Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly
2022; Springer Nature; Volume: 41; Issue: 11 Linguagem: Inglês
10.15252/embj.2022110891
ISSN1460-2075
AutoresSiu‐Shing Wong, Zachary M. Wilmott, Saroj Saurya, Ines Alvarez‐Rodrigo, Felix Zhou, Kwai‐Yin Chau, Alain Goriely, Jordan W. Raff,
Tópico(s)Mitochondrial Function and Pathology
ResumoArticle3 May 2022Open Access Transparent process Centrioles generate a local pulse of Polo/PLK1 activity to initiate mitotic centrosome assembly Siu-Shing Wong Siu-Shing Wong orcid.org/0000-0002-5327-8466 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Zachary M Wilmott Zachary M Wilmott orcid.org/0000-0001-6053-9874 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Mathematical Institute, University of Oxford, Oxford, UK Contribution: Conceptualization, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Saroj Saurya Saroj Saurya orcid.org/0000-0003-4057-0123 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Resources, Methodology Search for more papers by this author Ines Alvarez-Rodrigo Ines Alvarez-Rodrigo orcid.org/0000-0003-2181-5535 The Francis Crick Institute, London, UK Contribution: Conceptualization, Resources Search for more papers by this author Felix Y Zhou Felix Y Zhou orcid.org/0000-0003-4463-1165 Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Contribution: Software Search for more papers by this author Kwai-Yin Chau Kwai-Yin Chau orcid.org/0000-0001-9673-3783 Department of Computer Science, University of Bath, Bath, UK Contribution: Software Search for more papers by this author Alain Goriely Corresponding Author Alain Goriely [email protected] orcid.org/0000-0002-6436-8483 Mathematical Institute, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jordan W Raff Corresponding Author Jordan W Raff [email protected] orcid.org/0000-0002-4689-1297 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Siu-Shing Wong Siu-Shing Wong orcid.org/0000-0002-5327-8466 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Conceptualization, Data curation, Software, Formal analysis, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Zachary M Wilmott Zachary M Wilmott orcid.org/0000-0001-6053-9874 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Mathematical Institute, University of Oxford, Oxford, UK Contribution: Conceptualization, Formal analysis, Validation, Investigation, Visualization, Writing - original draft, Writing - review & editing Search for more papers by this author Saroj Saurya Saroj Saurya orcid.org/0000-0003-4057-0123 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Resources, Methodology Search for more papers by this author Ines Alvarez-Rodrigo Ines Alvarez-Rodrigo orcid.org/0000-0003-2181-5535 The Francis Crick Institute, London, UK Contribution: Conceptualization, Resources Search for more papers by this author Felix Y Zhou Felix Y Zhou orcid.org/0000-0003-4463-1165 Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK Contribution: Software Search for more papers by this author Kwai-Yin Chau Kwai-Yin Chau orcid.org/0000-0001-9673-3783 Department of Computer Science, University of Bath, Bath, UK Contribution: Software Search for more papers by this author Alain Goriely Corresponding Author Alain Goriely [email protected] orcid.org/0000-0002-6436-8483 Mathematical Institute, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Investigation, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Jordan W Raff Corresponding Author Jordan W Raff [email protected] orcid.org/0000-0002-4689-1297 Sir William Dunn School of Pathology, University of Oxford, Oxford, UK Contribution: Conceptualization, Supervision, Funding acquisition, Writing - original draft, Project administration, Writing - review & editing Search for more papers by this author Author Information Siu-Shing Wong1,†, Zachary M Wilmott1,2,†, Saroj Saurya1, Ines Alvarez-Rodrigo3, Felix Y Zhou4,6, Kwai-Yin Chau5, Alain Goriely *,2 and Jordan W Raff *,1 1Sir William Dunn School of Pathology, University of Oxford, Oxford, UK 2Mathematical Institute, University of Oxford, Oxford, UK 3The