Mitosis is not dramatically perturbed in <i>Plp</i> mutant SOPs, but centrioles can separate prematurely.

<p>(<b>A-C</b>) Images from videos of living WT (A) or <i>Plp</i> mutant (B,C) SOPs expressing Jupiter-mCherry to reveal the MTs (<i>magenta</i>) and Asl to reveal the centrioles (<i>green</i>). Time in minutes relative to nuclear envelope breakdown (NEB) (t = 0) is indicated. <i>White</i> arrowheads indicate when centriole separation is first detected; <i>yellow</i> arrowheads indicate an extra spindle pole; <i>red</i> arrowheads indicate instances where the centrioles in cells with extra centrioles separate. (<b>D-G</b>) Graphs compare various aspects of the behaviour of WT (<i>blue</i>) and <i>Plp</i> mutant (<i>red</i>) cells, as indicated. (<b>H</b>) Chart shows the division angle relative to the anterior/posterior axis of WT and Plp mutant SOPs. Distributions were assessed by the D'Agostino & Pearson normality test. Significance test for normal distributions was made by an unpaired two-tailed T-Students test and for non-normal distribution by Mann-Whitney ranking test. Bars indicate the mean +/- the SD. All significance tests shown in this and subsequent Figures were performed and presented in this manner, unless specified. Information on numbers analysed and biological repeats for these and all other experiments is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007198#pgen.1007198.s003" target="_blank">S1 Table</a>. In (G) a Wilcoxon signed-rank test was used to compare the median of <i>Plp</i> mutant to the WT value of 4. Scale bar = 2μm (A-C). * p < 0.05, *** p < 0.001.</p

<i>PLP</i> helps to recruit MTs to the interphase centriole in spermatocytes.

<p>(<b>A</b>) Graph quantifies the number of cytoplasmic MTs associated with the interphase centrioles in ETs of WT or <i>Plp</i> mutant spermatocytes. (<b>B-D</b>) Images from ETs of WT (B,C) or <i>Plp</i> mutant (D) spermatocytes: (<i>green</i> arrow, B) shows a MT with a “capped” minus end attached to an electron-dense region on the outer wall of a WT centriole; (C,D) shows traces of all the cytoplasmic MTs (highlighted in <i>green</i>) associated with WT or <i>Plp</i> mutant centrioles. Only one MT is closely associated with the <i>Plp</i> mutant centriole (<i>blue</i> arrow, D). (<b>E</b>) Images show fixed spermatocytes stained with anti-Asl antibodies to reveal the centrioles (<i>red</i>) and anti-PLP antibodies (<i>green</i>). The DNA is stained with DAPI (<i>blue</i>), and the insets illustrate how endogenous PLP is enriched at the proximal ends of the centrioles, while PLP-GFP is distributed more evenly along the entire length of the centriole. <b>(F,G)</b> Graphs quantify the number of MTs associated with the interphase centrioles (F), and the ratio of MTs emanating from the distal versus proximal end (G), in WT and PLP-GFP-expressing spermatocytes. <b>(H)</b> Image from an ET tracing the centriole associated MTs (<i>green</i>) in a spermatocyte expressing PLP-GFP. Scale bar = 100nm (C, D, E) or 10μm (1μm in insets) (E). * p < 0.05, ** p < 0.01.</p

<i>Plp</i> mutants assemble reduced “pericentriolar clouds” around the mother centriole.

<p>(<b>A-C</b>) Images from electron tomograms (ETs) of WT (A), <i>Plp</i> mutant (B) or <i>Plp</i> mutant rescued by PLP-GFP (C) centrioles in wing disc cells. Cytoplasmic MTs contacting the centrioles are highlighted by arrowheads, and electron-dense connections between the mother and engaged daughter are highlighted with arrows. (A’-C’) Images are the same as in (A-C) but with the electron dense pericentriolar clouds highlighted in <i>green</i>, with <i>red lines</i> illustrating how cloud length is measured, and <i>blue lines</i> illustrating how the distance between the cartwheel of the mother centriole and the edge of its engaged daughter is measured. Note that, for unknown reasons, we consistently found it difficult to clearly visualise the centriole MTs in <i>Plp</i> mutant cells that were rescued by PLP-GFP (C,C’), although the pericentriolar clouds were very obvious. (<b>D-G</b>) Graphs compare various aspects of centriole structure and behaviour in WT (<i>blue</i>), <i>Plp</i> mutant (<i>red</i>), and <i>Plp</i> mutant rescued by PLP-GFP (<i>green</i>) cells, as indicated. (<b>H)</b> Electron tomogram image from a <i>Plp</i> mutant wing disc centriole; arrowheads highlight missing outer B-MTs. Scale bars = 100nm; * p < 0.05, ** p < 0.01, *** p < 0.001 **** p < 0.0001.</p

<i>Plp</i> mutant centrioles/basal bodies can recruit TZ proteins.

