Supplemental Material for Al-Hashimi et al., 2018

<div>Supplemental data for Al-Hashimi <i>et al</i>, <i>Genetics</i>, 2018.</div><div>Manuscript #GENETICS/2018/301078<br></div><div><br></div><div>5 Figures, 2 Tables, 2 Video files.</div><div>Fig. S1. Phenotype of exc-2 alleles.</div><div>Fig. S2. Cloudmapping data output of <i>exc-2</i> whole-genome sequencing.</div><div>Fig. S3. RNAi-knockdown of <i>exc-2</i> isoforms A, B, and D.</div><div>Fig. S4. Location of <i>ifa-4</i> expression.</div><div>Fig. S5. Lethality of canal-specific <i>ifb-1</i> knockdown.</div><div>Table S1. Primer sequences used for RNAi experiments.</div><div>Table S2. Sequence mutations in <i>exc-2</i> and <i>ifa-4</i> alleles.</div><div>File S1. Video of GFP-labelled EXC-2 in <i>ifa-4</i> mutant.</div><div>File S2. Video of GFP-labelled EXC-2 in <i>ifb-1</i> RNAi knockdown animal.<br></div

The <i>nphp-2</i> and <i>arl-13</i> Genetic Modules Interact to Regulate Ciliogenesis and Ciliary Microtubule Patterning in <i>C. elegans</i>

<div><p>Cilia are microtubule-based cellular organelles that mediate signal transduction. Cilia are organized into several structurally and functionally distinct compartments: the basal body, the transition zone (TZ), and the cilia shaft. In vertebrates, the cystoprotein Inversin localizes to a portion of the cilia shaft adjacent to the TZ, a region termed the “Inversin compartment” (InvC). The mechanisms that establish and maintain the InvC are unknown. In the roundworm <i>C. elegans,</i> the cilia shafts of amphid channel and phasmid sensory cilia are subdivided into two regions defined by different microtubule ultrastructure: a proximal doublet-based region adjacent to the TZ, and a distal singlet-based region. It has been suggested that <i>C. elegans</i> cilia also possess an InvC, similarly to mammalian primary cilia. Here we explored the biogenesis, structure, and composition of the <i>C. elegans</i> ciliary doublet region and InvC. We show that the InvC is conserved and distinct from the doublet region. <i>nphp-2</i> (the <i>C. elegans</i> Inversin homolog) and the doublet region genes <i>arl-13</i>, <i>klp-11,</i> and <i>unc-119</i> are redundantly required for ciliogenesis. InvC and doublet region genes can be sorted into two modules—<i>nphp-2</i>+<i>klp-11</i> and <i>arl-13</i>+<i>unc-119—</i>which are both antagonized by the <i>hdac-6</i> deacetylase. The genes of this network modulate the sizes of the NPHP-2 InvC and ARL-13 doublet region. Glutamylation, a tubulin post-translational modification, is not required for ciliary targeting of InvC and doublet region components; rather, glutamylation is modulated by <i>nphp-2</i>, <i>arl-13</i>, and <i>unc-119</i>. The ciliary targeting and restricted localization of NPHP-2, ARL-13, and UNC-119 does not require TZ-, doublet region, and InvC-associated genes. NPHP-2 does require its calcium binding EF hand domain for targeting to the InvC. We conclude that the <i>C. elegans</i> InvC is distinct from the doublet region, and that components in these two regions interact to regulate ciliogenesis via cilia placement, ciliary microtubule ultrastructure, and protein localization.</p></div

NPHP-2 and ARL-13 do not require TZ-, doublet region-, and InvC-associated genes for ciliary targeting.

