Probing microtubule polymerisation state at single kinetochores during metaphase chromosome motion

Abstract

Kinetochores regulate the dynamics of attached microtubule bundles (kinetochore-fibres, K-fibres) to generate the forces necessary for chromosome movements in mitosis. Current models suggest that poleward-moving kinetochores are attached to depolymerising K-fibres and anti-poleward-moving kinetochores to polymerising K-fibres. How the dynamics of individual microtubules within the K-fibre relate to poleward and anti-poleward movements is poorly understood. To investigate this, we developed a live-cell imaging assay combined with computational image analysis that allows eGFP-tagged EB3 (also known as MAPRE3) to be quantified at thousands of individual metaphase kinetochores as they undergo poleward and anti-poleward motion. Surprisingly, we found that K-fibres are incoherent, containing both polymerising and depolymerising microtubules – with a small polymerisation bias for anti-poleward-moving kinetochores. K-fibres also display bursts of EB3 intensity, predominantly on anti-poleward-moving kinetochores, equivalent to more coherent polymerisation, and this was associated with more regular oscillations. The frequency of bursts and the polymerisation bias decreased upon loss of kinesin-13, whereas loss of kinesin-8 elevated polymerisation bias. Thus, kinetochores actively set the balance of microtubule polymerisation dynamics in the K-fibre while remaining largely robust to fluctuations in microtubule polymerisation.

Building an integrated model of chromosome congression

“…However, recent live-cell imaging in human cells (Armond et al, 2015) and electron microscopy studies (VandenBeldt et al, 2006) has demonstrated that both P and AP K-fibres are incoherent, i.e. contain both polymerising and depolymerising microtubules, with a small polymerisation bias towards the AP K-fibre (Armond et al, 2015) (Fig. 2B).…”

“…Experiments using fluorescent EB1 (also known as MAPRE1, a microtubule polymerisation marker) provided evidence that this bias is indeed strong, with high levels of polymerisation at the AP kinetochore relative to the P kinetochore (Tirnauer et al, 2002). However, recent live-cell imaging in human cells (Armond et al, 2015) and electron microscopy studies (VandenBeldt et al, 2006) has demonstrated that both P and AP K-fibres are incoherent, i.e. contain both polymerising and depolymerising microtubules, with a small polymerisation bias towards the AP K-fibre (Armond et al, 2015) (Fig.…”

“…As this could lead to the entire fibre switching to depolymerisation, the kinetochore must tightly control microtubule dynamics to maintain the observed incoherent state. In this regard, we know of several plus-end-tracking proteins and molecular motors that function as regulators of kinetochore-microtubule dynamics (Cross and McAinsh, 2014;Ferreira et al, 2014), but so far only the kinesin-8 Kif18A and kinesin-13 mitotic centromere-associated kinesin (MCAK, also known as Kif2C) have been shown to directly affect the balance of microtubule dynamics within the K-fibre (Armond et al, 2015). Kif18A is present in a comet-like gradient along kinetochore microtubules that is concentrated at the outer plate and proposed to be dependent on K-fibre length (Stumpff et al, 2008(Stumpff et al, , 2012.…”

“…Kinetochore-tracking experiments have shown that Kif18A depletion causes an increase in metaphase sister-kinetochore oscillation velocity (Jaqaman et al, 2010;Stumpff et al, 2011Stumpff et al, , 2008Stumpff et al, , 2012, a decrease in switching frequency (Stumpff et al 2008; although that observation is disputed, Jaqaman 2010), and loss of spatially controlled directional switching (Stumpff et al, 2012). These aberrant chromosome movements might reflect the reported increase in the microtubule polymerisation bias between AP and P kinetochores (Armond et al, 2015), a finding that is consistent with the idea that Kif18A normally acts to suppress microtubule dynamics (at the AP kinetochore) and stimulate directional switching in vivo (Du et al, 2010;Stumpff et al, 2008Stumpff et al, , 2012.…”

Measuring NDC80 binding reveals the molecular basis of tension-dependent kinetochore-microtubule attachments

“…The preferential binding is statistically significant (p<0.02, two-sided Z-test), 1 yet small, presumably because leading and trailing kinetochores have a mixture of both polymerizing and 2 depolymerizing MTs (Armond et al, 2015). This differential binding of NDC80 provides an explanation 3 for the higher detachment rate of depolymerizing microtubules from kinetochores in vitro (Akiyoshi et al, 4 2010), and may give insight into the nature of kMT attachments (Dumont et al, 2012).…”