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

Identification of an Actin Binding Surface on Vinculin that Mediates Mechanical Cell and Focal Adhesion Properties

Vinculin, a cytoskeletal scaffold protein essential for embryogenesis and cardiovascular function, localizes to focal adhesions and adherens junctions, connecting cell surface receptors to the actin cytoskeleton. While vinculin interacts with many adhesion proteins, its interaction with filamentous actin regulates cell morphology, motility, and mechanotransduction. Disruption of this interaction lowers cell traction forces and enhances actin flow rates. Although a model for the vinculin:actin complex exists, we recently identified actin-binding deficient mutants of vinculin outside sites predicted to bind actin, and developed an alternative model to better define this novel actin-binding surface, using negative-stain EM, discrete molecular dynamics, and mutagenesis. Actin-binding deficient vinculin variants expressed in vinculin knockout fibroblasts fail to rescue cell-spreading defects and reduce cellular response to external force. These findings highlight the importance of this new actin-binding surface and provide the molecular basis for elucidating additional roles of this interaction, including actin-induced conformational changes which promote actin bundling

Vinculin–actin interaction couples actin retrograde flow to focal adhesions, but is dispensable for focal adhesion growth

In migrating cells, integrin-based focal adhesions (FAs) assemble in protruding lamellipodia in association with rapid filamentous actin (F-actin) assembly and retrograde flow. How dynamic F-actin is coupled to FA is not known. We analyzed the role of vinculin in integrating F-actin and FA dynamics by vinculin gene disruption in primary fibroblasts. Vinculin slowed F-actin flow in maturing FA to establish a lamellipodium–lamellum border and generate high extracellular matrix (ECM) traction forces. In addition, vinculin promoted nascent FA formation and turnover in lamellipodia and inhibited the frequency and rate of FA maturation. Characterization of a vinculin point mutant that specifically disrupts F-actin binding showed that vinculin–F-actin interaction is critical for these functions. However, FA growth rate correlated with F-actin flow speed independently of vinculin. Thus, vinculin functions as a molecular clutch, organizing leading edge F-actin, generating ECM traction, and promoting FA formation and turnover, but vinculin is dispensible for FA growth

In Vivo Characterization of Interactions Among Dynein Complex Components at Microtubule Plus Ends

Dynein is a minus end directed molecular motor required for numerous cellular processes during intracellular transport and mitosis. Pac1/LIS1 and Bik1/CLIP-170 are two proteins required for targeting dynein to cytoplasmic microtubule plus ends in budding yeast. The lab previously proposed a model whereby Pac1/LIS1 binds to the motor domain of dynein heavy chain, Dyn1/HC, forming a complex that interacts with the +TIP protein Bik1/CLIP170 at plus ends. This project focused on using Bimolecular Fluorescence Complementation (BiFC) to visualize protein-protein interactions among dynein pathway components in vivo. Budding yeast, Saccharomyces cerevisiae is an ideal system to manipulate dynein as it is a non-essential protein in this system. The BiFC assay fuses two non-fluorescent halves of Venus, a YFP-derivative, to proteins of interest. If an interaction between the proteins occur, the two halves are brought to close proximity and the fluorophore is reconstituted. Cells co-expressing Dyn1-VN with Pac1-VC or Bik1-VC exhibited fluorescent foci associated with microtubule plus ends, the cell cortex and spindle pole bodies (SPBs). Additionally, cells co-expressing Pac1-VC with Bik1-VN exhibited fluorescent foci associated with microtubule plus ends. Cells coexpressing Tub1-VC and Bik1-VN or Dyn1-VN have BiFC signal indicating that both interact with the microtubule directly. Pac-1 coexpressed with Tub1 had no signal above background. These data support that these three components associate at microtubule plus ends. Dyn1 and Pac1 interact with Bik1 at microtubule plus ends. Bik1 serves as a docking platform for the two, but dynein is still able to interact with microtubules, while Pac1 is not

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

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

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

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 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