Mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos  Melissa Garner, Suzanne van Kreeveld,

Slides:



Advertisements
Similar presentations
Jeffrey T Wigle, Guillermo Oliver  Cell 
Advertisements

Carly I. Dix, Jordan W. Raff  Current Biology 
Volume 16, Issue 17, Pages (September 2006)
Two Phases of Astral Microtubule Activity during Cytokinesis in C
Volume 9, Issue 2, Pages (August 2005)
A Conserved Chromatin Architecture Marks and Maintains the Restricted Germ Cell Lineage in Worms and Flies  Christine E Schaner, Girish Deshpande, Paul.
Jeffrey T Wigle, Guillermo Oliver  Cell 
DNA Damage during Mitosis in Human Cells Delays the Metaphase/Anaphase Transition via the Spindle-Assembly Checkpoint  Alexei Mikhailov, Richard W Cole,
Caenorhabditis elegans TAC-1 and ZYG-9 Form a Complex that Is Essential for Long Astral and Spindle Microtubules  Martin Srayko, Sophie Quintin, Anne.
Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2
Inhibition of Aurora B Kinase Blocks Chromosome Segregation, Overrides the Spindle Checkpoint, and Perturbs Microtubule Dynamics in Mitosis  Marko J.
Volume 20, Issue 24, Pages (December 2010)
Vladic Mogila, Fan Xia, Willis X. Li  Developmental Cell 
The Spindle Checkpoint Kinase Bub1 and Cyclin E/Cdk2 Both Contribute to the Establishment of Meiotic Metaphase Arrest by Cytostatic Factor  Brian J Tunquist,
Condensin Is Required for Nonhistone Protein Assembly and Structural Integrity of Vertebrate Mitotic Chromosomes  Damien F. Hudson, Paola Vagnarelli,
Volume 8, Issue 1, Pages (January 2005)
Number of Nuclear Divisions in the Drosophila Blastoderm Controlled by Onset of Zygotic Transcription  Hung-wei Sung, Saskia Spangenberg, Nina Vogt, Jörg.
Nikhila S. Tanneti, Kathryn Landy, Eric F. Joyce, Kim S. McKim 
Kai Yuan, Patrick H. O’Farrell  Current Biology 
All Mouse Ventral Spinal Cord Patterning by Hedgehog Is Gli Dependent and Involves an Activator Function of Gli3  C.Brian Bai, Daniel Stephen, Alexandra.
Partner of Numb Colocalizes with Numb during Mitosis and Directs Numb Asymmetric Localization in Drosophila Neural and Muscle Progenitors  Bingwei Lu,
The Survivin-like C. elegans BIR-1 Protein Acts with the Aurora-like Kinase AIR-2 to Affect Chromosomes and the Spindle Midzone  Elizabeth K. Speliotes,
Sequential Protein Recruitment in C. elegans Centriole Formation
Flies without Centrioles
Overexpressing Centriole-Replication Proteins In Vivo Induces Centriole Overduplication and De Novo Formation  Nina Peel, Naomi R. Stevens, Renata Basto,
Drosophila fizzy-related Down-Regulates Mitotic Cyclins and Is Required for Cell Proliferation Arrest and Entry into Endocycles  Stephan J Sigrist, Christian.
Volume 26, Issue 18, Pages (September 2016)
Anterior-Posterior Gradient in Neural Stem and Daughter Cell Proliferation Governed by Spatial and Temporal Hox Control  Ignacio Monedero Cobeta, Behzad.