Francis Crick Institute, London, UK 4Ludwig Institute for Cancer Research, Nuffield Department of Clinical Medicine, University of Oxford, Oxford, UK 5Department of Computer Science, University of Bath, Bath, UK 6Present address: Lyda Hill Department of Bioinformatics, University of Texas Southwestern Medical Center, Dallas, TX, USA † These authors contributed equally to this work *Corresponding author. Tel: +44-1865275533; E-mail: [email protected] *Corresponding author. Tel: +44-1865615169; E-mail: [email protected] The EMBO Journal (2022)41:e110891https://doi.org/10.15252/embj.2022110891 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mitotic centrosomes are formed when centrioles start to recruit large amounts of pericentriolar material (PCM) around themselves in preparation for mitosis. This centrosome "maturation" requires the centrioles and also Polo/PLK1 protein kinase. The PCM comprises several hundred proteins and, in Drosophila, Polo cooperates with the conserved centrosome proteins Spd-2/CEP192 and Cnn/CDK5RAP2 to assemble a PCM scaffold around the mother centriole that then recruits other PCM client proteins. We show here that in Drosophila syncytial blastoderm embryos, centrosomal Polo levels rise and fall during the assembly process—peaking, and then starting to decline, even as levels of the PCM scaffold continue to rise and plateau. Experiments and mathematical modelling indicate that a centriolar pulse of Polo activity, potentially generated by the interaction between Polo and its centriole receptor Ana1 (CEP295 in humans), could explain these unexpected scaffold assembly dynamics. We propose that centrioles generate a local pulse of Polo activity prior to mitotic entry to initiate centrosome maturation, explaining why centrioles and Polo/PLK1 are normally essential for this process. Synopsis As cells prepare to enter mitosis, the centrioles recruit pericentriolar material (PCM) to form mitotic centrosomes. Here, experiments and modelling show centrioles to generate a pulse of Polo activity prior to mitosis, to stimulate the assembly of a Spd-2/Polo/Cnn scaffold that can recruit other PCM components. As Drosophila embryos prepare to enter mitosis, Ana1 is activated at centrioles, allowing it to recruit and activate Polo prior to the entry into mitosis. The Polo recruited by Ana1 phosphorylates Spd-2, allowing Spd-2 to form a scaffold that fluxes outwards and recruits and activates more Polo, and also Cnn. Polo phosphorylates Cnn, allowing it to assemble into a stronger scaffold that helps to support the weaker Spd-2 scaffold. Polo also inactivates Ana1, so centrosomal Polo and Spd-2 levels decline as embryos enter mitosis; the Cnn scaffold is maintained as Polo is globally activated in the mitotic cytoplasm. Introduction Centrosomes are important organisers of the cell that are formed when mother centrioles recruit a matrix of pericentriolar material (PCM) around themselves (Conduit et al, 2015; Bornens, 2021; Lee et al, 2021; Vasquez-Limeta & Loncarek, 2021; Woodruff, 2021). The PCM contains several hundred proteins (Alves-Cruzeiro et al, 2013), including many that help nucleate and organise microtubules (MTs), as well as many signalling molecules, cell cycle regulators and checkpoint proteins. In this way, the centrosomes function as major MT organising centres (MTOC) and also important coordination centres in many cell types (Arquint et al, 2014; Chavali et al, 2014). In interphase, most cells organise relatively little PCM, but there is a dramatic increase in PCM recruitment as cells prepare to enter mitosis—a process termed centrosome maturation (Palazzo et al, 2000; Conduit et al, 2015). Centrioles are required to initiate efficient mitotic PCM assembly (Bobinnec et al, 1998; Kirkham et al, 2003; Basto et al, 2006; Sir et al, 2013; Bazzi & Anderson, 2014; Wong et al, 2015), and, in worm embryos, centrioles are continuously required to promote the growth of the mitotic PCM—although they are not required to maintain the mitotic PCM once it has reached its full size (Cabral et al, 2019). The protein