FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2959619610> ?p ?o ?g. }

• W2959619610 endingPage "366.e6" @default.
• W2959619610 startingPage "355" @default.
• W2959619610 abstract "•A cascade of Aurora A/AIR-1, CEP192/SPD-2, and PLK-1 initiates PCM recruitment•SPD-2, PLK-1, and centrioles are required for mitotic PCM expansion•PLK-1 alone is required for PCM maintenance•Centrioles are required for centrosome structural integrity Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM. Centrosomes, the predominant sites of microtubule nucleation and anchorage, coordinate spindle assembly and cell division in animal cells. At the onset of mitosis, centrioles accumulate microtubule-organizing pericentriolar material (PCM) in a process termed centrosome maturation. To what extent centrosome maturation depends on the continued activity of mitotic regulators or the presence of centrioles has hitherto been unclear. Using the C. elegans early embryo, we show that PCM expansion requires the Polo-like kinase PLK-1 and CEP192 (SPD-2 in C. elegans), but not its upstream regulator Aurora A (AIR-1), while maintenance of the PCM polymer depends exclusively on PLK-1. SPD-2 and PLK-1 are highly concentrated at centrioles. Unexpectedly, laser microsurgery reveals that while centrioles are required for PCM recruitment and centrosome structural integrity they are dispensable for PCM maintenance. We propose a model whereby centrioles promote centrosome maturation by recruiting PLK-1, but subsequent maintenance occurs via PLK-1 acting directly within the PCM. Centrosomes are cytoplasmic structures that organize the microtubule network of animal cells, thereby directing the positioning of organelles, intracellular traffic, polarity, morphogenesis, and cell division. As the predominant sites of microtubule nucleation (Heald et al., 1997Heald R. Tournebize R. Habermann A. Karsenti E. Hyman A. Spindle assembly in Xenopus egg extracts: respective roles of centrosomes and microtubule self-organization.J. Cell Biol. 1997; 138: 615-628Crossref PubMed Scopus (291) Google Scholar), centrosomes are essential for the rapid divisions that characterize early embryogenesis (O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar, Stevens et al., 2007Stevens N.R. Raposo A.A. Basto R. St Johnston D. Raff J.W. From stem cell to embryo without centrioles.Curr. Biol. 2007; 17: 1498-1503Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, Yabe et al., 2007Yabe T. Ge X. Pelegri F. The zebrafish maternal-effect gene cellular atoll encodes the centriolar component sas-6 and defects in its paternal function promote whole genome duplication.Dev. Biol. 2007; 312: 44-60Crossref PubMed Scopus (61) Google Scholar) as well as for proper asymmetric cell division (Basto et al., 2006Basto R. Lau J. Vinogradova T. Gardiol A. Woods C.G. Khodjakov A. Raff J.W. Flies without centrioles.Cell. 2006; 125: 1375-1386Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar), a defining feature of stem cells. Centrosomes consist of a pair of centrioles surrounded by microtubule-organizing PCM. The PCM increases several-fold in size at the onset of mitosis, contributing to assembly of the mitotic spindle (Khodjakov and Rieder, 1999Khodjakov A. Rieder C.L. The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules.J. Cell Biol. 1999; 146: 585-596Crossref PubMed Scopus (298) Google Scholar). Lacking the layered structure found in interphase (Fu and Glover, 2012Fu J. Glover D.M. Structured illumination of the interface between centriole and peri-centriolar material.Open Biol. 2012; 2Crossref PubMed Scopus (176) Google Scholar, Lawo et al., 2012Lawo S. Hasegan M. Gupta G.D. Pelletier L. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material.Nat. Cell Biol. 2012; 14: 1148-1158Crossref PubMed Scopus (266) Google Scholar, Mennella et al., 2012Mennella V. Keszthelyi B. McDonald K.L. Chhun B. Kan F. Rogers G.C. Huang B. Agard D.A. Subdiffraction-resolution fluorescence microscopy reveals a domain of the centrosome critical for pericentriolar material organization.Nat. Cell Biol. 2012; 14: 1159-1168Crossref PubMed Scopus (269) Google Scholar), the mitotic PCM instead is organized around a proteinaceous scaffold that recruits γ-tubulin and other microtubule regulators (Moritz and Alberts, 1999Moritz M. Alberts B.M. Isolation of centrosomes from Drosophila embryos.Methods Cell Biol. 1999; 61: 1-12PubMed Google Scholar, Schnackenberg et al., 1998Schnackenberg B.J. Khodjakov A. Rieder C.L. Palazzo R.E. The disassembly and reassembly of functional centrosomes in vitro.Proc. Natl. Acad. Sci. USA. 1998; 95: 9295-9300Crossref PubMed Scopus (147) Google Scholar). Functional studies have put forward the coil-coiled proteins SPD-5 and Cnn as the main components of the PCM scaffold in C. elegans and Drosophila, respectively (Hamill et al., 2002Hamill D.R. Severson A.F. Carter J.C. Bowerman B. Centrosome maturation and mitotic spindle assembly in C. elegans Require SPD-5, A protein with multiple coiled-coil domains.Dev. Cell. 2002; 3: 673-684Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, Megraw et al., 1999Megraw T.L. Li K. Kao L.R. Kaufman T.C. The centrosomin protein is required for centrosome assembly and function during cleavage in Drosophila.Development. 1999; 126: 2829-2839Crossref PubMed Google Scholar). The homolog of Cnn, CDK5RAP2, likely forms the PCM scaffold in vertebrates, potentially together with another protein, Pericentrin (Lawo et al., 2012Lawo S. Hasegan M. Gupta G.D. Pelletier L. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material.Nat. Cell Biol. 2012; 14: 1148-1158Crossref PubMed Scopus (266) Google Scholar). Loss or mutation of SPD-5 and Cnn strongly impairs mitotic PCM assembly, while recent in vitro reconstitution experiments found them capable of oligomerizing into micron-scale assemblies dynamically concentrating PCM ‘client’ proteins (Feng et al., 2017Feng Z. Caballe A. Wainman A. Johnson S. Haensele A.F.M. Cottee M.A. Conduit P.T. Lea S.M. Raff J.W. Structural basis for mitotic centrosome assembly in flies.Cell. 2017; 169: 1078-1089Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, Woodruff et al., 2015Woodruff J.B. Wueseke O. Viscardi V. Mahamid J. Ochoa S.D. Bunkenborg J. Widlund P.O. Pozniakovsky A. Zanin E. Bahmanyar S. et al.Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.Science. 2015; 348: 808-812Crossref PubMed Scopus (119) Google Scholar, Woodruff et al., 2017Woodruff J.B. Gomes B.F. Widlund P.O. Mahamid J. Honigmann A. Hyman A.A. The centrosome is a selective condensate that nucleates microtubules by concentrating tubulin.Cell. 2017; 169: 1066-1077Abstract Full Text Full Text PDF PubMed Scopus (328) Google Scholar). PCM assembly must be tightly regulated to ensure the formation of two equally sized spindle poles in mitosis. Central to PCM assembly is the Polo-like kinase PLK-1 (Sunkel and Glover, 1988Sunkel C.E. Glover D.M. Polo, a mitotic mutant of Drosophila displaying abnormal spindle poles.J. Cell Sci. 1988; 89: 25-38Crossref PubMed Google Scholar). Localized to centrosomes and required for centrosome maturation across species (Decker et al., 2011Decker M. Jaensch S. Pozniakovsky A. Zinke A. O'Connell K.F. Zachariae W. Myers E. Hyman A.A. Limiting amounts of centrosome material set centrosome size in C. elegans embryos.Curr. Biol. 2011; 21: 1259-1267Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Dobbelaere et al., 2008Dobbelaere J. Josué F. Suijkerbuijk S. Baum B. Tapon N. Raff J. A genome-wide RNAi screen to dissect centriole duplication and centrosome maturation in Drosophila.PLoS Biol. 2008; 6: e224Crossref PubMed Scopus (175) Google Scholar, Lane and Nigg, 1996Lane H.A. Nigg E.A. Antibody microinjection reveals an essential role for human polo-like kinase 1 (Plk1) in the functional maturation of mitotic centrosomes.J. Cell Biol. 1996; 135: 1701-1713Crossref PubMed Scopus (596) Google Scholar), PLK-1 acts, at least in part, by phosphorylating the PCM matrix components SPD-5 and Cnn, which enhances their intrinsic