Jianjun Sun, Wu-Min Deng  Developmental Cell 
Spindle Pole Regulation by a Discrete Eg5-Interacting Domain in TPX2
Christopher B. O'Connell, Anne K. Warner, Yu-li Wang  Current Biology 
Volume 27, Issue 9, Pages (May 2017)
From Stem Cell to Embryo without Centrioles
Naoyuki Fuse, Kanako Hisata, Alisa L. Katzen, Fumio Matsuzaki 
Ruth Grosskortenhaus, Frank Sprenger  Developmental Cell 
Volume 105, Issue 4, Pages (May 2001)
Volume 12, Issue 4, Pages (April 2007)
Edan Foley, Frank Sprenger  Current Biology 
Differential Activation of the DNA Replication Checkpoint Contributes to Asynchrony of Cell Division in C. elegans Embryos  Michael Brauchle, Karine Baumer,
Volume 15, Issue 4, Pages (October 2008)
MIX-1: An Essential Component of the C
Axel Dienemann, Frank Sprenger  Current Biology 
Yumi Uetake, Greenfield Sluder  Current Biology 
Mark Van Doren, Anne L. Williamson, Ruth Lehmann  Current Biology 
Epicardial Spindle Orientation Controls Cell Entry into the Myocardium
Volume 15, Issue 8, Pages (April 2005)
Volume 6, Issue 4, Pages (April 2004)
The Mitotic Arrest in Response to Hypoxia and of Polar Bodies during Early Embryogenesis Requires Drosophila Mps1  Matthias G. Fischer, Sebastian Heeger,
S. Chodagam, A. Royou, W. Whitfield, R. Karess, J.W. Raff 
Joshua N. Bembenek, John G. White, Yixian Zheng  Current Biology 
Volume 3, Issue 5, Pages (November 2002)
Volume 16, Issue 17, Pages (September 2006)
Volume 19, Issue 8, Pages (April 2009)
Volume 12, Issue 10, Pages (May 2002)
Volume 9, Issue 1, Pages (January 2002)
Volume 13, Issue 3, Pages (February 2003)
A Genetic Link between Morphogenesis and Cell Division during Formation of the Ventral Furrow in Drosophila  Jörg Großhans, Eric Wieschaus  Cell  Volume.
Volume 8, Issue 9, Pages (April 1998)
Volume 24, Issue 13, Pages (July 2014)
Volume 11, Issue 20, Pages (October 2001)
Volume 22, Issue 22, Pages (November 2012)
Volume 5, Issue 2, Pages (August 2003)
Inscuteable and Staufen Mediate Asymmetric Localization and Segregation of prosperoRNA during Drosophila Neuroblast Cell Divisions  Peng Li, Xiaohang.
A Cyclin-Dependent Kinase Inhibitor, Dacapo, Is Necessary for Timely Exit from the Cell Cycle during Drosophila Embryogenesis  Joriene C de Nooij, Mary.
Apical Complex Genes Control Mitotic Spindle Geometry and Relative Size of Daughter Cells in Drosophila Neuroblast and pI Asymmetric Divisions  Yu Cai,
TAC-1, a Regulator of Microtubule Length in the C. elegans Embryo
Novel Functions for Integrins in Epithelial Morphogenesis
Frank Sprenger, Nikita Yakubovich, Patrick H. O’Farrell 
Markus Kaspar, Axel Dienemann, Christine Schulze, Frank Sprenger 
Melina Schuh, Christian F. Lehner, Stefan Heidmann  Current Biology 
Presentation transcript:

mei-41 and bub1 block mitosis at two distinct steps in response to incomplete DNA replication in Drosophila embryos  Melissa Garner, Suzanne van Kreeveld, Tin Tin Su  Current Biology  Volume 11, Issue 20, Pages 1595-1599 (October 2001) DOI: 10.1016/S0960-9822(01)00483-3

Figure 1 dupa3 mutants delay entry into M16 in a mei-41-dependent manner. Embryos were fixed and stained with PH3, an antibody to phosphorylated histone H3 [red in (a)–(f); white in (g) and (h)], to identify mitotic cells and with an antibody to α-tubulin [blue in (a)–(f)] and DNA (not shown) to reveal morphology that aid in embryo staging, as in [7]. CyO refers to dupa3/CyO heterozygotes or CyO/CyO that are wt with respect to the dup gene, identified by staining for β-galactosidase encoded by the CyO balancer chromosome. a1 indicates dupa1 homozygotes, a3 indicates dupa3 homozygotes, and mei-41 a3 indicates homozygous for dupa3 and either homozygous or hemizygous for mei-41. mei-41 mutants were identified by a lack of antibody staining for GFP encoded by the FM7 balancer chromosome. Embryos are shown with anterior to the left and dorsal side up. (a,b) In heterozygous or wt embryos, epidermal cells are in M16 in stage 11, and most have finished M16 by stage 13. (c) dupa1 mutant cells enter M16 concurrently with controls [e.g., (a)]. As previously described, arrest in M16 lasts for at least 2 hr; (d) by stage 13, the number of mitotic cells are reduced. (e) dupa3 mutant cells are delayed in entering M16 such that most are still in interphase and lack PH3 stain in stage 11 embryos. (f) By stage 13, most have entered M16 and are arrested there. (g) The delay in entry into M16 in dupa3 single mutants [e.g., (e)] is abolished in dupa3mei-41 double mutants. (h) Most cells have escaped the arrest by stage 13, similar to dupa1 mutants seen in (d). dup and mei-41D3 mutants and antibodies to PH3 (Upstate Biotechnologies), β-galactosidase (Chemicon International), and α-tubulin (Sigma) have been described before [1, 8, 11]. A rabbit polyclonal antibody to GFP was a gift from J. Kahana and P. Silver. Detailed methods and stock information are in Supplementary materials Current Biology 2001 11, 1595-1599DOI: (10.1016/S0960-9822(01)00483-3)

Figure 2 S phase in dup mutants as revealed by incorporation of BrdU. Genotype denotations are as in Figure 1. Embryos were pulsed with BrdU for 13 min and fixed, and incorporation was detected by immunostaining as described before [21] [red in (a) and (b) and white in (c)–(h)]. Embryos were also stained for (a,b) β-galactosidase and for (i–k) DNA. Detailed methods are in Supplementary materials. Amnioserosa in each case is enclosed by broken lines. At these stages, the dorsal epidermis lies adjacent to the amnioserosa on both sides, followed by the ventral epidermis. The scale bar equals 32 μm in (c)–(k). (a,b) Whole embryos illustrate β-galactosidase stain [cyan stripes present in (a) and absent in (b)]. Brackets depict an area similar to those magnified and shown in (c)–(h). (c–e) In late stage 10/early stage 11 embryos, cells of the dorsal epidermis initiate cycle 16 while those in the ventral epidermis are still in cycle 15 (all staging as in [7]). dup mutants complete cycle 15 [1]. Therefore, ventral epidermis (vEpi) incorporates BrdU in all genotypes and serves as a positive control. In these embryos, dorsal epidermal cells (dEpi) of heterozygous or wt embryos are in S16 and (c) show robust BrdU signal. This signal (d) is diminished in the corresponding cells of dupa3 mutants and (e) is absent in the corresponding cells of dupa1 mutants. (f) In stage 11 embryos that are presumably older, ventral epidermis is finishing S16 in controls. (g,h) At similar stages, little or no BrdU incorporation is detected throughout dupa1 and dupa3 mutant embryos. Thus, dupa1 mutants forgo S16 as reported previously [1], whereas dupa3 mutants initiate but do not complete S16 (summarized in Figure 4f). (i), (j), and (k) show DNA signal corresponding to (f), (g), and (h), respectively Current Biology 2001 11, 1595-1599DOI: (10.1016/S0960-9822(01)00483-3)