kinase Polo/PLK1 is also required for the assembly of the mitotic PCM in most, if not all, systems (Sunkel & Glover, 1988; Lane & Nigg, 1996; Dobbelaere et al, 2008; Haren et al, 2009; Lee & Rhee, 2011; Conduit et al, 2014a; Woodruff et al, 2015b; Ohta et al, 2021). PLK1 performs many functions during mitosis (Archambault & Glover, 2009; Colicino & Hehnly, 2018), and it is recruited to different locations within the cell via its Polo-Box-Domain (PBD), which binds to phosphorylated S-S(P)/T(P) motifs on various scaffolding proteins (Song et al, 2000; Seong et al, 2002; Elia et al, 2003; Reynolds & Ohkura, 2003). Importantly, PBD binding to these scaffolding proteins helps to activate PLK1 by relieving an inhibitory interaction between the PBD and the kinase domain (Xu et al, 2013), although full activation also requires phosphorylation (Archambault & Glover, 2009; Colicino & Hehnly, 2018). PLK1 is recruited to centrosomes by the scaffolding protein CEP192 in vertebrates (Joukov et al, 2010, 2014; Meng et al, 2015), and by the CEP192 homologues Spd-2/SPD-2 in flies and worms (Decker et al, 2011; Alvarez-Rodrigo et al, 2019; Ohta et al, 2021). In these species, the Polo/PLK-1 recruited by Spd-2/SPD-2 can then phosphorylate Cnn/SPD-5 (flies/worms), which allows these large helical proteins to assemble into macromolecular PCM-scaffolds that help recruit the many other PCM "client" proteins (Conduit et al, 2014a; Woodruff et al, 2015a, 2017; Feng et al, 2017; Cabral et al, 2019; Ohta et al, 2021). Here, we focus on the kinetics of mitotic PCM scaffold assembly in living Drosophila syncytial blastoderm embryos—where we can simultaneously track the behaviour of tens to hundreds of centrosomes as they rapidly and near-synchronously assemble over several nuclear division cycles that occur in a common cytoplasm. Surprisingly, we observe that the centrosomal levels of Polo rise and fall during the assembly process, with centrosomal levels peaking, and then starting to decline, even as the Cnn scaffold continues to grow. Mathematical modelling and further experiments indicate that an interaction between Polo and its centriole receptor Ana1 (CEP295 in vertebrates) could generate a local pulse of centriolar Polo activity, and that such a mechanism could explain the unexpected assembly kinetics of the PCM scaffold. We propose that centrioles generate a local pulse of Polo activity that initiates mitotic centrosome assembly in syncytial fly embryos prior to mitotic entry. We speculate that the ability of centrioles to locally activate Polo/PLK1 prior to mitosis may be a conserved feature of mitotic centrosome assembly—explaining why centrioles and Polo/PLK1 are both normally required to initiate this process. Results PCM-scaffold proteins exhibit distinct assembly dynamics To better understand how Spd-2, Polo and Cnn cooperate to assemble the PCM scaffold, we quantified their recruitment dynamics in syncytial Drosophila embryos during nuclear cycles 11–13 (Fig 1). Note that we have not attempted to quantify (nor model—see below) the dramatic disassembly of the mitotic PCM that occurs at the end of mitosis, as in fly and worm embryos this is a complicated process in which large "packets" or "flares" of the mitotic PCM are mechanically removed from the PCM in a MT-dependent manner (Megraw et al, 2002; Magescas et al, 2019; Mittasch et al, 2020). In the experiments reported here, we used fluorescent reporters fused to several different fluorescent tags—Neon Green (NG), GFP, RFP or mCherry—and expressed from several different promoters (see Appendix Table S2). Most importantly, the expression levels of the Spd-2- and Cnn-fusion proteins used to measure recruitment dynamics were similar to endogenous levels (Fig EV1A and B), while the Polo-GFP fusion was expressed from a GFP-insertion into the endogenous Polo gene (Buszczak et al, 2007). Figure 1. Analysis of PCM scaffold assembly dynamics during nuclear cycles 11–13 A. Graphs show the average centrosomal fluorescence intensity of NG-Cnn, Spd-2-GFP and Polo-GFP—dark lines (± SD for each individual