Overlapping expression patterns and functions of three paralogous P5B ATPases in Caenorhabditis elegans

P5B ATPases are present in the genomes of diverse unicellular and multicellular eukaryotes, indicating that they have an ancient origin, and that they are important for cellular fitness. Inactivation of ATP13A2, one of the four human P5B ATPases, leads to early-onset Parkinson's disease (Kufor-Rakeb Syndrome). The presence of an invariant PPALP motif within the putative substrate interaction pocket of transmembrane segment M4 suggests that all P5B ATPases might have similar transport specificity;however, the identity of the transport substrate(s) remains unknown. Nematodes of the genus Caenorhabditis possess three paralogous P5B ATPase genes, catp-5, catp-6 and catp-7, which probably originated from a single ancestral gene around the time of origin of the Caenorhabditid clade. By using CRISPR/Cas9, we have systematically investigated the expression patterns, subcellular localization and biological functions of each of the P5B ATPases of C. elegans. We find that each gene has a unique expression pattern, and that some tissues express more than one P5B. In some tissues where their expression patterns overlap, different P5Bs are targeted to different subcellular compartments (e.g., early endosomes vs. plasma membrane), whereas in other tissues they localize to the same compartment (plasma membrane). We observed lysosomal co-localization between CATP-6::GFP and LMP-1::RFP in transgenic animals;however, this was an artifact of the tagged LMP-1 protein, since anti-LMP-1 antibody staining of native protein revealed that LMP-1 and CATP-6::GFP occupy different compartments. The nematode P5Bs are at least partially redundant, since we observed synthetic sterility in catp-5(0);catp-6(0) and catp-6(0) catp-7(0) double mutants. The double mutants exhibit defects in distal tip cell migration that resemble those of ina-1 (alpha integrin ortholog) and vab-3 (Pax6 ortholog) mutants, suggesting that the nematode P5Bs are required for ina-1and/or vab-3 function. This is potentially a conserved regulatory interaction, since mammalian ATP13A2, alpha integrin and Pax6 are all required for proper dopaminergic neuron function

The role of the calmodulin-binding and calmodulin-like domains of the epidermal growth factor receptor in tyrosine kinase activation

The epidermal growth factor receptor (EGFR) harbors a calmodulin (CaM)-binding domain (CaM-BD) and a CaM-like domain (CaM-LD) upstream and downstream, respectively, of the tyrosine kinase (TK) domain. We demonstrate in this paper that deletion of the positively charged CaM-BD (EGFR/CaM-BD∆) inactivated the TK activity of the receptor. Moreover, deletion of the negatively charged CaM-LD (EGFR/CaM-LD∆), leaving a single negative residue (glutamate), reduced the activity of the receptor. In contrast, substituting the CaM-LD with a histidine/valine-rich peptide (EGFR/InvCaM-LD) caused full inactivation. We also demonstrated using confocal microscopy and flow cytometry that the chimera EGFR-green fluorescent protein (GFP)/CaM-BD∆, the EGFR/CaM-LD∆, and EGFR/InvCaM-LD mutants all bind tetramethylrhodamine-labelled EGF. These EGFR mutants were localized at the plasma membrane as the wild-type receptor does. However, only the EGFR/CaM-LD∆ and EGFR/InvCaM-LD mutants appear to undergo ligand-dependent internalization, while the EGFR-GFP/CaM-BD∆ mutant seems to be deficient in this regard. The obtained results and in silico modelling studies of the asymmetric structure of the EGFR kinase dimer support a role of a CaM-BD/CaM-LD electrostatic interaction in the allosteric activation of the EGFR TK.Consejería de Educación, Juventud y Deportes–Comunidad de Madrid,Grant/Award Number: B2017/BMD‐36involving contributions from the EuropeanFunds for Regional Development (EFRD) andthe Social European Fund (SEF); ConsejoSuperior de Investigaciones Científicas, Grant/Award Number: COOPA20053;Secretaría de Estado de Investigación, Desarrollo e Innovación, Grant/Award Number: SAF2014‐52048‐R; Agencia Española de Cooperación Internacional para el Desarrollo, Grant/Award Numbers: A/019018/08,A/5444/06, A/8197/0

Estimated growth rate.