tendency to oligomerize in vitro (Conduit et al., 2014Conduit P.T. Feng Z. Richens J.H. Baumbach J. Wainman A. Bakshi S.D. Dobbelaere J. Johnson S. Lea S.M. Raff J.W. The centrosome-specific phosphorylation of Cnn by Polo/Plk1 drives Cnn scaffold assembly and centrosome maturation.Dev. Cell. 2014; 28: 659-669Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar, Feng et al., 2017Feng Z. Caballe A. Wainman A. Johnson S. Haensele A.F.M. Cottee M.A. Conduit P.T. Lea S.M. Raff J.W. Structural basis for mitotic centrosome assembly in flies.Cell. 2017; 169: 1078-1089Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, Woodruff et al., 2015Woodruff J.B. Wueseke O. Viscardi V. Mahamid J. Ochoa S.D. Bunkenborg J. Widlund P.O. Pozniakovsky A. Zanin E. Bahmanyar S. et al.Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.Science. 2015; 348: 808-812Crossref PubMed Scopus (119) Google Scholar, Wueseke et al., 2016Wueseke O. Zwicker D. Schwager A. Wong Y.L. Oegema K. Jülicher F. Hyman A.A. Woodruff J.B. Polo-like kinase phosphorylation determines Caenorhabditis elegans centrosome size and density by biasing SPD-5 toward an assembly-competent conformation.Biol. Open. 2016; 5: 1431-1440Crossref PubMed Scopus (34) Google Scholar). Another centrosome-localized kinase implicated in mitotic PCM assembly is Aurora A/AIR-1 (Hannak et al., 2001Hannak E. Kirkham M. Hyman A.A. Oegema K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.J. Cell Biol. 2001; 155: 1109-1116Crossref PubMed Scopus (366) Google Scholar). Depletion or mutation of Aurora A/AIR-1 impairs recruitment of Cnn and SPD-5 and consequently centrosome maturation (Berdnik and Knoblich, 2002Berdnik D. Knoblich J.A. Drosophila aurora-A is required for centrosome maturation and actin-dependent asymmetric protein localization during mitosis.Curr. Biol. 2002; 12: 640-647Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, Hamill et al., 2002Hamill D.R. Severson A.F. Carter J.C. Bowerman B. Centrosome maturation and mitotic spindle assembly in C. elegans Require SPD-5, A protein with multiple coiled-coil domains.Dev. Cell. 2002; 3: 673-684Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). Aurora A has been shown to phosphorylate and activate PLK-1 (Macůrek et al., 2008Macůrek L. Lindqvist A. Lim D. Lampson M.A. Klompmaker R. Freire R. Clouin C. Taylor S.S. Yaffe M.B. Medema R.H. Polo-like kinase-1 is activated by aurora A to promote checkpoint recovery.Nature. 2008; 455: 119-123Crossref PubMed Scopus (520) Google Scholar, Seki et al., 2008Seki A. Coppinger J.A. Jang C.Y. Yates J.R. Fang G. Bora and the kinase Aurora a cooperatively activate the kinase Plk1 and control mitotic entry.Science. 2008; 320: 1655-1658Crossref PubMed Scopus (450) Google Scholar), placing Aurora A upstream of PLK-1 in the PCM assembly pathway. PCM assembly is also regulated by the centrosomal protein SPD-2/CEP192 (Kemp et al., 2004Kemp C.A. Kopish K.R. Zipperlen P. Ahringer J. O'Connell K.F. Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2.Dev. Cell. 2004; 6: 511-523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, Pelletier et al., 2004Pelletier L. Ozlü N. Hannak E. Cowan C. Habermann B. Ruer M. Müller-Reichert T. Hyman A.A. The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication.Curr. Biol. 2004; 14: 863-873Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). In C. elegans, SPD-2 was proposed to set PCM size by controlling the recruitment of PLK-1 to centrosomes (Decker et al., 2011Decker M. Jaensch S. Pozniakovsky A. Zinke A. O'Connell K.F. Zachariae W. Myers E. Hyman A.A. Limiting amounts of centrosome material set centrosome size in C. elegans embryos.Curr. Biol. 2011; 21: 1259-1267Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). CEP192 in vertebrates was similarly shown to initiate a cascade of events that lead to PCM maturation. By recruiting Aurora A and PLK-1 to centrosomes, CEP192 increases the local concentration of the kinases leading to their activation (Joukov et al., 2010Joukov V. De Nicolo A. Rodriguez