embryo indicated in reduced opacity; N ≥ 15 embryos)—over time during nuclear cycles 11, 12 and 13. The white parts of the graphs indicate S-phase and the grey parts mitosis. All individual embryo tracks were aligned to the start of mitosis (NEB; t = 0). B. Scatter plots show the correlation between the centrosome growth period and S-phase length for the embryos analysed in (A). Lines indicate mathematically regressed fits. The goodness of fit (R2) was assessed in GraphPad Prism. The bivariate Gaussian distribution of the data was confirmed by Henze-Zirkler test, and the strength of correlation (r) and the statistical significance (P-value) were calculated using Pearson correlation test. C, D. Graphs show the average centrosomal fluorescent intensity over time during nuclear cycles 11, 12 and 13 for embryos (N ≥ 8 embryos) co-expressing Spd-2-mCherry (orange) with either GFP-Cnn (blue) (C) or Polo-GFP (green) (D). Fluorescence intensity was rescaled to between 0 and 1 in each cycle. (C',D') Dot plots compare the time difference between the peak Spd-2-mCherry levels and the peak GFP-Cnn (C') or peak Polo-GFP (D') levels in each embryo. Data are presented as Mean ± SD. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Analysing the relative expression levels of the fluorescent fusion-proteins used to quantify centrosome recruitment dynamics A, B. Western blots compare the relative expression levels in syncytial embryos of the GFP- and NG-fusion proteins (FP) used here to quantify the centrosomal recruitment dynamics of Cnn (A) or Spd-2 (B). This analysis reveals that GFP-Cnn and NG-Cnn expressed transgenically from the ubiquitin promoter (u) are present at slightly higher levels than the endogenous untagged Cnn. In contrast, Spd-2-GFP expressed transgenically from the ubiquitin promoter (u) and Spd-2-NG expressed as a CRISPR knock-in at the endogenous Spd-2 locus (e) are both present at slightly lower levels than the endogenous protein. Western blots of serial-dilutions of these samples indicate that the uNG-Cnn and uGFP-Cnn are overexpressed by ~2–3 fold compared to the endogenous protein, and that uSpd-2-GFP and eSpd-2-NG are underexpressed by ~2 fold. These blots were also probed with anti-GAGA factor antibodies as a loading control (Raff et al, 1994). The brightness and contrast were adjusted for optimal display. C. Images show the recruitment of either mNG-Cnn, Spd-2-GFP or Polo-GFP at an exemplar centrosome during nuclear cycles 11, 12 and 13. All images are aligned to nuclear envelope breakdown (NEB; t = 0). The white parts of the graphs indicate S-phase and the grey parts mitosis. The first (most leftward) image in each series is taken when the two centrosomes associated with each nucleus at the end of mitosis have first completely separated from one another in early S-phase; because the Cnn scaffold is significantly larger than the Spd-2 or Polo scaffold, it takes longer for the two centrosomes to fully separate, so there are less images of Cnn in S-phase. Scale bar = 1 μm. Download figure Download PowerPoint The rapid nuclear cycles in these embryos comprise alternating periods of S- and M-phase without intervening Gap periods, and S-phase gradually lengthens at each successive cycle (Foe & Alberts, 1983). Perhaps surprisingly, the centrosomal recruitment dynamics of Cnn were quite distinct from Spd-2 and Polo (Figs 1A and EV1C). In all the nuclear cycles, the centrosomal levels of NG-Cnn increased through most of S-phase, the period when centrosomes grow in preparation for mitosis in these rapidly cycling embryos. In cycle 11, however, NG-Cnn levels continued to increase even after the embryos had entered mitosis—scored by nuclear envelope breakdown (NEB; t = 0 in Fig 1A) and indicated by the grey shading in the graphs in Fig 1A—while in cycles 12 and 13 centrosomal levels peaked and then largely plateaued at about the time (cycle 12), or a few minutes before (cycle 13), the embryos entered mitosis. In contrast, the centrosomal levels of Spd-2-GFP and Polo-GFP peaked in mid-late S-phase and then started to decline