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

Abstract

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...

Mps1 promotes rapid centromere accumulation of Aurora B

“…U2OS cells were transfected with a pIRESpuro2b vector containing H2B-targeted FRET sensor for Aurora B activity [24] and selected with 1 mg/ml puromycin (Sigma-Aldrich) for stable expression. HeLa and U2OS cells were grown in DMEM supplemented with 6% fetal bovine serum, pen/strep and l-glutamine (2 mM).…”

Condensin Regulates the Stiffness of Vertebrate Centromeres

“…Thus, the reduced stiffness of centromeric chromatin in condensindepleted cells could create a gradient of tension: lowest near the surface of the centromere and progressively higher as the kinetochore is stretched further poleward. Because Aurora B activity is high on the chromosomes (Fuller et al, 2008), regions of lowered tension might be expected to favor kMT detachment and spindle checkpoint activation (King and Nicklas, 2000). Indeed, kinetochores undergoing excursions might be predicted to have particularly stable MT attachments, because they are located furthest from the Aurora B in the inner centromere (Cimini et al, 2006).…”

Dynein prevents erroneous kinetochore-microtubule attachments in mitosis

“…[36][37][38] Once chromosomes become bi-oriented and tension is applied between sister kinetochores, Ndc80 moves away from Aurora B, resulting in kinetochoremicrotubule attachment stabilization. 39,40 Here we elaborate on recent studies that demonstrated a role of Dynein, 5 chromokinesins 5,41 and Aurora A kinase 5,41,42 in the prevention and correction of erroneous kinetochore-microtubule attachments during chromosome congression.…”