Figure 3 Mitosis 16 in dup mutants. Embryos were fixed and stained with various antibodies and for DNA: in (a)–(c) and red in (d)–(f) for PH3, cyan in (d)–(f) for α-tubulin, cyan in (g)–(i) for Bub1, cyan in (j) for Cyclin A, cyan in (k) for Cyclin B, and red in (g)–(i) for DNA. Genotype denotations are as in Figure 1. Antibodies to cyclins have been described before [22]. A polyclonal anti-Drosophila Bub1 antibody was a gift from C. Sunkel. Detailed methods are in Supplementary materials. The scale bar equals 11 μm in (a)–(j). (a), (b), and (c) show PH3 stain corresponding to (d), (e), and (f), respectively. Chromosomes compact into metaphase plates midway between spindle poles in controls [arrow in (a) and (d); magnified 2× in insets). In dup mutants (b,c,e,f), chromosomes are spread along the spindle axis, often as far as the spindle poles (e.g., arrows; magnified 2× in insets). (g–i) Bub1 (cyan, may be white when present on red background) is present on chromosomes (red) in a fraction of metaphase cells in heterozygotes or wt, consistent with previous data [16]. One of three metaphase figures in (g) show Bub1 staining and is circled. In dup mutants, Bub1 signal is found on chromosomes of most mitotic cells (h,i; those with Bub1 foci clearly in this plane of focus are circled). (j) Two embryos indicated by arrowheads are of similar age (stage 13). In the control (CyO), most epidermal cells have exited M16 and lack Cyclin A, while CNS and PNS cells that remain proliferative retain this protein. Cyclin A persists throughtout dupa1 mutant embryos (a1), although the staining intensity varies from cell to cell (e.g., Figure S1 in Supplementary material). Similar data were obtained with dupa3 mutants (Figure S1 in Supplementary materials). (k) Two embryos indicated by arrowheads are of similar age (stage 14); Cyclin B levels are higher in the dupa3 mutant. Similar data were obtained for dupa1 mutants (Figure S1 in Supplementary materials) Current Biology 2001 11, 1595-1599DOI: (10.1016/S0960-9822(01)00483-3)

Figure 4 dupa3bub1 double mutants are unable to sustain arrest in M16. Embryos were fixed and stained for [(a,b), red in (c,d), cyan in (e)] PH3, [cyan in (c,d)] α-tubulin, and [red in (e)] DNA. a3 bub1 are double homozygotes of dupa3bub1. Other genotype denotations are as in legend to Figure 1. bub1K03113 mutants have been described before [16]. Detailed methods and stock information are in Supplementary materials. The scale bar equals 32 μm in (c)–(e). (a,b) dupa3bub1 double mutants have fewer mitotic cells than dupa3 mutants. Stage 13 embryos are shown with dorsal up and anterior to the left. The dorsal side of the embryo in (a) is tilted toward the viewer; consequently, amnioserosa (region without mitosis in the middle of the embryo) appears larger in (a) than in (b). (c–e) dupa3bub1 mutants show more telophase cells [brackets in (d)] than dupa3 mutants (c). Epidermal cells of stage 12 embryos are shown. In dupa3 mutants in embryonic stages 12 and 13, the ratio of telophase cells to cells arrested with bipolar spindles is 0.03 (±0.01) to 1; for every 33 cells arrested in M16, one was found to be exiting mitosis. The corresponding ratio is 0.35 (±0.28) to 1 in dupa3bub1 double mutants. The variation in this number is likely due to the fact that anaphase and telophase are transient states, and the fraction of cells caught in these states at the time of fixing may vary from embryo to embryo. (e) Separating nuclei in dupa3bub1 double mutants show chromosome bridges (arrows; magnified 2× in inset), which retain PH3, consistent with incomplete DNA replication. (f) A summary of dup phenotypes. Bars represent the predicted length of Dup protein encoded by each allele. (g) A model for regulation of mitosis in the presence of incompletely replicated DNA, such as in dupa3 mutants. mei-41 and bub1 inhibit the entry into and the exit from mitosis, respectively. The inhibition, although depicted as a single step, is likely to be indirect and to involve many steps and molecules. The inhibition of mitotic exit is seen also in dupa1 mutants. Therefore, this regulation may also occur when cells fail to initiate DNA replication, as well as when cells fail to complete DNA replication. We caution, however, that dupa1 mutants may synthesize a small amount of DNA that is below the level of detection in this study Current Biology 2001 11, 1595-1599DOI: (10.1016/S0960-9822(01)00483-3)