well before NEB (Figs 1A and EV1C). In these syncytial embryos, S-phase length is determined by the activity of the core Cdk/Cyclin cell cycle oscillator (CCO) that drives progression through these early nuclear cycles (Farrell & O'Farrell, 2014; Liu et al, 2021), and there was a strong correlation (r ~ 0.96; P < 0.0001) between S-phase length and the Spd-2 and Polo growth period (measured as the time it takes for Spd-2 and Polo levels to peak in S-phase) (Fig 1B). This suggests that CCO activity influences the kinetics of centrosomal Polo and Spd-2 recruitment. Spd-2/CEP192 is thought to be the major protein that recruits Polo into the assembling mitotic PCM in vertebrates (Joukov et al, 2010, 2014; Meng et al, 2015), worms (Decker et al, 2011) and flies (Alvarez-Rodrigo et al, 2019), but the shapes of the Spd-2 and Polo centrosomal growth curves were quite distinct, particularly during cycles 11 and 12 (Fig 1A). Moreover, we noticed that during each cycle centrosomal Polo levels peaked slightly before Spd-2 levels peaked, and the centrosomal levels of both Polo and Spd-2 peaked before the levels of Cnn peaked—meaning that the Cnn scaffold could continue to grow and/or plateau even as the centrosomal levels of Polo and Spd-2 declined (Fig 1A). As these measurements were taken from different sets of embryos expressing each protein individually, we confirmed these relative timings in embryos co-expressing Spd-2-mCherry with either Polo-GFP or GFP-Cnn (Fig 1C and D). An underlying pulse of Polo activity could explain the observed kinetics of PCM scaffold assembly As the rise and fall in centrosomal Polo levels appeared to precede the rise and fall in centrosomal Spd-2 levels (Fig 1D), we wondered whether the centrosomes might generate a pulse of Polo activity to initiate the assembly of the Spd-2/Cnn scaffold. We have previously developed a molecular model to explain how Spd-2, Polo and Cnn cooperate to assemble a mitotic PCM scaffold in Drosophila embryos (Fig 2A). In this scheme, Spd-2 and Polo are recruited to centrioles, and Spd-2 becomes phosphorylated at centrioles as cells prepare to enter mitosis—allowing Spd-2 to form a scaffold that fluxes outwards away from the centriole (Conduit et al, 2014b). This scaffold is structurally weak, but it can bind Polo and Cnn from the cytoplasm, which stabilises the scaffold (indicated by the dotted line in Fig 2A). This pool of Polo can then phosphorylate the Cnn to generate an independent Cnn scaffold which is structurally strong and can flux outwards from the Spd-2 scaffold along the centrosomal MTs (Conduit et al, 2014a; Feng et al, 2017) (see Fig EV2 for a cartoon illustration of this scheme). Figure 2. Mathematical modelling of PCM scaffold assembly A schematic summary of the putative molecular interactions that drive the assembly of a Spd-2/Polo/Cnn mitotic PCM scaffold in Drosophila (see main text for details). Schematic illustrates a version of the molecular model of PCM scaffold assembly that can be formulated as a series of ODEs (see Materials and Methods), allowing us to calculate how the levels of each component in the system changes over time. See main text for the meaning of the various symbols. Graph shows the output from the model depicted in (B), illustrating how the centrosomal levels of the various PCM scaffold components change over time if a centriolar pulse of Polo activity (solid green line) is imposed on the system. Total Polo (dotted green line) represents the sum of the P* generated at the centriole surface and the P* bound to the S ¯ scaffold; Total Spd-2 (dotted orange line) represents the sum of Spd-2 in S* and S ¯ ; Total Cnn (dotted blue line) represents the sum of Cnn in S ¯ and C*. To better reflect the situation in vivo—where the centrosomes start each cycle already associated with some PCM scaffold acquired from the previous cycle (Conduit et al, 2010)—we allow the model to run for a complete initial cycle (where the levels of all scaffolding components start at zero), divide the final amount of PCM between two new centrosomes, and then graph the behaviour of the system starting from this point during a second cycle. Thus, the pulse of centriolar Polo activity starts from zero at the start of the cycle, but some Polo, Spd-2 and Cnn recruited in the previous cycle are already present at the centrosome. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. A molecular model of how Spd-2, Polo and Cnn cooperate to form a mitotic PCM scaffold Cartoon illustrates the assembly of the Spd-2/Polo/Cnn mitotic PCM scaffold in Drosophila. During interphase (i), Spd-2, Polo and Cnn are recruited to a toroid that surrounds the mother centriole (Fu & Glover, 2012). Polo is presumably inactive, and Spd-2 and Cnn are presumably not phosphorylated. As cells prepare to enter mitosis (ii), Polo is activated at the centriole and the centrosomal Spd-2 becomes phosphorylated, allowing it to assemble into a scaffold that can flux outwards away from the centriole. The phosphorylated Spd-2 scaffold (equivalent to S ∗ in Fig 2B) is structurally weak, but it can recruit Polo—via phosphorylated S-S(P)/T(P) motifs (Alvarez-Rodrigo et al, 2019)—and also Cnn (Conduit et al, 2014b) to form the more stable S ¯ scaffold depicted in Fig 2B. The Polo recruited by Spd-2 is activated and can phosphorylate Cnn, allowing Cnn to assemble into a strong macromolecular scaffold ( C ∗ in Fig 2B) (Conduit et al, 2014a; Feng et al, 2017). Cnn itself cannot recruit more Spd-2 or Polo, but it stabilises the expanding Spd-2 scaffold, so allowing Spd-2 to accumulate around the mother centriole (iii) (Conduit et al, 2014b). Download figure Download PowerPoint We turned to mathematical modelling to test whether imposing an underlying pulse of centriolar Polo activity on these proposed molecular interactions could explain the observed kinetics of PCM scaffold assembly. In this model (Model 1; Fig 2B), we assume that a pulse of active Polo (P*) is generated at the surface of mother centrioles, with levels peaking at mid-S-phase (we explore below how this pulse might be generated). We allow centrosomal receptors ( R S ) to recruit cytoplasmic Spd-2 (S) to the centriole to form the complex R ¯ S . The Spd-2 bound to this complex can be phosphorylated by P ∗ and converted to a form that can form a scaffold ( S ∗ ) that is released from R ¯ S to flux outwards. This scaffold is unstable and can be rapidly converted back to S by a phosphatase, which we allow to be active in the cytoplasm at a constant level. However, S ∗ can also bind cytoplasmic Polo ( P) and Cnn ( C), to form a more stable scaffold ( S ¯ ) that converts back to S relatively slowly. When bound to S ¯ , Polo is activated so that it can phosphorylate the S ¯ -bound Cnn and convert it into a form ( C ∗ ) that can form a scaffold and be released from S ¯ to flux further outwards. In this way, the Spd-2 scaffold acts to convert catalytically C into the scaffold C ∗ . The C ∗ scaffold disassembles when it is dephosphorylated by a cytoplasmic phosphatase (PPTase), which we allow to be active in the cytoplasm at a constant level. Note that this PPTase activity drives a low-level of Cnn scaffold disassembly during the assembly process, but it is not intended to mimic the high levels of PPTase activity that are thought to drive the rapid disassembly of the PCM scaffold at the end of mitosis (Enos et al, 2018; Magescas et al, 2019; Mittasch et al, 2020). As explained above, this rapid disassembly is a complex process that we do not attempt to measure or model here. We also allow the rate of C ∗ disassembly to increase as the size of the C ∗ scaffold increases, which appears to be the case in these embryos (see Materials and Methods). We modelled these reactions as a system of ordinary differential equations (ODEs, detailed in Appendix Supplementary Methods for Mathematical Modelling) and estimated values for each of the 12 model parameters (see "Justification of Model Parameters" in Materials and Methods). Encouragingly, the output of this model recapitulated two of the most surprising features of scaffold assembly dynamics that we observed in vivo (Fig 2C): (1) The imposed