A. Walter J.C. Livingston D.M. Centrosomal protein of 192 kDa (Cep192) promotes centrosome-driven spindle assembly by engaging in organelle-specific Aurora A activation.Proc. Natl. Acad. Sci. USA. 2010; 107: 21022-21027Crossref PubMed Scopus (79) Google Scholar, Joukov et al., 2014Joukov V. Walter J.C. De Nicolo A. The cep192-organized aurora a-Plk1 cascade is essential for centrosome cycle and bipolar spindle assembly.Mol. Cell. 2014; 55: 578-591Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Whether SPD-2 and AIR-1 merely trigger PCM recruitment by activating PLK-1 or also participate in downstream events remains unclear. However, it should be noted that SPD-2 potentiated SPD-5 scaffold expansion in vitro independently of the presence of PLK-1 (Woodruff et al., 2015Woodruff J.B. Wueseke O. Viscardi V. Mahamid J. Ochoa S.D. Bunkenborg J. Widlund P.O. Pozniakovsky A. Zanin E. Bahmanyar S. et al.Centrosomes. Regulated assembly of a supramolecular centrosome scaffold in vitro.Science. 2015; 348: 808-812Crossref PubMed Scopus (119) Google Scholar), suggesting a role that goes beyond the recruitment and activation of PLK-1. Centrioles are absolutely essential to initiate PCM assembly. In the absence of centrioles centrosomes do not form (Basto et al., 2006Basto R. Lau J. Vinogradova T. Gardiol A. Woods C.G. Khodjakov A. Raff J.W. Flies without centrioles.Cell. 2006; 125: 1375-1386Abstract Full Text Full Text PDF PubMed Scopus (537) Google Scholar), and centriole duplication is a pre-requisite for centrosome duplication (O'Connell et al., 2001O'Connell K.F. Caron C. Kopish K.R. Hurd D.D. Kemphues K.J. Li Y. White J.G. The C. elegans zyg-1 gene encodes a regulator of centrosome duplication with distinct maternal and paternal roles in the embryo.Cell. 2001; 105: 547-558Abstract Full Text Full Text PDF PubMed Scopus (238) Google Scholar). Both SPD-2/CEP192 and PLK-1 strongly localize to centrioles (Fu and Glover, 2012Fu J. Glover D.M. Structured illumination of the interface between centriole and peri-centriolar material.Open Biol. 2012; 2Crossref PubMed Scopus (176) Google Scholar, Lawo et al., 2012Lawo S. Hasegan M. Gupta G.D. Pelletier L. Subdiffraction imaging of centrosomes reveals higher-order organizational features of pericentriolar material.Nat. Cell Biol. 2012; 14: 1148-1158Crossref PubMed Scopus (266) Google Scholar, Pelletier et al., 2004Pelletier L. Ozlü N. Hannak E. Cowan C. Habermann B. Ruer M. Müller-Reichert T. Hyman A.A. The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication.Curr. Biol. 2004; 14: 863-873Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar), providing a potential molecular mechanism for localized PCM assembly. To what extent centrioles contribute to further mitotic centrosome growth and maintenance is not clear. Spatial analysis of Cnn and SPD-5 recruitment during centrosome maturation in Drosophila neuroblasts and C. elegans embryos revealed that new scaffold incorporation does not occur specifically at centrioles but rather isotropically throughout the existing PCM (Conduit and Raff, 2015Conduit P.T. Raff J.W. Different Drosophila cell types exhibit differences in mitotic centrosome assembly dynamics.Curr. Biol. 2015; 25: R650-R651Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, Laos et al., 2015Laos T. Cabral G. Dammermann A. Isotropic incorporation of SPD-5 underlies centrosome assembly in C. elegans.Curr. Biol. 2015; 25: R648-R649Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Centrioles, therefore, do not directly participate in PCM expansion by acting as docking sites for new scaffold protein incorporation. However, this does not exclude an indirect, signaling role, perhaps mediated by SPD-2 and PLK-1 (Alvarez Rodrigo et al., 2018Alvarez Rodrigo I. Conduit P.T. Baumbach J. Novak Z.A. Aydogan M.G. Wainman A. Raff J.W. A positive feedback loop drives centrosome maturation in flies.bioRxiv. 