<p>(<b>A,B</b>) Micrographs show images from fixed WT (A) or <i>Plp</i> mutant (B) spermatocytes stained with anti-Asl antibodies to reveal the centrioles (<i>red</i>), and anti-GFP antibodies (<i>green</i>) to reveal the distribution of GFP-fusions to the TZ proteins MKS1, Cby, Cep290 and CC2D2A (as indicated). Although EM studies show that the vast majority of <i>Plp</i> mutant centrioles are not connected to the PM, all of the centrioles appear to organise TZ proteins in a manner that appears to be very similar to the WT centrioles that are forming a cilium. Note that in some instances, the clustering of the centrioles can make this difficult to visualise, as multiple, prematurely separated, centrioles can be clustered one on top of the other, as appears to be the case in the panel showing the localisation of Cby-GFP in the mutant cells (B). Scale bar = 1μm.</p

Centrioles and cilia exhibit a variety of defects in <i>Plp</i> mutant spermatocytes.

<p>(<b>A</b>) Images from fixed WT or <i>Plp</i> mutant spermatocytes stained with anti-Asl antibodies to reveal the centrioles (<i>red</i>) and DAPI to reveal the nuclei (<i>blue</i>). (<b>B,C</b>) Graphs compare the number of centrioles per cell, or per cell per cyst (a cyst contains the 16 primary spermatocytes derived from the 4 rounds of division of the original spermatocyte gonialblast) in WT or <i>Plp</i> mutant testes. This analysis indicates that centrioles tend to overduplicate (as the number of centrioles per cell per cyst increases) but that centrioles can also mis-segregate, leading to individual cells with either too many or too few centrioles. (<b>D-H</b>) Micrographs show images from electron tomograms (ETs) of WT (D) or <i>Plp</i> mutant (E-H) spermatocytes. Inset and arrows in (D) highlight an electron-dense region that we often observed connecting the centriole/basal body to the plasma membrane at the base of the cilium. Arrows in (F) highlight electron-dense regions that seem to connect the side of this mis-oriented centriole to the plasma membrane (PM). Arrowheads in G and H highlight areas where the centrioles appear to be forming an axoneme-like structure, as the centriole MT triplets transition to axoneme-like doublets; arrows in G and H highlight regions where the clustered centrioles appear to connect to each other. Scale bar = 5μm (A) or 100 nm (E-H). * p < 0.05.</p

Centrioles are not positioned properly relative to the cortex in <i>Plp</i> mutant nota.

<p>(<b>A,B</b>) Images from videos of living WT (A) or <i>Plp</i> mutant (B) SOPs expressing Jupiter-mCherry to reveal the MTs (<i>magenta</i>) and Asl to reveal the centrioles (<i>green</i>). Time in minutes relative to nuclear envelope breakdown (NEB) (t = 0) is indicated. These images are taken from the same videos shown in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007198#pgen.1007198.g001" target="_blank">Fig 1A and 1B</a>, but shown from a side-on view to the spindle. <i>White</i> arrow indicates a centriole pair separating prematurely. (<b>C,D</b>) Graphs compare the behaviour of WT (<i>blue</i>) and <i>Plp</i> mutant (<i>red</i>) cells, as indicated. (<b>E,F</b>) Images from electron tomograms (ETs) of WT (E) or <i>Plp</i> mutant (F) pupal notum cells, highlighting the position of the centrioles (arrows) relative to the cell cortex (dotted <i>green</i> line). (<b>G</b>) Graph quantifies the centriole-to-cortex distance in pupal notum cells. Scale bar = 2μm (A, B) or 100 nm (E, F) * p < 0.05.</p

Basal bodies appear to be specified properly in <i>Plp</i> mutant sensory neurons, but they are mis-positioned.

<p><b>(A-C)</b> Images from videos of living WT or <i>Plp</i> mutant sensory organs, as indicated, expressing Jupiter-mCherry to reveal the MTs (<i>magenta</i>) and Cep-104-GFP to reveal the centrioles and basal bodies (<i>green</i>). Time in hours:minutes after puparium formation (APF) (t = 0) is indicated. <b>(D)</b> Graph charts the position of the brightest Cep-104-GFP containing centriole (that will become the basal body) relative to the cortex in a single WT (<i>blue</i>) or <i>Plp</i> mutant (<i>red</i>) sensory organ, illustrating how the centriole appears to gradually drift away from the cortex. <b>(E)</b> Graph quantifies the distance of the brightest Cep-104-GFP containing centriole from the cortex at 30:00h AFP (when ciliogenesis is normally complete) in WT (<i>blue</i>), <i>Plp</i> mutant (<i>red</i>) or <i>Plp</i> mutant rescued by PLP-GFP (<i>green</i>) nota. (F,G) Images show 3D-reconstructions from SBF-SEM data of the cells in a WT (F) or <i>Plp</i> mutant (G) pupal notum sensory organ. The Sensory Neuron (<i>blue</i>) sends an extension (that would normally contain the axoneme close to its tip) through the cell body of the Bristle Cell (<i>green</i>); the Support Cell (<i>magenta</i>) is illustrated in the images, on the left, but not on the right, which are also rotated by 90^o. The overall organisation of the <i>Plp</i> mutant organ is not detectably perturbed. Scale bar = 2μm (A, B) or 5μm (C) or 500 nm (F,G).</p