Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function

Cnn and PLP directly interact at two defined sites to coordinate the cell cycle–dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability.Pericentriolar material (PCM) mediates the microtubule (MT) nucleation and anchoring activity of centrosomes. A scaffold organized by Centrosomin (Cnn) serves to ensure proper PCM architecture and functional changes in centrosome activity with each cell cycle. Here, we investigate the mechanisms that spatially restrict and temporally coordinate centrosome scaffold formation. Focusing on the mitotic-to-interphase transition in Drosophila melanogaster embryos, we show that the elaboration of the interphase Cnn scaffold defines a major structural rearrangement of the centrosome. We identify an unprecedented role for Pericentrin-like protein (PLP), which localizes to the tips of extended Cnn flares, to maintain robust interphase centrosome activity and promote the formation of interphase MT asters required for normal nuclear spacing, centrosome segregation, and compartmentalization of the syncytial embryo. Our data reveal that Cnn and PLP directly interact at two defined sites to coordinate the cell cycle–dependent rearrangement and scaffolding activity of the centrosome to permit normal centrosome organization, cell division, and embryonic viability

Two Polo-like kinase 4 binding domains in Asterless perform distinct roles in regulating kinase stability

Plk4 (Polo-like kinase 4) and its binding partner Asterless (Asl) are essential, conserved centriole assembly factors that induce centriole amplification when overexpressed. Previous studies found that Asl acts as a scaffolding protein; its N terminus binds Plk4’s tandem Polo box cassette (PB1-PB2) and targets Plk4 to centrioles to initiate centriole duplication. However, how Asl overexpression drives centriole amplification is unknown. In this paper, we investigated the Asl–Plk4 interaction in Drosophila melanogaster cells. Surprisingly, the N-terminal region of Asl is not required for centriole duplication, but a previously unidentified Plk4-binding domain in the C terminus is required. Mechanistic analyses of the different Asl regions revealed that they act uniquely during the cell cycle: the Asl N terminus promotes Plk4 homodimerization and autophosphorylation during interphase, whereas the Asl C terminus stabilizes Plk4 during mitosis. Therefore, Asl affects Plk4 in multiple ways to regulate centriole duplication. Asl not only targets Plk4 to centrioles but also modulates Plk4 stability and activity, explaining the ability of overexpressed Asl to drive centriole amplification

Does spell-checking software need a warning label?

The problems faced by the users in the language-checking software are discussed. There are false negatives, where the language-checking software fails to detect true errors, and false positives, where the software detects problems that are not errors. False negatives are troublesome because they might allow users to overlook problems that could be obvious to the human reader. False positives are also troublesome, although this issue has not been studied extensively in a usage context. The level of trust that users attribute to language-checking software may not always commensurate with the software's ability to do the job without errors

Actin-based Motility during Endocytosis in Budding Yeast

Actin assembly nucleated by Arp2/3 complex has been implicated in the formation and movement of endocytic vesicles. The dendritic nucleation model has been proposed to account for Arp2/3-mediated actin assembly and movement. Here, we explored the model by examining the role of capping protein in vivo, with quantitative tracking analysis of fluorescence markers for different stages of endocytosis in yeast. Capping protein was most important for the initial movement of endocytic vesicles away from the plasma membrane, which presumably corresponds to vesicle scission and release. The next phase of endosome movement away from the plasma membrane was also affected, but less so. The results are consistent with the dendritic nucleation model's prediction of capping protein as important for efficient actin assembly and force production. In contrast, the movement of late-stage endocytic vesicles, traveling through the cytoplasm en route to the vacuole, did not depend on capping protein. The movement of these vesicles was found previously to depend on Lsb6, a WASp interactor, whereas Lsb6 was found here to be dispensable for early endosome movement. Thus, the molecular requirements for Arp2/3-based actin assembly differ in early versus later stages of endocytosis. Finally, acute loss of actin cables led to increased patch motility

Newly Characterized Region of CP190 Associates with Microtubules and Mediates Proper Spindle Morphology in <i>Drosophila</i> Stem Cells