2018; Google Scholar). Mathematical modeling of centrosome dynamics in the C. elegans embryo argues that the observed kinetics of PCM growth can be explained by autocatalytic assembly coupled with a degree of catalytic activity of the centrioles suppressing the unequal growth of the two centrosomes (Zwicker et al., 2014Zwicker D. Decker M. Jaensch S. Hyman A.A. Jülicher F. Centrosomes are autocatalytic droplets of pericentriolar material organized by centrioles.Proc. Natl. Acad. Sci. USA. 2014; 111: E2636-E2645Crossref PubMed Scopus (121) Google Scholar). This model also accounts for the unequal growth of centrosomes around normal and structurally defective centrioles following partial depletion of the centriolar component SAS-4 (Kirkham et al., 2003Kirkham M. Müller-Reichert T. Oegema K. Grill S. Hyman A.A. SAS-4 is a C. elegans centriolar protein that controls centrosome size.Cell. 2003; 112: 575-587Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar). However, direct experimental evidence for or against centriole involvement in PCM growth or maintenance is currently lacking. Here, we use the C. elegans early embryo as an experimental model to dissect the mechanisms underlying mitotic centrosome assembly and in particular the role of centrioles in this process. We present evidence that SPD-2 and PLK-1, but not the upstream regulator AIR-1, are continually required for PCM recruitment, with centrioles actively contributing to PCM expansion. In contrast, PLK-1 alone is required for maintenance of the mitotic PCM scaffold, which furthermore is independent of the continued presence of centrioles. Centrioles do, however, contribute to centrosome structural integrity. Centrioles, then, are not merely initiators of PCM recruitment, but are critically involved in further growth as well as centrosome stability. Progression through the cell cycle is marked by the cyclical growth and disassembly of the mitotic PCM. The C. elegans one-cell embryo provides an excellent model to study this process. A pair of centrioles devoid of PCM is introduced at fertilization; these centrioles separate, duplicate, and recruit PCM, with centrosomes reaching their maximum size in late mitosis (Decker et al., 2011Decker M. Jaensch S. Pozniakovsky A. Zinke A. O'Connell K.F. Zachariae W. Myers E. Hyman A.A. Limiting amounts of centrosome material set centrosome size in C. elegans embryos.Curr. Biol. 2011; 21: 1259-1267Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar; Figures 1 and S1A; Video S1). Like other cellular processes in C. elegans, the centrosome cycle is highly stereotypical, facilitating quantitative analysis (Dammermann et al., 2008Dammermann A. Maddox P.S. Desai A. Oegema K. SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the gamma-tubulin-mediated addition of centriolar microtubules.J. Cell Biol. 2008; 180: 771-785Crossref PubMed Scopus (92) Google Scholar, Decker et al., 2011Decker M. Jaensch S. Pozniakovsky A. Zinke A. O'Connell K.F. Zachariae W. Myers E. Hyman A.A. Limiting amounts of centrosome material set centrosome size in C. elegans embryos.Curr. Biol. 2011; 21: 1259-1267Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Laos et al., 2015Laos T. Cabral G. Dammermann A. Isotropic incorporation of SPD-5 underlies centrosome assembly in C. elegans.Curr. Biol. 2015; 25: R648-R649Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). Previous genome-wide screens identified three conserved regulators of PCM assembly, AIR-1, SPD-2 and PLK-1 (Decker et al., 2011Decker M. Jaensch S. Pozniakovsky A. Zinke A. O'Connell K.F. Zachariae W. Myers E. Hyman A.A. Limiting amounts of centrosome material set centrosome size in C. elegans embryos.Curr. Biol. 2011; 21: 1259-1267Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, Hannak et al., 2001Hannak E. Kirkham M. Hyman A.A. Oegema K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.J. Cell Biol. 2001; 155: 1109-1116Crossref PubMed Scopus (366) Google Scholar, Kemp et al., 2004Kemp C.A. Kopish K.R. Zipperlen P. Ahringer J. O'Connell K.F. Centrosome maturation and duplication in C. elegans require the coiled-coil protein