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 minutes2
. While mitotic chromosomes are thought to generate gradients of RanGTP
that self-organize the prometaphase spindle3
, this cannot be the only self-organizing signal in anaphase because cytokinesis can occur in the absence of chromatin4,5
. Instead, the location of the cleavage furrow is coupled to the position of the spindle midzone where the Chromosome Passenger Complex (CPC) containing Aurora B kinase is localized. How signals are transmitted over micron length-scales between midzone microtubules and the cell cortex is unknown.
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 design6
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 family7
(). To mimic localizations of endogenous Aurora B substrates8
, sensors were targeted to centromeres (CENP-B fusion), to chromatin (histone H2B fusion), or to cytosol (lacking targeting sequences) (Fig. S1a
). To examine the sensor response to changes in Aurora B activity in living cells, we first imaged mitotic cells before and after kinase inhibition. Second, we imaged cells through anaphase, when endogenous Aurora B substrates are dephosphorylated9
. For each sensor the YFP/CFP emission ratio increased both after inhibitor treatment and in anaphase, consistent with dephosphorylation for this sensor design6
. The maximal increase in emission ratio following chemical inhibition is similar to the increase during anaphase for each sensor (Fig. S1b
), indicating that the measured FRET changes correspond to full dephosphorylation of the sensor. To test specificity for Aurora B, the cytosolic sensor was treated with a Polo-like kinase (Plk) inhibitor, which did not cause an increase in the emission ratio (Fig. S2a
). In addition, the cytosolic sensor was not phosphorylated in mitotic cells after Aurora B depletion by RNAi (Fig. S2b
). Together, these data validate the sensors as reporters of Aurora B activity.
A FRET-based sensor of Aurora B kinase activity demonstrates a spatial phosphorylation gradient during anaphase
To map Aurora B kinase activity during anaphase, we examined the kinetics of changes in sensor phosphorylation at different sites. Dephosphorylation of all three Aurora B sensors begins immediately after sister chromosome separation and is complete within 8 min for the centromere and chromatin targeted sensors, compared to 30 min for the cytosolic sensor (). This analysis indicates that dephosphorylation kinetics of Aurora B substrates in anaphase depend on substrate localization. Mutation of the substrate threonine to alanine, using the chromosomal sensor, eliminated the change in emission ratio (Fig. S3
The rapid dephosphorylation kinetics of the chromosome-targeted sensors are remarkably similar to the kinetics of chromosome segregation, suggesting that phosphorylation changes may be linked to chromosome position during anaphase. To test this possibility we calculated both the YFP/CFP emission ratio at each centromere () and its position along the division axis in cells expressing the centromere-targeted sensor. Analysis of single time points early in anaphase, when variance in centromere position is maximal, consistently revealed a correlation between position and sensor phosphorylation (Fig. S4a
, Table S1
). These results indicate that while dephosphorylation occurs at all centromeres over time, phosphorylation differences between individual centromeres depend on centromere position.
To determine the length scale over which position influences sensor phosphorylation, we depleted Mad2 by RNAi to inhibit the spindle checkpoint and increase the variance in centromere positions during anaphase. The sensor is dephosphorylated within 8 minutes of anaphase onset on centromeres that segregate normally in Mad2-depleted cells, but remains phosphorylated for up to 10 minutes on centromeres that remain in the center (). Quantitative analyses demonstrate that changes in sensor phosphorylation depend primarily on centromere position along the division axis, over ~6 µm distance from the center rather than on time (, Fig. S4, Table S1
We next examined the chromatin-targeted and cytoplasmic sensors. Spatial phosphorylation patterns were not detected using the cytoplasmic sensor, possibly because rapid diffusion of cytosolic proteins may degrade any spatial patterns so that they are not detected by our methods. The chromatin-targeted sensor revealed a clear phosphorylation gradient. Early in anaphase sensor phosphorylation is highest on chromatin near the spindle midzone and lowest near the spindle poles (). Chromosomes segregated normally in these experiments, indicating that microtubule attachments are not perturbed. As the phosphorylation gradient is not restricted to a few individual chromosomes, it is unlikely to reflect differences in chromosome-spindle attachments. A Plk sensor did not reveal spatial phosphorylation patterns in anaphase (Fig. S5
), which indicates that the phosphorylation gradient is specific for Aurora B substrates.