<div><p>CP190 is a large, multi-domain protein, first identified as a centrosome protein with oscillatory localization over the course of the cell cycle. During interphase it has a well-established role within the nucleus as a chromatin insulator. Upon nuclear envelope breakdown, there is a striking redistribution of CP190 to centrosomes and the mitotic spindle, in addition to the population at chromosomes. Here, we investigate CP190 in detail by performing domain analysis in cultured <i>Drosophila</i> S2 cells combined with protein structure determination by X-ray crystallography, <i>in vitro</i> biochemical characterization, and <i>in vivo</i> fixed and live imaging of <i>cp190</i> mutant flies. Our analysis of CP190 identifies a novel N-terminal centrosome and microtubule (MT) targeting region, sufficient for spindle localization. This region consists of a highly conserved BTB domain and a linker region that serves as the MT binding domain. We present the 2.5 Å resolution structure of the CP190 N-terminal 126 amino acids, which adopts a canonical BTB domain fold and exists as a stable dimer in solution. The ability of the linker region to robustly localize to MTs requires BTB domain-mediated dimerization. Deletion of the linker region using CRISPR significantly alters spindle morphology and leads to DNA segregation errors in the developing <i>Drosophila</i> brain neuroblasts. Collectively, we highlight a multivalent MT-binding architecture in CP190, which confers distinct subcellular cytoskeletal localization and function during mitosis.</p></div

CP190-F1 is enriched at the plus ends of growing MTs.

<p><i>A</i>, Images of S2 cells expressing GFP-F1 and TagRFP-Tubulin. Box indicates inset for zoom in C. <i>B</i>, Schematic of the CP190-F1 sub-fragments F1, BTB and Linker domain (F1-L) analyzed for MT association. <i>C</i>, Live-cell imaging of F1 reveals enrichment at the plus end of MTs. Yellow arrowhead indicates a growing MT plus end. <i>D</i>, Graphs indicate percent of cells with MT and Nuclear localization. <i>E</i>, Graphs shown to the far right are line scans along the MTs indicated in frames 0:04 (red dashed line) in C. X-axes are arbitrary fluorescence units (a.u.) and y-axis is distance in microns from the MT +tip end (coordinate x = 0). F1 linescan indicates its enrichment at the MT +ends (grey area on graph). In contrast, the BTB domain and F1-L show very weak localization to some MTs (pink arrowhead, grey box on graph). Scale bars = 5μm. Time = min:s.</p

CP190-F1 binds directly to MTs.

<p>MT co-sedimentation assay with purified components. <i>A</i>, Coomassie stained gel shows CP190-Linker (F1-L), CP190-BTB domain and CP190-F1 fragment incubated without (-) and with (+) MTs followed by high-speed centrifugation. Supernatant (S) and pellet (P) fractions are run separately. F1 co-sediments with MTs (red dotted box), while neither the F1-L linker nor the BTB domain show MT-binding. <i>B</i>, Coomassie stained gel shows the F1-L linker artificially dimerized as a GST fusion (GST-F1-L) and GST alone incubated without (-) and with (+) MTs followed by high-speed centrifugation. Supernatant (S) and pellet (P) fractions are run separately. Although we were not able to fully recapitulate F1 MT binding, dimerized linker (GST-F1-L) is able to weakly associate with MTs (purple dotted box).</p

CP190-L is important for spindle formation in developing brain.

<p><i>Drosophila</i> NBs were fixed and stained as indicated. <i>A</i>. Metaphase NBs are shown with CP190 in red, Asl to mark the centrosome in green, and DAPI in blue. WT control (<i>cp190</i>^<i>ΔL</i><i>/TM6</i>) is in the top row and mutant <i>cp190</i>^<i>ΔL</i><i>/ Df</i>^<i>p11</i> in the bottom row. Boxed regions indicating centrosomes are magnified and shown on the far right panels (numbers next to centrosome indicate which zoomed centrosome is displayed). Yellow arrows point out centrosomes in the merged channel. White dotted line indicates NB outline. <i>B</i>.Fixed anaphase NBs. Labeling is the same as in <i>A</i>. Note a lagging chromosome in <i>cp190</i>^<i>ΔL</i><i>/ Df</i>^<i>p11</i> (yellow arrowhead), which is never seen in WT (frequency indicated in DAPI channel). Scale bar for <i>A</i> and <i>B</i> = 5μm, zoom = 1μm. <i>C</i>, Live imaging of MTs in WT (<i>cp190</i>^<i>ΔL</i><i>/TM6</i>) and <i>cp190</i>^<i>ΔL</i><i>/ Df</i>^<i>p11</i>mutant NBs, note bent spindle (frequency indicated in right most image) and detached centrosome (yellow arrows) in mutant cell. Scale bar = 5μm. Panels in C from top to bottom correspond to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144174#pone.0144174.s007" target="_blank">S1 Movie</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144174#pone.0144174.s008" target="_blank">S2 Movie</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144174#pone.0144174.s009" target="_blank">S3 Movie</a>, respectively.</p