SPD-2.Dev. Cell. 2004; 6: 511-523Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar, Pelletier et al., 2004Pelletier L. Ozlü N. Hannak E. Cowan C. Habermann B. Ruer M. Müller-Reichert T. Hyman A.A. The Caenorhabditis elegans centrosomal protein SPD-2 is required for both pericentriolar material recruitment and centriole duplication.Curr. Biol. 2004; 14: 863-873Abstract Full Text Full Text PDF PubMed Scopus (181) Google Scholar). Depletion of all three proteins strongly impairs the mitotic accumulation of the PCM scaffold component SPD-5, while leaving interphase levels largely unchanged (Figures 1B and 1C). This is in contrast to depletion of SPD-5 itself, which entirely eliminates PCM assembly (Figure S1B). Notably, both SPD-2 and PLK-1 are highly concentrated at centrioles, while AIR-1 localizes throughout the PCM and on astral microtubules (Figure 1A; see also Figure S1B). https://www.cell.com/cms/asset/b29e45b0-c054-4d36-918c-3a342f18737e/mmc2.mp4Loading ... Download .mp4 (1.85 MB) Help with .mp4 files Video S1. Control Embryo Expressing GFP:SPD-5 and mCherry:Histone, Related to Figures 1 and S1One-cell-stage C. elegans embryo co-expressing GFP:SPD-5 and mCherry:Histone H2B to mark PCM and chromosomes, respectively, imaged on a spinning disk confocal microscope. Sequence begins in early S phase and ends after cytokinesis onset. Single-plane fluorescence images used to generate each video frame. Time is min:s. While AIR-1, SPD-2, and PLK-1 are essential for centrosome maturation, it is possible that some or all of these regulators merely initiate mitotic PCM assembly, but are dispensable for the maintenance of already assembled PCM. To test this hypothesis, we arrested embryos in metaphase with the proteasome inhibitor clasto-lactacystin-β-lactone, which results in stable levels of mitotic PCM (Laos et al., 2015Laos T. Cabral G. Dammermann A. Isotropic incorporation of SPD-5 underlies centrosome assembly in C. elegans.Curr. Biol. 2015; 25: R648-R649Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar; Video S2A). We then treated embryos with specific inhibitors to acutely inactivate AIR-1 and PLK-1 (Lénárt et al., 2007Lénárt P. Petronczki M. Steegmaier M. Di Fiore B. Lipp J.J. Hoffmann M. Rettig W.J. Kraut N. Peters J.M. The small-molecule inhibitor BI 2536 reveals novel insights into mitotic roles of polo-like kinase 1.Curr. Biol. 2007; 17: 304-315Abstract Full Text Full Text PDF PubMed Scopus (558) Google Scholar, Manfredi et al., 2011Manfredi M.G. Ecsedy J.A. Chakravarty A. Silverman L. Zhang M. Hoar K.M. Stroud S.G. Chen W. Shinde V. Huck J.J. et al.Characterization of alisertib (MLN8237), an investigational small-molecule inhibitor of aurora A kinase using novel in vivo pharmacodynamic assays.Clin. Cancer Res. 2011; 17: 7614-7624Crossref PubMed Scopus (228) Google Scholar, Noatynska et al., 2010Noatynska A. Panbianco C. Gotta M. SPAT-1/Bora acts with Polo-like kinase 1 to regulate PAR polarity and cell cycle progression.Development. 2010; 137: 3315-3325Crossref PubMed Scopus (40) Google Scholar, Sumiyoshi et al., 2015Sumiyoshi E. Fukata Y. Namai S. Sugimoto A. Caenorhabditis elegans Aurora A kinase is required for the formation of spindle microtubules in female meiosis.Mol. Biol. Cell. 2015; 26: 4187-4196Crossref PubMed Scopus (17) Google Scholar). Importantly, inhibition of either AIR-1 (MLN8237) or PLK-1 (BI2536) prior to mitosis disrupted centrosomal accumulation of SPD-5 in embryos, phenocopying the effect of RNAi (Figures S2A–S2D). When applied to mitotically arrested embryos, PLK-1 inhibition induced a marked loss of centrosomal SPD-5, with levels dropping to those found in S phase (Figures 2A and 2B ; Video S2B), revealing an ongoing requirement for PLK-1 activity to maintain the PCM polymer. This is not due to a lack of centriole integrity, as centrioles remain stable even when PLK-1 is depleted by RNAi (Figure S2E). In contrast, AIR-1 inhibition of arrested embryos resulted in spindle collapse (Figure S2F), a signature air-1 phenotype (Hannak