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. 2016 Oct 25;17(5):1399-1413.
doi: 10.1016/j.celrep.2016.09.089.

Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina

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Free PMC article

Loss of MACF1 Abolishes Ciliogenesis and Disrupts Apicobasal Polarity Establishment in the Retina

Helen L May-Simera et al. Cell Rep. .
Free PMC article

Abstract

Microtubule actin crosslinking factor 1 (MACF1) plays a role in the coordination of microtubules and actin in multiple cellular processes. Here, we show that MACF1 is also critical for ciliogenesis in multiple cell types. Ablation of Macf1 in the developing retina abolishes ciliogenesis, and basal bodies fail to dock to ciliary vesicles or migrate apically. Photoreceptor polarity is randomized, while inner retinal cells laminate correctly, suggesting that photoreceptor maturation is guided by polarity cues provided by cilia. Deletion of MACF1 in adult photoreceptors causes reversal of basal body docking and loss of outer segments, reflecting a continuous requirement for MACF1 function. MACF1 also interacts with the ciliary proteins MKKS and TALPID3. We propose that a disruption of trafficking across microtubles to actin filaments underlies the ciliogenesis defect in cells lacking MACF1 and that MKKS and TALPID3 are involved in the coordination of microtubule and actin interactions.

Keywords: actin; basal body; centrosome; cilia; ciliogenesis; ciliopathy; microtubule; polarity; retina; retinal degeneration.