<p>The progeny (1 h layoff) from 30 worms for each genotype were scored after 48 h according to their developmental stage. L3 = Larval stage 3. L4 = Larval stage 4. Both presented as percentage. n = Total number of scored worms. Groups that share the same letter (x) do not statistically differ from each other. Groups with different letters are significantly different from each other (x and y). Fisher's exact test. Adj. <i>p</i>-value threshold < 0.05.</p

Phylogenetic analysis of paralogous P5B ATPases.

<p>Maximum Likelihood tree of 9 nematode species (including 6 Caenorhabditis species) and <i>Drosophila melanogaster</i> as an outgroup. Statistical model: LG+G. Bootstrap values for each node are indicated (100 replicates).</p

Overlapping expression patterns in developing reproductive tissues.

<p>(A) Native CATP-6::mKate2 vs. native GFP::CATP-7 in living spermatheca. <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP]) catp-7(dx193[gfp</i>::<i>catp-7 + loxP])</i> IV. (B) Native GFP::CATP-5 vs. native mCherry::CATP-7 in living spermatheca. <i>catp-7(dx191[mcherry</i>::<i>catp-7 + loxP])</i> IV; <i>catp-5(dx187[gfp</i>::<i>catp-5 + loxP])</i> X. Arrowhead: Membrane bound mCherry signal. Arrow: mCherry aggregates. (C) Native CATP-6::mKate2 vs. native GFP::CATP-5 in living spermatheca. <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP])</i> IV; <i>catp-5(dx187[gfp</i>::<i>catp-5 + loxP])</i> X. Arrowhead: Apical luminal membrane. Arrow: Apical lateral borders. (D-G) Native CATP-6::mKate2 vs. native GFP::CATP-7 in living sheath cell (adult) and developing gonad (L2), DTC (late L4) and vulva morphogenesis (early L4). <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP]) catp-7(dx193[gfp</i>::<i>catp-7 + loxP])</i> IV. Arrowheads: (E and F): DTCs. Note: the DTCs extend long processes to cover nearby germ cells, even in early larvae (G): Colocalization of CATP-6::mKate2 and GFP in central somatic gonad (arrow) and developing vulva (arrowhead).</p

<i>C</i>. <i>elegans</i> P5B sequence alignment.

<p>Protein sequence alignment of <i>C</i>. <i>elegans</i> P5B ATPases in comparison with Human ATP13A2. Blue: Putative membrane associated domain Ma [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194451#pone.0194451.ref009" target="_blank">9</a>] and putative transmembrane domains M1–M10 (TMHMM v1.6). Yellow: A (actuator) domain. Red: P (phosphorylation) domain. Green: N (nucleotide binding) domain [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194451#pone.0194451.ref008" target="_blank">8</a>]. Orange: Putative kink in Ma through conserved glycine. Pink: Putative lipid binding site. Purple: P-type ATPase motifs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194451#pone.0194451.ref009" target="_blank">9</a>]. Green triangle: Fluorescent protein insertion site via CRISPR/Cas9. Dashed grey boxes: <i>catp-5(tm4481)</i>, <i>catp-6(ok3473)</i> and <i>catp-7(tm4438)</i> respectively. Dotted grey box: <i>catp-7(dx189)</i> CRISPR/Cas9 mediated deletion.</p

Phenotypic analysis of single, double and triple null mutants.