et al., 2001Hannak E. Kirkham M. Hyman A.A. Oegema K. Aurora-A kinase is required for centrosome maturation in Caenorhabditis elegans.J. Cell Biol. 2001; 155: 1109-1116Crossref PubMed Scopus (366) Google Scholar, Ozlü et al., 2005Ozlü N. Srayko M. Kinoshita K. Habermann B. O’toole E.T. Müller-Reichert T. Schmalz N. Desai A. Hyman A.A. An essential function of the C. elegans ortholog of TPX2 is to localize activated aurora A kinase to mitotic spindles.Dev. Cell. 2005; 9: 237-248Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). However, centrosomal SPD-5 accumulation remained similar to controls, indicating that AIR-1 is dispensable for maintenance of already assembled PCM (Figures 2A and 2B; Video S2C). https://www.cell.com/cms/asset/aac58786-8c2c-46cb-bc03-4589a8273d45/mmc3.mp4Loading ... Download .mp4 (4.47 MB) Help with .mp4 files Video S2. Acute Inhibitions in Embryos Expressing GFP:SPD-5 and mCherry:Histone Arrested in Metaphase with Clasto-lactacystin-ß-lactone, Related to Figures 2 and 3(A) Treatment with the proteasome inhibitor clasto-lactacystin-ß-lactone results in prolonged mitotic arrest. PCM is maintained for the duration of arrest. Video starts at prometaphase and continues through metaphase arrest.(B) Embryo arrested in metaphase with clasto-lactacystin-ß-lactone and subsequently treated with the PLK-1 inhibitor BI2536. Acute inhibition of PLK-1 results in PCM disassembly. Video starts at prometaphase before addition of BI2536 and continues through metaphase arrest.(C) Embryo arrested in metaphase with clasto-lactacystin-ß-lactone and subsequently treated with the AIR-1 inhibitor MLN8237. Acute inhibition of AIR-1 results in spindle collapse but does not affect PCM maintenance. Video starts at prometaphase before addition of MLN8237 and continues through metaphase arrest.(D) Embryo arrested in metaphase with clasto-lactacystin-ß-lactone and subsequently treated with the CDK-1 inhibitor flavopiridol. Acute inhibition of CDK-1 results in typical events of forced cell cycle exit, including chromosome decondensation without sister chromatid segregation as well as PCM disassembly. Video starts at prometaphase before addition of flavopiridol.(E) Control for temperature upshift experiment. Wild-type embryo treated with the proteasome inhibitor clasto-lactacystin-ß-lactone and allowed to develop at 16°C until prometaphase, at which point temperature was shifted to 26°C. Video starts before NEBD just prior to temperature upshift and continues through metaphase arrest. Temperature upshift accelerates cell division timing in cycling embryos but has no effect on arrested embryos.(F) Temperature-sensitive spd-2 mutant embryo treated as in (E). Acute temperature upshift does not affect PCM maintenance. Maximum intensity projections of 3D z-stacks used to generate each video frame. Time is min:s. Finally, to test the role of SPD-2 in PCM maintenance, we utilized a previously described fast-acting temperature-sensitive mutant allele, spd-2(or293) (O'Rourke et al., 2011O'Rourke S.M. Carter C. Carter L. Christensen S.N. Jones M.P. Nash B. Price M.H. Turnbull D.W. Garner A.R. Hamill D.R. et al.A survey of new temperature-sensitive, embryonic-lethal mutations in C. elegans: 24 alleles of thirteen genes.PLoS One. 2011; 6: e16644Crossref PubMed Scopus (43) Google Scholar). Remarkably, we could not even detect centrosomal SPD-2 1.5 min after embryos were shifted to the restrictive temperature (Figure S2G). Prolonged inactivation of SPD-2 starting at early stages of the cell cycle resulted in a phenotype similar to the one observed after" @default.
• W2959619610 created "2019-07-23" @default.
• W2959619610 creator A5002172208 @default.
• W2959619610 creator A5024214871 @default.
• W2959619610 creator A5032004677 @default.
• W2959619610 creator A5069824308 @default.
• W2959619610 date "2019-08-01" @default.
• W2959619610 modified "2023-09-30" @default.
• W2959619610 title "Differential Requirements for Centrioles in Mitotic Centrosome Growth and Maintenance" @default.
