Exit from mitosis in<i>Drosophila</i> syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation

Proper partitioning of the contents of a cell between two daughters requires integration of spatial and temporal cues. The anaphase array of microtubules that self-organize at the spindle midzone contributes to positioning the cell division plane midway between the segregating chromosomes 1 . How this signaling occurs over micron length-scales, from the midzone to the cell cortex, is not known. Here we examine the anaphase dynamics of protein phosphorylation by Aurora B kinase, a key mitotic regulator, using FRET-based sensors in living cells and immunofluorescence of native Aurora B substrates. Quantitative analysis of phosphorylation dynamics, using chromosome and centromere targeted sensors, reveals that changes are due primarily to position along the division axis rather than time. These dynamics result in the formation of a spatial phosphorylation gradient early in anaphase that is centered at the spindle midzone. This gradient depends on Aurora B targeting to a subpopulation of microtubules that activate it. Aurora kinase activity organizes the targeted microtubules to generate a structure based feedback loop. We propose that feedback between Aurora B kinase activation and midzone microtubules generates a gradient of posttranslational marks that provides spatial information for events in anaphase and cytokinesis. Keywordsanaphase; gradient; FRET; Aurora B; INCENP; histone H3; serine 10; mitotic spindle; Hesperadin; monopolar It is believed that self-organizing systems position the cleavage furrow, since experimental displacement of the anaphase spindle results in repositioning of the cleavage furrow within minutes 2 . While mitotic chromosomes are thought to generate gradients of Ran GTP that selforganize the prometaphase spindle 3 , this cannot be the only self-organizing signal in anaphase because cytokinesis can occur in the absence of chromatin 4,5 . Instead, the location of the cleavage furrow is coupled to the position of the spindle midzone where the Chromosome *Correspondence to: PTS (e-mail pts7h@virginia.edu) or MAL (e-mail lampson@sas.upenn.edu). $ Both authors contributed equally to this work Author Information: Development of the Aurora B and Plk phosphorylation sensors and FRET imaging and analysis were done in the Kapoor lab by MA Lampson, together with EA Foley. BG Fuller performed immunofluorescence experiments. SR Nitcher and P Tobelman performed the kinase assays and P-Lisa experiments, respectively. KV Le performed live imaging of Aurora B-GFP. BG Fuller and MA Lampson wrote the paper. To examine spatial patterns of Aurora B signaling during anaphase, we developed a strategy using FRET-based sensors that report quantitative changes in substrate phosphorylation in living cells. We adapted a sensor design 6 in which changes in intramolecular CFP-YFP FRET depend on changes in phosphorylation of an Aurora B substrate peptide, which is conserved among members of the kinesin-13 family 7 (Fig. 1a). To mimic localizations of endogenous Aurora B substrates 8 , sensors were targeted to ce...

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“…S6a-d). A similar result was reported in drosophila syncitial embryos 10 . Second, we analyzed another Aurora B substrate, MCAK Ser-196 7 .…”

supporting

confidence: 76%

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient

Fuller

Lampson

Foley

et al. 2008

Nature

336

380

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“…The activity of APC-Cdc20 appears to be regulated in a partially cell cycle-dependent manner (Su et al, 1998). In the present study, HSF1 was found to interact directly with Cdc20, and this interaction appeared to be responsible for aneuploidy production by HSF1, because transfection of a regulatory domain-deficient HSF1 mutant, which could not bind to Cdc20, also failed to produce aneuploidy in RIF cells (Figure 4).…”

Section: Discussionsupporting

confidence: 50%

A novel function for HSF1-induced mitotic exit failure and genomic instability through direct interaction between HSF1 and Cdc20

Lee

et al. 2007

Oncogene

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Although heat-shock factor (HSF) 1 is a known transcriptional factor of heat-shock proteins, other pathways like production of aneuploidy and increased protein stability of cyclin B1 have been proposed. In the present study, the regulatory domain of HSF1 (amino-acid sequence 212-380) was found to interact directly with the amino-acid sequence 106-171 of Cdc20. The association between HSF1 and Cdc20 inhibited the interaction between Cdc27 and Cdc20, the phosphorylation of Cdc27 and the ubiquitination activity of anaphase-promoting complex (APC). The overexpression of HSF1 inhibited mitotic exit and the degradations of cyclin B1 and securin, which resulted in production of aneuploidy and multinucleated cells, but regulatory domain-deficient HSF1 did not. Moreover, HSF1-overexpressing cells showed elevated levels of micronuclei and genomic alteration. The depletion of HSF1 from cells highly expressing HSF1 reduced nocodazole-mediated aneuploidy in cells. These findings suggest a novel function of HSF1 frequently overexpressed in cancer cells, to inhibit APC/C activity by interacting with Cdc20, and to result in aneuploidy development and genomic instability.

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“…Characteristically, these early cycles have only S and M phases (Murray and Hunt 1993). Both biochemical and morphological studies have documented that sequential activation and inactivation of Cdk1-CycB play a major role in regulating these biphasic cycles (Edgar et al 1994(Edgar et al , 1995Su et al 1998;Ji et al 2004).…”

Section: E Mbryonic Development Except In Mammals Ismentioning

confidence: 99%

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

Crest

Oxnard

et al. 2007

Genetics

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The Drosophila embryo is a promising model for isolating gene products that coordinate S phase and mitosis. We have reported before that increasing maternal Cyclin B dosage to up to six copies (six cycB) increases Cdk1-Cyclin B (CycB) levels and activity in the embryo, delays nuclear migration at cycle 10, and produces abnormal nuclei at cycle 14. Here we show that the level of CycB in the embryo inversely correlates with the ability to lengthen interphase as the embryo transits from preblastoderm to blastoderm stages and defines the onset of a checkpoint that regulates mitosis when DNA replication is blocked with aphidicolin. A screen for modifiers of the six cycB phenotypes identified 10 new suppressor deficiencies. In addition, heterozygote dRPA2 (a DNA replication gene) mutants suppressed only the abnormal nuclear phenotype at cycle 14. Reduction of dRPA2 also restored interphase duration and checkpoint efficacy to control levels. We propose that lowered dRPA2 levels activate Grp/Chk1 to counteract excess Cdk1-CycB activity and restore interphase duration and the ability to block mitosis in response to aphidicolin. Our results suggest an antagonistic interaction between DNA replication checkpoint activation and Cdk1-CycB activity during the transition from preblastoderm to blastoderm cycles.

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Exit from mitosis inDrosophila syncytial embryos requires proteolysis and cyclin degradation, and is associated with localized dephosphorylation

Cited by 142 publications

References 44 publications

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient

Midzone activation of aurora B in anaphase produces an intracellular phosphorylation gradient

A novel function for HSF1-induced mitotic exit failure and genomic instability through direct interaction between HSF1 and Cdc20

Onset of the DNA Replication Checkpoint in the Early Drosophila Embryo

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