The anaphase phosphorylation gradient is observed for multiple Aurora B substrates
We next examined phosphorylation of endogenous Aurora B substrates by immunofluorescence, using phospho-specific antibodies. First we analyzed histone H3 Ser-10 (H3(S10)) phosphorylation, which was highest towards the spindle midzone and lower towards the poles (). H3(S10) phosphorylation increased 1.5–2.6 fold from pole to midzone in 78% of anaphase cells (60–120 cells per experiment, n=6). This anaphase H3(S10) phosphorylation gradient was verified in multiple cell types and using a second phospho-specific antibody (Fig. S6a–d
). A similar result was reported in drosophila syncitial embryos10
. Second, we analyzed another Aurora B substrate, MCAK Ser-1967
. During anaphase MCAK localizes throughout the cell with highest concentrations at the spindle poles, while phospho-MCAK(S196) appears highest in the spindle midzone (). Together, these data demonstrate phosphorylation gradients for endogenous and exogenous (FRET sensor) Aurora B substrates on chromosomes or the cytoskeleton during anaphase.
To determine whether Aurora B localization contributes to formation of the phosphorylation gradient, we used three different perturbations. First, brief (8 min) nocodazole treatment led to microtubule disassembly, spindle midzone disorganization, and dispersion of Aurora B throughout the cytoplasm11
). We observed loss of the normal H3(S10) phosphorylation gradient in 76% of nocodazole-treated HeLa cells (n=110) (; S8d
). Slight increases in H3(S10) phosphorylation were sometimes apparent on chromatin near the spindle midzone, most likely reflecting incomplete microtubule disruption (Fig. S7c
). Second, we depleted the kinesin MKLP-2 with shRNAi (Fig. S8b–c
), leading to loss of midzone localization of Aurora B in 63% of anaphase cells (n=27)12
and absence of the H3(S10) phosphorylation gradient in 70% of these cells (). Third, following expression of non-degradable cyclin B, Aurora B remained on chromosome arms in anaphase11,13
, and the H3(S10) phosphorylation gradient was disrupted (Fig. S7e
). Together, these findings indicate that the anaphase phosphorylation gradient depends on Aurora B localization to the spindle midzone.
The phosphorylation gradient requires Aurora B localization to the midzone
We next addressed how a gradient might be established. One of the best examples occurs during development, when morphogen gradients are produced by self-organizing systems that require localization of an activator and positive feedback14
. To determine where Aurora B is activated during anaphase, we analyzed INCENP Ser-850 phosphorylation in Xenopus
cells (Table S2
) and Aurora B Thr-232 phosphorylation in HeLa cells. Both modifications are associated with full Aurora B activation15,16
. Using phospho-specific antibodies, we find that both INCENP(S850) and Aurora B(T232) phosphorylation are limited to the spindle midzone (, Fig. S6d
), indicating that Aurora B activation is restricted to this site. Brief (8 min) treatment with an Aurora B inhibitor, Hesperadin17
, led to disruption of midzone microtubule organization (, Fig. S9a
) and reduction of total phospho-INCENP(S850) staining by 88% (, Table S3
). Loss of phospho-INCENP(S850) is not caused by a decrease in INCENP protein, as Hesperadin treatment increased total INCENP staining during anaphase by over 70% (, Fig. S9a, S9d–e
). Together, these data suggest that Aurora B must be continuously activated during anaphase, and that active kinase localizes to the spindle midzone.