• W2959619610 cites W1139420029 @default.
• W2959619610 cites W1548984069 @default.
• W2959619610 cites W1606375448 @default.
• W2959619610 cites W1792058463 @default.
• W2959619610 cites W1924895133 @default.
• W2959619610 cites W1966015910 @default.
• W2959619610 cites W1967284833 @default.
• W2959619610 cites W1969117210 @default.
• W2959619610 cites W1969761431 @default.
• W2959619610 cites W1976311440 @default.
• W2959619610 cites W1977560942 @default.
• W2959619610 cites W1980493259 @default.
• W2959619610 cites W1992062482 @default.
• W2959619610 cites W1992692440 @default.
• W2959619610 cites W1993860951 @default.
• W2959619610 cites W1998678044 @default.
• W2959619610 cites W1999353962 @default.
• W2959619610 cites W2000481871 @default.
• W2959619610 cites W2000654425 @default.
• W2959619610 cites W2001971083 @default.
• W2959619610 cites W2007496445 @default.
• W2959619610 cites W2012229898 @default.
• W2959619610 cites W2012950009 @default.
• W2959619610 cites W2016552601 @default.
• W2959619610 cites W2017914952 @default.
• W2959619610 cites W2021002630 @default.
• W2959619610 cites W2026504472 @default.
• W2959619610 cites W2028984447 @default.
• W2959619610 cites W2029355706 @default.
• W2959619610 cites W2031069616 @default.
• W2959619610 cites W2031805729 @default.
• W2959619610 cites W2035842805 @default.
• W2959619610 cites W2040511696 @default.
• W2959619610 cites W2042146481 @default.
• W2959619610 cites W2042901082 @default.
• W2959619610 cites W2044862111 @default.
• W2959619610 cites W2046347502 @default.
• W2959619610 cites W2047029050 @default.
• W2959619610 cites W2054146568 @default.
• W2959619610 cites W2056627868 @default.
• W2959619610 cites W2058367570 @default.
• W2959619610 cites W2058727957 @default.
• W2959619610 cites W2059706603 @default.
• W2959619610 cites W2060297895 @default.
• W2959619610 cites W2065961884 @default.
• W2959619610 cites W2067499825 @default.
• W2959619610 cites W2067518266 @default.
• W2959619610 cites W2071209248 @default.
• W2959619610 cites W2072153102 @default.
• W2959619610 cites W2076883097 @default.
• W2959619610 cites W2079458003 @default.
• W2959619610 cites W2084173199 @default.
• W2959619610 cites W2087009915 @default.
• W2959619610 cites W2099878697 @default.
• W2959619610 cites W2101216488 @default.
• W2959619610 cites W2103390035 @default.
• W2959619610 cites W2106148947 @default.
• W2959619610 cites W2116619702 @default.
• W2959619610 cites W2123587496 @default.
• W2959619610 cites W2124533742 @default.
• W2959619610 cites W2127776124 @default.
• W2959619610 cites W2133702185 @default.
• W2959619610 cites W2139844764 @default.
• W2959619610 cites W2141229359 @default.
• W2959619610 cites W2143720287 @default.
• W2959619610 cites W2153036107 @default.
• W2959619610 cites W2153081604 @default.
• W2959619610 cites W2157443774 @default.
• W2959619610 cites W2157448853 @default.
• W2959619610 cites W2158438290 @default.
• W2959619610 cites W2161659315 @default.
• W2959619610 cites W2163975232 @default.
• W2959619610 cites W2166080650 @default.
• W2959619610 cites W2175915328 @default.
• W2959619610 cites W2276514545 @default.
• W2959619610 cites W2422465532 @default.
• W2959619610 cites W2471407418 @default.
• W2959619610 cites W2501215022 @default.
• W2959619610 cites W2513367803 @default.
• W2959619610 cites W2606833132 @default.
• W2959619610 cites W2620662403 @default.
• W2959619610 cites W2620664852 @default.
• W2959619610 cites W2622426729 @default.
• W2959619610 cites W2767123039 @default.
• W2959619610 cites W2950343526 @default.
• W2959619610 doi "https://doi.org/10.1016/j.devcel.2019.06.004" @default.
• W2959619610 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/31303441" @default.
• W2959619610 hasPublicationYear "2019" @default.
• W2959619610 type Work @default.

• next