<p>(A) In wild type (WT), NPHP-2 is restricted to the proximal cilium. In both <i>nphp-4</i> and <i>mks-3</i> mutants NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. Several NPHP-2::GFP puncta were visible in the periciliary compartment. (B) In doublet region and InvC mutants, NPHP-2::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium in <i>klp-11</i>, <i>arl-13</i>, and <i>unc-119</i> mutants. <i>arl-13</i> and <i>klp-11</i> mutants exhibited periciliary NPHP-2::GFP puncta, and <i>unc-119</i> mutants exhibited distal dendritic accumulation of NPHP-2::GFP. (C) In WT, ARL-13::GFP localizes to the proximal cilium. In both <i>nphp-4</i> and <i>mks-3</i> mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. ARL-13::GFP also mislocalized to the periciliary membrane compartment in TZ mutants. (D) In <i>nphp-2</i> and <i>klp-11</i> mutants, ARL-13::GFP was targeted to the cilium and restricted to the post-TZ proximal cilium. In these mutants, ARL-13::GFP also mislocalized to the periciliary membrane compartment, and in <i>unc-119</i> mutants, ARL-13::GFP mislocalized to the distal dendrite. Periciliary puncta – arrowheads, periciliary membrane – white arc, distal dendrite/periciliary accumulation – white bar. Periciliary membrane localization was judged by a visible enrichment of ARL-13::GFP on the edges of the periciliary compartment without a concomitant enrichment in the interior lumen of the periciliary region.</p

The synthetic dye-filling defective phenotype of <i>arl-13; nphp-2</i> mutants is modulated by <i>hdac-6</i>.

<p><i>hdac-6</i> deletion suppresses <i>arl-13; nphp-2</i> double mutant phenotypes. In phasmids, <i>nphp-2</i> and <i>arl-13</i> single mutants are mildly Dyf, which is not suppressed by <i>hdac-6</i> deletion. In both amphids and phasmids, <i>arl-13; nphp-2</i> is severely SynDyf and was suppressed by <i>hdac-6</i>. Data in both panels was analyzed with pairwise Mann-Whitney U-test between all groups, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Groups from either panel sharing a capital letter are not significantly different, whereas groups from either panel with different capital letters do differ significantly.</p

The EF hand is necessary for proper NPHP-2 localization and function.

<p>(A) Diagram of the domains present in Inversin, NPHP-2, and each of the NPHP-2 domain deletion constructs. (B) Localization of GFP-tagged NPHP-2 domain deletion constructs. In the head, full length NPHP-2::GFP localizes to the InvC of amphid channel cilia, to the base of IL2 cilia, and to the base of either OLQ or CEP cilia. In the tail (outlined by dashes), NPHP-2::GFP localizes to the InvC of phasmid cilia. Localization of both NPHP-2-NLS1Δ::GFP and NPHP-2-NLS2Δ::GFP appeared roughly wild-type in all cell types, though both constructs appeared enriched in AWC wing cilia. NPHP-2-EFΔ::GFP is present in IL2 and CEP cilia but fails to localize to amphid channel and phasmid cilia. (C) NPHP-2-NLS1Δ::GFP, NPHP-2-NLS2Δ::GFP, and NPHP-2-EFΔ::GFP can rescue <i>nphp-2 nphp-4</i> amphid and phasmid dye-filling defects, but NPHP-2-EFΔ::GFP rescue is significantly worse than full length NPHP-2::GFP in both amphid and phasmid neurons. Yellow arrowheads - IL2 cilia, red arrowheads - OLQ, blue arrowheads - CEP cilia, white bar - amphid/phasmid bundle. Letters indicate statistically distinct groups. Data was analyzed with pairwise Mann-Whitney U-test against both positive and negative controls, followed by the Holm-Bonferroni multiple comparison adjustment with a total alpha of 0.01. Non-transgene expressing siblings were used as negative controls in rescue experiments.</p

Elegance screenshot.

<p>Cell profiles are marked by open blue squares. Different chains of connected objects are labeled with a colored number for ease of recognition. The pink 215 neuron branches between the central and left-hand images. Synapses are marked by red circles.</p

<i>nphp-2</i>, <i>arl-13</i>, and <i>hdac-6</i> regulate glutamylation in head and tail cilia.