To test the possibility that Aurora B activation depends on microtubule association in anaphase, INCENP(S850) phosphorylation was examined following nocodazole treatment, which led to 85% reduction in phospho-INCENP(S850) (). Brief nocodazole treatment did not depolymerize all microtubules, and residual phospho-INCENP(S850) was confined to the remaining midzone microtubules. Nocodazole treatment also reduced anaphase H3(S10) phosphorylation by approximately 50% (Table S4
). Microtubules can directly stimulate Aurora B kinase activity in vitro
), consistent with previous results18
. To determine if Aurora B directly contacts microtubules during anaphase, we performed a proximity ligation in situ assay19
(P-LISA). The P-LISA product was detected primarily within the spindle midzone, consistent with a direct interaction between midzone microtubules and Aurora B (). This signal co-localized with both markers of Aurora B activation, phospho-INCENP(S850) and phospho-Aurora B(T232), but not the bulk of tubulin (Fig. S10c–d
). Together, these data indicate that Aurora kinase activity at the spindle midzone is continuously maintained through local interactions with microtubules.
Formation of a phosphorylation gradient centered at the spindle midzone suggests a mechanism to communicate the position of the midzone to the cortex. Although inhibition of Aurora B or of midzone components such as MKLP-2 perturbs cytokinesis, it is difficult to separate the function of the gradient from other functions of these proteins. To test whether the gradient may provide spatial information to position the cleavage furrow, we changed the shape of the gradient by perturbing the spatial organization of the anaphase spindle. In the presence of a kinesin-5 inhibitor, spindles are monopolar but anaphase still occurs if the spindle checkpoint is inhibited. Chromosomes are pulled to one side of the cell, followed by microtubule stabilization and cell cleavage on the opposite side20
. This assay introduces a dramatic spatial change without directly inhibiting Aurora B or other midzone or furrow components. We observe a phosphorylation gradient within 1.5 minutes (±0.5 sem, N=6) of chromosome movement in monopolar anaphase. The gradient is oriented with maximal phosphorylation opposite the direction of chromosome movement (N=9 out of 12 cells examined) (). This result demonstrates that gradient formation is robust to changes in spindle geometry. Although we do not always observe a cleavage furrow in monopolar anaphase, furrows that do form are positioned in the direction of maximal phosphorylation in the gradient (Fig. S11
). We also find that Aurora B disappears from centromeres in a monopolar anaphase and subsequently redistributes to the cortex where the cleavage furrow forms (), beginning 3.1 min (±0.2 sem, N=5) after chromosome movement. As the gradient precedes both cortical Aurora B localization and furrow ingression, these data suggest that the anaphase phosphorylation gradient provides spatial information to position the cleavage furrow.
The phosphorylation gradient in a monopolar anaphase predicts the cleavage site
Formation of the cleavage furrow depends on signals from the spindle midzone, but how the midzone is initially established in unknown. We propose that release of active Aurora B from centromeres establishes a phosphorylation gradient early in anaphase (), so that substrates known to regulate microtubule organization1
are preferentially phosphorylated at the center of the anaphase spindle. The phosphorylation gradient is maintained through a positive feedback loop in which Aurora B activity organizes midzone microtubules, and the midzone catalyzes local Aurora B auto-phosphorylation of its own regulatory sites. Active Aurora B diffuses away from the midzone where it is inactivated by cytosolic phosphatases (). Many Aurora B substrates are localized to structures (chromosomes or the cytoskeleton) that would limit diffusion and maintain gradient information. While we favor this model, we cannot exclude alternatives, for example involving spatial patterns of phosphatase activity.
We have shown that perturbations that block cytokinesis (nocodazole21
, MKLP-2 siRNA22
, non-degradable cyclinB13
) also inhibit gradient formation (; Fig. S7c–e,g
; Fig. S8d
). Moreover, the relationship between gradient direction and furrow location persists in monopolar anaphase. We propose that the anaphase phosphorylation gradient, which extends over micron length-scales, provides a signaling mechanism to communicate the location and orientation of the spindle midzone to the cell cortex to position the cleavage furrow. Our molecular dissection has uncovered the underlying regulatory basis for an anaphase phosphorylation gradient, and our quantitative analysis of phosphorylation dynamics will lend itself to future mathematical modeling of spatial patterning in anaphase.