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

The BTB domain exists as a dimer and is critical for F1 MT binding.

<p><i>A</i>, Space-filling model showing homodimeric mates in the crystal lattice with one molecule colored purple, the other grey. The β1 strands wrap along the length of the opposing molecule. <i>B</i>, Zoom of the dimer interface shows the hydrophobic leucine (L20) residue that was mutated to a charged glutamic acid residue (L20E). This mutation destabilizes the BTB domain at the dimer interface. <i>C</i>, CP190’s BTB domain exists as a stable dimer in solution as shown by SEC-MALS. The x-axis is the time of the run in minutes, the left y-axis is the MW (black: kDa), and the right y-axis is the Raleigh ratio (grey). The predicted CP190 BTB domain monomer and dimer molecular weights are indicated by dotted lines (16 and 32 kDa respectively). The experimentally determined mass of the eluted CP190 BTB domain is plotted as a black line and corresponds to a homodimer.</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

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-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

Interphase cells serve as an excellent model to study CP190 MT association.

<p><i>A</i>, <i>Drosophila</i> S2 cells expressing GFP-CP190 constructs and TagRFP-Tubulin. Box in GFP channel is zoomed to highlight GFP localization to MTs (right column). F1 localizes to centrosomes and is unique in its localization to MTs during interphase, similar to what we document in mitosis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144174#pone.0144174.g001" target="_blank">Fig 1<i>B</i></a>). <i>B</i>, Quantification of the percent of cells in which CP190 constructs co-localize with MTs or show nuclear (Nuc) localization. FL and F2 are localized to the nucleus during interphase. F3 is cytoplasmic during interphase. F1 localizes to MTs robustly during interphase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0144174#pone.0144174.s001" target="_blank">S1 Fig</a>). Scale bars = 5 μm.</p

The CP190 BTB domain is highly conserved across species.

<p>CP190 F1 region sequence alignment across six species shows a high level of conservation within the CP190 BTB domain. Residue numbers correspond to <i>Drosophila melanogaster</i> CP190. Conservation is mapped on the alignment. Residues with 100% identity are mapped in green and residues with 100% similarity are mapped in yellow. Below the CP190 sequences (last row) are displayed residues that are highly conserved among other <i>Drosophila melanogaster</i> BTB domain containing proteins (not CP190) and are likely involved in the BTB domain fold. Residues from this set that are divergent in CP190 are indicated above the <i>Drosophila</i> CP190 sequence by a red rectangle. This alignment suggests that there are residues important for the BTB domain structural fold and others that are specifically unique to CP190. Secondary structure is mapped on the alignment. Arrows indicate β-sheets and rectangles indicate α-helices. Red letters in the linker region highlight basic (R and K) residues. The SGLP motif is underlined. Solvent accessible surface area (SASA) of a theoretical monomer as well as the buried surface area (BSA) of the homodimer is plotted above the alignment. <i>B</i>, The BTB domain adopts a dimeric fold and makes extensive structural contacts with its dimeric mate. One BTB domain is shown in color with secondary structure elements colored as in Figure 5<i>A</i>. The dimeric mate is colored grey. The β1 and β6’ strands from dimeric mates form an antiparallel, two-stranded β-sheet. <i>C</i>, The BTB domain shown in surface representation, rotated 90° about the x-axis relative to the orientation shown in B. Conservation is colored on the structure (above) following the scheme in A. Electrostatics are indicated on the structure below.</p

2D ¹⁵N-HSQC spectra of monomeric CM2 show a calcium sensitive interaction of calmodulin.

<p>(A) 2D ¹⁵N-HSQC spectra of monomeric CM2 alone (gray) overlaid with the spectra after the addition of 18μM CaM with 2mM CaCl₂ (pink) shows significant shifts. While 18μM CaM in the presence of 2mM EGTA (purple) shows more minimal shifts. Inset: Shifts on leucine 1131 and leucine 1137 are apparent. (B) Quantitation of shifts from a CaM titration (2mM CaCl₂) where several shifts are greater than 0.01ppm (dashed line) on addition of 5.3μM CaM. Residues that align with the predicted CaM binding site in mammalian CDK5RAP2 are shown in red. The star indicated residue N1135 that is not visible in the HSQC spectra while the gray shading indicates residues that are not modeled in the 5MWE crystal structure. (C) Mapping of chemical shift changes (blue-red) onto the 5MWE crystal structure (CNNLZ is shown in gray) reveals shifts along the length of the helices including the region where dimer contacts are made.</p

2D ¹⁵N-HSQC spectra of assigned monomeric CM2 shows interaction along three patches of residues.