<p>(A-C) 1 day old adult gonad of: <i>catp-6(0)</i>; <i>catp-5(0)</i> double mutants (<i>catp-6(ok3473)</i> IV; <i>catp-5(tm4481)</i> X), <i>catp-6(0) catp-7(dx189)</i> double mutants (<i>catp-6(ok3473) catp-7(dx189[delta 1492 bp Ma-M3 + gfp + loxP]</i> IV) and <i>catp-6(0) catp-7(dx189)</i>; <i>catp-5(0)</i> triple mutants (<i>catp-6(ok3473) catp-7(dx189[delta 1492 bp Ma-M3 + gfp + loxP]</i> IV; <i>catp-5(tm4481)</i> X). (D) 1 day old adult gonad of wt N2. (E) Quantification of phenotypic segregants. (Ste (Vul)): Sterile Vulvaless; Ste (other): Sterile Evl, Pvl or Muv. Fertile: Able to produce progeny (normal vulva). Asterisk indicates that animals were obtained from a hermaphrodite heterozygous for the mutant chromosome. Fisher's exact test and subsequent Holm’s correction for multiple comparison, adj. <i>p</i>-value: ** ≤ 0.01, *** ≤ 0.001.</p

Schematic overview of P5B ATPase expression in somatic gonad and germ line.

<p>(A) CATP-5, CATP-6 and CATP-7 expression in somatic gonad and germ line in a young adult hermaphrodite. Most of the ovary is contiguously covered by somatic sheath cells; however, there this coverage ceases prior to the distal tip cell. In addition, the proximal sheath cells are perforated by "sheath pores" 100–200 nm in diameter, allowing contact between oocytes that have pinched off the syncytium and the pseudocoelomic fluid [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194451#pone.0194451.ref046" target="_blank">46</a>]. Dashed lines: 2 paralogs expressed in the same subcellular compartment. (B) Schematic of P5B localization in the distal oviduct (pachytene) region of the gonad.</p

Tissue-specific expression patterns of CATP-5, CATP-6 and CATP-7.

<p>Transgene CATP-X::FP indicates expression from an extrachromosomal array. Native CATP-X::FP indicates CRISPR/Cas9 tagged endogenous locus. (A) Representative tissues in which CATP-5 is expressed. <i>Ex [P</i>_{<i>catp-5</i>}<i>catp-5</i>::<i>gfp;rol-6(d)]</i> and <i>catp-5(dx187[gfp</i>::<i>catp-5 + loxP])</i> X. (B) Representative tissues in which CATP-6 is expressed. <i>catp-6(dx183[catp-6</i>::<i>gfp</i>::<i>3xFlag + loxP])</i> IV and <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP])</i> IV. (C) Representative tissues in which CATP-7 is expressed. <i>catp-7(dx185[gfp</i>::<i>catp-7 + loxP])</i> IV. Arrowheads indicate fluorescence signal (B1 and B3 GFP panels contain extensive autofluorescence signal from intestine). (C.3) Arrowhead: Sheath cell with an engulfed an apoptotic germ cell. Arrow: Sheath cell. (C.4) Arrowhead: Spermatocyte. Arrow: Spermatid. Pentagram: Residual body. (C.5) Arrowhead: Hyp10.</p

Overlapping expression in the germ line.

<p>(A, C and E) Native CATP-6::mKate2 vs. native GFP::CATP-7 in germ line during spermatogenesis (L4, A and C) and in spermatheca of young adults (E). <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP]) catp-7(dx193[gfp</i>::<i>catp-7 + loxP])</i> IV. (B) Co-localization of native CATP-6 vs. mCherry PH. <i>catp-6(dx183[catp-6</i>::<i>gfp</i>::<i>3xFlag + loxP])</i> IV; <i>ltIs44(P</i>_<i>pie-1</i><i>mCherry</i>::<i>ph</i>^<i>PLCδ</i><i>)</i> V. (D) Native GFP::CATP-5 in pachytene region of adults. <i>catp-5(dx187[gfp</i>::<i>catp-5 + loxP])</i> X. (F) Native CATP-6::mKate2 localizes to the plasma membrane of diakinesis stage oocytes (arrowhead); cytoplasmic signal is autofluorescence. <i>catp-6(dx179[catp-6</i>::<i>degron</i>::<i>mKate2</i>::<i>3xFlag + loxP])</i>.</p