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

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles.

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix

The centrosomin CM2 domain is a multi-functional binding domain with distinct cell cycle roles.

The centrosome serves as the main microtubule-organizing center in metazoan cells, yet despite its functional importance, little is known mechanistically about the structure and organizational principles that dictate protein organization in the centrosome. In particular, the protein-protein interactions that allow for the massive structural transition between the tightly organized interphase centrosome and the highly expanded matrix-like arrangement of the mitotic centrosome have been largely uncharacterized. Among the proteins that undergo a major transition is the Drosophila melanogaster protein centrosomin that contains a conserved carboxyl terminus motif, CM2. Recent crystal structures have shown this motif to be dimeric and capable of forming an intramolecular interaction with a central region of centrosomin. Here we use a combination of in-cell microscopy and in vitro oligomer assessment to show that dimerization is not necessary for CM2 recruitment to the centrosome and that CM2 alone undergoes significant cell cycle dependent rearrangement. We use NMR binding assays to confirm this intramolecular interaction and show that residues involved in solution are consistent with the published crystal structure and identify L1137 as critical for binding. Additionally, we show for the first time an in vitro interaction of CM2 with the Drosophila pericentrin-like-protein that exploits the same set of residues as the intramolecular interaction. Furthermore, NMR experiments reveal a calcium sensitive interaction between CM2 and calmodulin. Although unexpected because of sequence divergence, this suggests that centrosomin-mediated assemblies, like the mammalian pericentrin, may be calcium regulated. From these results, we suggest an expanded model where during interphase CM2 interacts with pericentrin-like-protein to form a layer of centrosomin around the centriole wall and that at the onset of mitosis this population acts as a nucleation site of intramolecular centrosomin interactions that support the expansion into the metaphase matrix

A molecular mechanism for the procentriole recruitment of Ana2

During centriole duplication, a preprocentriole forms at a single site on the mother centriole through a process that includes the hierarchical recruitment of a conserved set of proteins, including the Polo-like kinase 4 (Plk4), Ana2/STIL, and the cartwheel protein Sas6. Ana2/STIL is critical for procentriole assembly, and its recruitment is controlled by the kinase activity of Plk4, but how this works remains poorly understood. A structural motif called the G-box in the centriole outer wall protein Sas4 interacts with a short region in the N terminus of Ana2/STIL. Here, we show that binding of Ana2 to the Sas4 G-box enables hyperphosphorylation of the Ana2 N terminus by Plk4. Hyperphosphorylation increases the affinity of the Ana2-G-box interaction, and, consequently, promotes the accumulation of Ana2 at the procentriole to induce daughter centriole formation.Open access articleThis item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

A centrosome interactome provides insight into organelle assembly and reveals a non-duplication role for Plk4

The centrosome is the major microtubule-organizing centre of many cells, best known for its role in mitotic spindle organization. How the proteins of the centrosome are accurately assembled to carry out its many functions remains poorly understood. The non-membranebound nature of the centrosome dictates that protein-protein interactions drive its assembly and functions. To investigate this massive macromolecular organelle, we generated a `domain-level' centrosome interactome using direct protein-protein interaction data from a focused yeast two-hybrid screen. We then used biochemistry, cell biology and the model organism Drosophila to provide insight into the protein organization and kinase regulatory machinery required for centrosome assembly. Finally, we identified a novel role for Plk4, the master regulator of centriole duplication. We show that Plk4 phosphorylates Cep135 to properly position the essential centriole component Asterless. This interaction landscape affords a critical framework for research of normal and aberrant centrosomes.Division of Intramural Research at the NIH/NHLBI [1ZIAHL006104]; NIH/NIGMS [R01GM110166, R01GM094415]; NSF [MCB1158151]This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]

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

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

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