Figures

Figure 1
Figure 1. Macf1 is expressed throughout vertebrate retina development
A) RNA sequencing data from developing mouse retina and flow-sorted photoreceptors. FPKM (fragments per kilobase of transcript per million fragments mapped) indicates the level of transcription. Schematic of Macf1a protein domains and localization of antibody epitope. B) Immunoblots of control (Ctrl) and Macf1-excised (cKO;Six3) mouse retinal lysates probed with Macf1 antibody show a reduction in expression in the mutant. C) Immunohistochemistry of retinal sections using antibodies against Macf1 in control and Macf1-excised sections. To visualize relevant structures in each time point, F-actin was stained with phalloidin, the emerging connecting cilium with GT335, and the base of the cilium with pericentrin. D) Immuno-EM using an antibody against Macf1. Immuno-gold particles were concentrated near the basal body in the photoreceptor inner segment (3 months). SB C:50 μm; D:100 nm.
Figure 2
Figure 2. Loss of Macf1 impairs retinal function and disrupts retinal lamination
A) Representative ERG traces from control (Macf1+/+;Six3-Cre/Macf1fl/+;Six3-Cre) and mutant (Macf1fl/fl;Six3-Cre) mice at six weeks of age (n=5–6 mice). Mean a- and b-wave amplitudes at six different light intensities are plotted with errors bars indicating +/− standard error of the mean (SEM). Both rod and cone function were reduced in Macf1 homozygous mutants. B) Haematoxylin and eosin staining of developing retina showed severe retinal dysplasia in Macf1 mutant mice (Macf1fl/fl;Six3-Cre) predominantly affecting the outer retina by P5. OS/IS, outer/inner segments; ONL/INL, outer/inner nuclear layer; OPL/IPL, outer/inner plexiform layer; GCL, ganglion cell layer. SB 50 μm.
Figure 3
Figure 3. Photoreceptors differentiate and inner retinal neuronal morphologies are preserved in Macf1 mutant retina
A) Immunohistochemistry on retina sections (P21) stained with antibodies against rhodopsin, blue opsin, and green opsin.showing normal photoreceptor differentiation in Macf1-null retina (cKO; Six3-Cre) B) Staining for calretinin and calbindin showed that amacrine, horizontal and ganglion cells were relatively unaffected in Macf1-null retina. C) Staining for PKCα to label bipolar cells (PKCα) showed increased disruption with age (P10 vs. P21) and predominantly affected the scleral side of the retina. ONL, outer nuclear layer; INL, inner nuclear layer. SB 25 μm.
Figure 4
Figure 4. Polarity, basal body docking, and cilia extension are disrupted in Macf1 mutant retina
Immunohistochemistry on control (Macf1fl/+;Six3-Cre) and Macf1 mutant (Macf1fl/fl;Six3-Cre) retina sections. A) Ciliary rootlets (rootletin) were aligned along the apical edge of the neuroepithelium in control retina but abnormally distributed in Macf1 mutants. B) Compared to control, Macf1 mutant retina showed discontinuous localization of polarity marker Crb1 and failed alignment of the ciliary connecting cilium (GT335). C) As early as P0, Crb1 and additional polarity markers Pals1 and Par3 were abnormally distributed in Macf1 mutant retina. D) Mislocalization of basal bodies (rootletin) was observed in mutant retina at P5 and E) P0 and the emergence of the transition zone (GT335) was only observed in controls. F) Electron micrographs of basal bodies at the apical edge of the neuroblast layer in P1 retina. Images from control retina show a docked ciliary vesicle (left), initiation of ciliary axoneme extension (middle), and fusion with the apical membrane (right). G) These events were quantified and were less readily observed in Macf1 mutant retina. Lower panel shows examples of basal body profiles found in mutant. ONL, outer nuclear layer; CC, connecting cilia. SB A:50 μm; B–D:25 μm; D:10 μm; F:500 nm
Figure 5
Figure 5. Macf1 is required for photoreceptor homeostasis in mature retina
A) Whole retina cross sections of Macf1fl/+(Ctrl) and Macf1fl/fl(cKO;AAV-Rk-Cre) mice four-six weeks post co-injection of AAV-Rk-Cre and AAV8-RKp-EGFP. Co-transduced regions are visualized by GFP expression. B) Mean ERG a- and b-wave amplitudes (n=4 mice) are plotted with error bars indicating +/− standard error of the mean. C) Confocal and corresponding DIC images show elevated GFAP expression extending into a thinning ONL in the mutant. D) Vibratome sections show rhodopsin mislocalization in the ONL of mutant retina. E, F) TUNEL positive nuclei (red) were readily observed in mutant but not control retinas. G) Electron micrographs of the basal body at the photoreceptor transition zone. H) Quantification of ‘dropped’ basal bodies in control and mutant photoreceptors, measured as the distance between the basal body profile and start of the outer segment (n>10 basal bodies per treatment group). ONL, outer nuclear layer; INL, inner nuclear layer. SB C–E: 50 μm; G 500 nm.
Figure 6
Figure 6. Cilia fail to extend in multiple Macf1-null cell types
A) Scanning electron micrographs (SEM) of exposed lateral ventricle in Macf1fl/+;Foxg1-Cre (Ctrl) and Macf1fl/fl;Foxg1-Cre (cKO;Foxg1) brain show diminished ventricle size (upper panels) in the mutant. Ventricle epithelial cells have a primary cilium extending into the ventricle (lower panels, red arrows), which were scarce and stumpy in the mutant compared to control. B) SEM and C) immunohistochemistry of the cochlea basal turn. One row of inner hair cells (IHC) is separated from three rows of outer hair cells (OHC) in both control and mutant (B, upper panel). Higher magnification images revealed shorter outer hair cells in the mutant (lower panel). The microtubule-based kinocilium at the vertex of each stereocilia bundle was also slightly shorter (inset cartoon; pink= actin bundles, blue= kinocilium). Schematic representation of hair cells (round circles, blue kinocilia) and support cells (gray ovals, gray primary cilia). Hair cell kinocilia were shortened in Macf1 mutants, support cell cilia were unaffected. F) Kinocilium length was quantified using Arl13b and acetylated tubulin stained IHC images. D) Staining of confluent serum-starved MEFs with ciliary markers Arl13b and acetylated tubulin showed failure of ciliary axoneme extension in Macf1-null MEFs. Tubulin was increasingly dispersed in the mutant, and ciliary rootlets (rootletin) were still present in mutant cells. E) In heterozygous cells, the ciliary transition zone (GT335) extended past the basal body, unlike in mutant where it was confined to the basal body (top panel confocal, lower panel super resolution) G) Quantification of ciliation in Macf1 heterozygous and null MEFs cells (n>400 cells counted in four separate experiments). SB A:0.5 mm; B–E: 5 μm.
Figure 7
Figure 7. Loss of Macf1 disrupts microtubule anchoring to subdistal appendages
a) In control MEFs, microtubules (tubulin) extended radially from an anchored centrosome (pericentrin), which was lost in mutant. B) Outer hair cells of control cochlea showed microtubules (acetylated tubulin) emanating from the base of the kinocilium (Arl13b) and extending across the cell. In the mutant, microtubules were disconnected from the kinocilium and abnormally bundled in the cell center. C) Immunohistochemistry of stable microtubules (acetylated tubulin) and the centrosome (pericentrin) in MEFs at 0, 5 and 10 minutes post-nocodazole treatment. Although microtubules began to nucleate from the centrosome in both control and mutant cells, within 10′ microtubules were no longer anchored to the centrosome in mutant cells. D,E) Pericentriolar material (PCM1) was more dispersed around the centrosome in control cells compared to mutant. F) DNAH5 (red) localized to the basal body (green) in control cells, but was missing from the mutant. G) Schematic representation of loss of microtubule anchoring and vesicle trafficking at the basal body resulting in lack of ciliary extension in the mutant. SB A,D: 5 μm; B,C: 2 μm; D,F 10 μm.

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