0001 (C) Schematic depicting ciliary motors on the axoneme. Colored bars and arrows depict protein localization and direction of movement (anterograde to the right, retrograde to the left) in a C. elegans cilium. (D) Representative kymographs of KAP-1, OSM-3, and XBX-1 in Control, maph-9(0) (‘m9(0)’) mutant, or other indicated mutant backgrounds. Scale bar, 5 μm (x); 1s (y). (E) Quantification of motor velocity (nm/s). KAP-1::GFP [Control: 655.4±169.7 n=614. maph-9(0): 731.9±206.3 n=478; osm-3(0): 397.6±120.9 n=289; osm-3(0); maph-9(0): 440.9±128.7 n=287]; OSM-3::GFP [Control: 781.9±181.7 n=319; maph-9(0): 609.1±186.7 n=217; kap-1(0): 1128.4±216.5 n=765; kap-1(0); maph-9(0): 1128.4±280.4 n=673]; XBX-1::RFP [Control: 850.8±255.8 n=177; maph-9(0): 973.4±282.0 n=203]. *: p<0.0001 (F) Quantification of number of motors/second. KAP-1::GFP [Control: 0.367±0.11 n=27; maph-9(0): 0.588±0.152 n=19; osm-3(0): 0.28±.054 n=25; osm-3(0); maph-9(0): 0.202±0.052 n=21]; OSM-3::GFP [Control: 0.682±0.15 n=16; maph-9(0): 0.459±0.12 n=29; kap-1(0): 0.541±0.108 n=23; kap-1(0); maph-9(0): 0.316±0.088 n=20]; XBX-1::RFP [Control: 1.02±0.0917 n=10; maph-9(0): 0.996±0.157 n=11]. *: p<0.0001 (G) Localization of endogenous GFP::MAPH-9 (green) and RFP::SPD-5, TBB-4::RFP (magenta) in an adult male tail. Right Insets display endogenous GFP::MAPH-9 localization in the cilium of an individual ray. Scale bar, 5 μm. (H) Mating efficiency: Quantification of percent of progeny from mating (cross-progeny) or from self-progeny with wild-type (‘WT’) or maph-9(0) (‘m9(0)’) mutant or Control (‘C’) worms as indicated (Male x Hermaphrodite): Control x wild-type: 65±12.49% n=3; Control x maph-9(0): 33±5.69% n=3; maph-9(0) x wild-type: 16±28.29% n=3; maph-9(0) x maph-9(0): 2±3.46% n=3 Values are mean±SD. p-values are calculated by Student’s t-test. Graphs present individual data points with horizontal bar representing the mean.

Figure 3.. Loss of MAPH-9 causes ultrastructural microtubule doublet defects in the axoneme

(A) Representative electron microscopy (EM) images of amphid middle segment axonemes in wild-type and maph-9(0) mutant worms with ADL neurons indicated in green. Scale bar, 100 nm. (B) Representative EM images of amphid ADL neuron axonemes in indicated genotype. Arrowheads indicate representative singlet (magenta) or incomplete doublet (orange) microtubule in ADL and non-ADL MTD (blue). Scale bar, 100 nm. (C) Quantification of MTD (blue), incomplete doublets (orange), and singlet (magenta) microtubules in ADL axonemes in sections in the middle segment going proximal (left) to distal (right). Sections were aligned (0 position) using the autojunction (AJ) of the amphid socket. Control: 3 ADL neurons, 6 cilia, 2 worms, n = 1408 microtubules; maph-9(0): 3 ADL 6 cilia, 2 worms, n =1431 microtubules. (D) Top: Representative EM images of cross sections through adult phasmid neurons of indicated genotypes. Scale bar, 100 nm. Bottom: Examples of individual phasmid MTDs with genotype and roundness measurement (O) indicated. Scale bar, 10 nm. (E) Roundness measurement (O) of MTDs in wild-type and maph-9(0) mutant phasmid axonemes: A-tubule [wild-type (‘WT’): 0.903±0.03; maph-9(0) (‘m9(0)’): 0.902±0.037 p>0.05]; B-tubule [wild-type: 0.85±0.04; maph-9(0): 0.81±0.07 p<0.001]; wild-type n=3 worms,148 sections; maph-9(0) n=4 worms, 305 sections. (F) Representative phasmid MTD from indicated genotype (top) with protofilaments labeled (bottom). (G) Quantification of protofilament (PF) number: A-tubule [wild-type: 13PF – 100% n=37; maph-9(0): 13PF – 100% n=50]; B-tubule [wild-type: 10PF – 81% 9PF – 19% n=37; maph-9(0): 10PF – 50% 9PF – 36%; 8PF – 14% n =50]. B-tubule average [wild-type: 9.81; maph-9(0): 9.36 p<0.001] Values are mean±SEM. p-values are calculated by Student’s t-test.

Figure 4.. MAP9 localization is conserved in mammalian cells

(A) MAP9 homologs with a consistent main alpha-helix length are present throughout Metazoa. (B) Aligned AlphaFold structural predictions for mouse MAP9 (blue) and C. elegans MAPH-9 (green) Scale bar, 100Å. Root mean square deviation = 9.390. (C) Isotropically expanded hTERT RPE-1 cell stained with antibodies against MAP9 (green), acetylated tubulin (‘Ac. Tub.’, magenta), and polyglutamylated tubulin (‘PolyGlut.’, cyan). Basal bodies (BB, orange) and axoneme (yellow dashed line) indicated in merge. Note that MAP9 localizes to the proximal axoneme and dimly to the basal bodies. Scale bar, 1 μm. (D) Percent of max fluorescence of background subtracted MAP9, polyglutamylated tubulin, and acetylated tubulin binned by percent of the cilium length determined by acetylated tubulin staining in hTERT RPE-1 cells (n=18). Error bars = standard deviation from the mean. (E) MDCK-II cell cultured using the same protocol as for existing MDCK-II MTD measurement stained with indicated antibodies. Axoneme (‘ax.’, yellow dashed line) indicated in merge. Scale bar, 1 μm. (F) Percent of max fluorescence of MAP9, polyglutamylated tubulin, and acetylated tubulin binned by cilium length determined by acetylated tubulin staining in MDCK-II cells (n=87). Error bars = 95% confidence interval. Grey box represents MTD region previously observed by EM with B-tubules variably terminating between ~2–5 μm. Note that the x-axis has been cropped to 20 μm. (G) Indicated mouse tissues fixed and stained with antibodies against MAP9 (green), acetylated tubulin (magenta), and DAPI (blue). Kidney collecting duct: Scale bar, 5 μm. Eye photoreceptor: outer segment (OS) connecting cilium (CC) outer nuclear layer (ONL). Scale bar, 5 μm. Spermatozoon: Scale bar, 10 μm.