centriolar P ∗ pulse (solid green line, Fig 2B) generated a subsequent pulse in centrosomal Spd-2 levels (dotted orange line, Fig 2C); (2) the system generated the assembly of a Cnn scaffold (dotted blue line, Fig 2C) that could continue to grow and then plateau even as centrosomal Polo and Spd-2 levels declined. To assess the robustness of this model, we tested the effect of individually halving or doubling each of the reaction rate parameters. Although the precise shapes of the curves varied, these two key features were recapitulated in all cases (Fig EV3). Thus, this simple model can robustly explain the basic dynamic features of PCM scaffold assembly kinetics that we observe in vivo in the parameter regime we consider. Click here to expand this figure. Figure EV3. Model predictions are relatively robust to changes in parameter values Graphs show the computed output of Models 1 and 2 when each of the 13 reaction rate parameters is either doubled or halved (as indicated above each graph). The qualitative behaviour of the model is consistent in all cases, demonstrating the model's robustness in the parameter regime considered. Download figure Download PowerPoint Spd-2 and Ana1 help to generate the centrosomal Polo pulse How might the centrioles generate such a pulse of Polo activity? This pulse of activity is unlikely to simply reflect the general activity of Polo in the embryo, which, like Cdk/Cyclin activity (Deneke et al, 2016), peaks during mitosis (Stefano Di Talia, Duke University (USA), personal communication). Thus, the centrioles must generate a local pulse of Polo activity well before Polo is maximally activated in the rest of the embryo more generally. Polo/PLK1 is known to be recruited to mitotic centrosomes by its Polo-box domain (PBD) that binds to phosphorylated S-S(P)/T(P) motifs (Song et al, 2000; Seong et al, 2002; Elia et al, 2003; Reynolds & Ohkura, 2003); this recruitment is sufficient to at least partially activate the kinase (Xu et al, 2013). In fly embryos, the Polo required for mitotic PCM assembly appears to be recruited to centrosomes via the sequential phosphorylation of S-S(P)/T(P) motifs first in Ana1 (that helps recruit Polo to mother centrioles) (Alvarez-Rodrigo et al, 2021) and then in Spd-2 (which helps recruit Polo into the expanding mitotic PCM) (Alvarez-Rodrigo et al, 2019). To test the potential role of these proteins in generating the Polo pulse, we examined Polo-GFP recruitment during nuclear cycle 12 in embryos expressing a mutant form of either Ana1 (Ana1-S34T-mCherry) (Alvarez-Rodrigo et al, 2021) or Spd-2 (Spd-2-S16T-mCherry—previously called Spd-2-CONS-mCherry) (Alvarez-Rodrigo et al, 2019) in which multiple S-S/T motifs (34 for Ana1 and 16 for Spd-2) were mutated to T-S/T (Fig 3). These conservative substitutions severely impair the ability of the mutant proteins to recruit Polo, seemingly without perturbing other aspects of their function (Alvarez-Rodrigo et al, 2019, 2021). These experiments were performed in the presence of endogenous, untagged, Spd-2 or Ana1 because embryos laid by females co-expressing Polo-GFP in the presence of only Ana1-S34T or Spd-2-S16T die very early in development due to centrosome defects (Alvarez-Rodrigo et al, 2019, 2021)—as centrosomes are essential for early embryo development (Stevens et al, 2007; Varmark et al, 2007), but not for the rest of development in Drosophila (Basto et al, 2006). Figure 3. Perturbing the ability of Ana1 or Spd-2 to recruit Polo perturbs the pulse of Polo activity A–D. Graphs in (A) and (C) show how the average fluorescent intensity (±SD) of Polo-GFP changes over time at OM (dark green squares) or NM (light green triangles) centrosomes during nuclear cycle 12 in embryos (N = 12) laid by WT females expressing either Ana1-mCherry, Ana1-S34T-mCherry, Spd-2-mCherry, or Spd-2-S16T-mCherry. In this experiment, embryos (N = 12) were aligned to the start of S-phase (t = 0), which was scored by centriole separation. (B, D) Bar charts compare various growth parameters (indicated
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