<p>(A) Overlays of 2D ¹⁵N-HSQC spectra and assignments of CM2 monomer (gray) and dimer (pink) showing the peaks corresponding to residues F1105-L1148 have identical chemical shifts. All backbone monomer peaks are assigned. Though N1135 does appear in backbone tracing experiments, it does not appear in HSQC spectra. (B) Mapping of the combined chemical shift differences (blue to red) between the monomer and dimer HSQC spectra onto the crystal structure of CM2 (pdb:5MWE). Peaks that were not present in the dimer HSQC spectra were arbitrarily colored as a combined chemical shift difference of 0.1ppm for visualization purposes. (C) Overlay of monomer CM2 (gray) with 200μM PLPMD (pink) and 150μM of CNNLZ (purple) illustrates chemical shift changes, particularly for L1131 and L1137 (inset). (D) Quantitation of the combined chemical shift changes of CNNLZ (purple) compared to monomeric CM2 alone reveals three regions with greatest shift changes. Dashed line indicates a combined chemical shift change of 0.0025ppm. The star indicates residue N1135 that is not visible in the HSQC spectra. Gray shading indicates the residues that are not modeled in the crystal structure (pdb:5MWE). (E) Quantitation of chemical shift changes of PLPMD (pink) compared to monomeric CM2. (F) Combined chemical shift changes of CM2 in the presence of CNNLZ mapped onto the crystal structure of CM2 with CNNLZ (pdb:5MWE). CM2 is colored blue to red in proportion to chemical shift changes. CNNLZ is shown in gray. Inset shows the interface between CM2 and CNNLZ and the proximity of I1130 to L539 and L1137 to L532.</p

Conserved domain, CM2, of centrosomin characterized by SEC-MALS, SIM, and yeast-two-hybrid experiments.

<p>(A) Schematic of Centrosomin with coiled-coil prediction shown in blue. CM1 and CM2 are highlighted in red. Alignment of <i>D</i>. <i>Melanogaster’s</i> CM2 with the zebra fish, mouse and human (CDK5RAP2) homologues, with residue numbering according to <i>D</i>. <i>Melanogaster</i> residues. Higher conservation can be seen starting at residue E1090 in line with the start of the predicted coiled coil region. The predicted calmodulin binding site in human CDK5RAP2 is shown in red. (B) Molecular weights (left axis) measured with SEC-MALS are consistent with aa1064-1148 forming a dimer of CM2 (red) with a predicted monomer weight of 11kDa and a calculated weight of 22.4kDa. The construct aa1090-1148 exists as monomer (blue) with a predicted monomer weight of 7kDa and a calculated weight of 7.7kDa. (C) SEC-MALS of a more minimal dimer construct aa1074-1148 (blue) that has a calculated molecular weight of 19.4kDa and predicted monomer weight of 9kDa. A construct with the final 18 residues truncated (Δ1130–1148) maintains a dimeric oligomer state (red) with a calculated molecular weight of 21.6kDa and predicted monomer weight of 8.8kDA. (D) Z-projections of the aligned and averaged PLP and GFP distributions for both the monomer and dimer GFP-CM2 fusion constructs in interphase S2 cells. Scale bar represents 100nm. (E) Z-projections of the monomer and dimer GFP-CM2 fusion constructs in mitotic S2 cells. Scale bar represents 100nm (F) Radial averages of the data represented in figures D and E with vertical bars indicating the average and horizontal bars representing the standard deviation of the Gaussian fit. (G) Schematic summaries of yeast-two-hybrid experiments showing the CM2 construct (aa1087-1148) interacting with a minimal domain of PLP(aa583-740) and the middle domain of CNN (aa454-556). Yeast plates of the interaction test are shown with red boxes indicating plating on selection media.</p