<p>(A) Worms stained with GT335 anti-glutamylated tubulin antibody. In WT background, GT335 labels amphid and phasmid doublet region microtubules, and CEP doublet region and singlet region microtubules consistently, and OLQ cilia inconsistently. CEP distal microtubule singlets were also labelled by GT335. Inner labial cilia were also glutamylated, but specific IL1 and IL2 identification was not possible. <i>nphp-2</i> mutants showed characteristic posterior shifted cilia, and glutamylation in the head looked similar to WT, with infrequent weak inner labial cilia staining. Phasmids exhibited an elongated glutamylation signal. <i>arl-13</i> mutants exhibited elongated staining of amphid bundle, CEP, and OLQ cilia. Amphid staining in <i>unc-119</i> mutants was extremely shortened and cilia were angled inwards. <i>unc-119</i> mutants exhibited almost no staining of CEP, IL, and OLQ cilia. <i>hdac-6</i> mutants displayed shorter amphid bundle staining, and reduced IL and OLQ staining. (B) In <i>ttll-4</i> mutants, NPHP-2::GFP and GFP::UNC-119 localize similarly to WT. (C) In <i>ccpp-1</i> mutants, both NPHP-2::GFP and GFP::UNC-119 localize similarly to in WT, and GFP::UNC-119 is present in the TZ and accumulates at the distal dendrite. Yellow arrowheads – IL2 cilia, red arrowheads - OLQ cilia, blue arrowheads – CEP cilia, white bar – amphid/phasmid bundle.</p

UNC-119 localizes to the proximal cilium in phasmids and does not require DR and InvC genes to target the cilium.

<p>(A) N-terminal GFP tagged UNC-119 localizes to dendrites and cilia of amphid channel and phasmid neurons. The TZ gap (arrow) can be discerned in the cilia of amphid channel neurons. In cilia of the CEP and OLQ neurons, GFP::UNC-119 fluorescence was faint and infrequently visible. In phasmid cilia, GFP::UNC-119 localizes to the doublet region, and is excluded from the TZ (arrow) and singlet region. (B) GFP::UNC-119 localization in doublet region- and InvC-associated mutants. GFP::UNC-119 localizes to the proximal cilium in all mutant backgrounds examined.</p

Model of the composition of the proximal cilium.

<p>ARL-13 is depicted as membrane associated based on published characterizations <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen.1004866-Cevik2" target="_blank">[37]</a>. NPHP-2 is depicted as membrane associated based on the membrane association of Inversin in mammalian primary cilia, and because in <i>C. elegans</i> NPHP-2 reporters appear membrane associated by casual observation <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen.1004866-Shiba1" target="_blank">[12]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen.1004866-WarburtonPitt1" target="_blank">[17]</a>. Kinesin-II is microtubule associated, and UNC-119 is depicted nonspecifically because of the diffuse localization of GFP::UNC-119. Poly-glutamylated tubulin is depicted as a modification of the B-tubule, as reported <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen.1004866-Lechtreck1" target="_blank">[45]</a>.</p

<i>klp-11</i> and <i>unc-119</i> genetically interact with <i>arl-13</i> and <i>nphp-2</i> in an <i>hdac-6</i> dependent manner.

<p>(A) <i>klp-11</i> single mutants are not Dyf. <i>klp-11</i> is SynDyf with <i>arl-13</i>, which is partially suppressed by deletion of <i>hdac-6</i>. (B) <i>unc-119</i> single mutants are moderately Dyf. <i>unc-119</i> is SynDyf with both <i>klp-11</i> and <i>nphp-2</i>. <i>unc-119; hdac-6; nphp-2</i> triple mutants exhibit suppression of the <i>unc-119</i> Dyf phenotype. (C) Diagram of interactions between <i>klp-11</i>, <i>arl-13</i>, <i>nphp-2</i>, <i>unc-119</i>, and <i>hdac-6</i>, and between DR and TZ genes, based on SynDyf phenotypes presented in panels A and B and in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen-1004866-g001" target="_blank">Fig. 1</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen.1004866.s005" target="_blank">S5 Figure</a>. The T-bar indicates <i>hdac-6</i> mediated suppression of SynDyf phenotypes. Data was analyzed with pairwise Mann-Whitney U-test between wild type, double mutants, and their respective single mutants, followed by the Holm-Bonferroni multiple comparison adjustment. *, significant versus single mutants at a total alpha of 0.05. **, significant versus single mutants at a total alpha of 0.01. <i>nphp-2</i>, <i>arl-13</i>, <i>hdac-6</i>, and <i>arl-13; hdac-6</i> Dyf data is presented in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004866#pgen-1004866-g001" target="_blank">Fig. 1</a>.</p