Desempeño de la Probabilidad de Error

1.5.1 Lateral sliding

The formation a bi-oriented attachment was originally thought to be an essential prerequisite for congression. On the contrary, congression can also occur before bi-orientation, via the motor dependent sliding of

kinetochores along the microtubule sidewall (Fig 5). Chromosomes that are located in the spindle periphery following NEB initially migrate to their proximal pole prior to ejection towards the spindle equator. This movement is driven by the kinetochore-bound dynein, a minus-end directed motor that steps towards the pole (Barisic et al., 2014; Li et al., 2007; Savoian et al., 2000; Yang et al., 2007) (Fig. 5, Step 1). Subsequent equatorially directed motion is driven CENP-E, a high processive plus-end directed motor that is enriched at polar chromosomes (Gudimchuk et al., 2013; Kapoor et al., 2006; Kim et al., 2008; Wood et al., 1997) (Fig. 5, Steps 2 and 3). The lateral sliding of chromosomes was first demonstrated in a study that used single kinetochore tracking and correlative EM to analyse the behaviour and attachment state of congressing sister-pairs in PtK1 cells. Here, kinetochore-pairs were observed to glide toward the spindle equator

Slide: CENP-E Slide: Dynein (HSET?) spindle _equator -

3

4

1

5

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F

Away-from-pole (AP) kinetochore

kinetochore movement direction + + + ATP hydrolysis

2

Figure 5: Congression of laterall attached kinetochores

Congression can take place prior to bi-orientation, mediated by the motor proteins dynein and CENP-E. These motors form lateral attachments with the microtubule lattice, and walk along the microtubule toward the minus-end (dynein, steps 1 and 2) or plus-end (CENP-E, steps 3 and 4). HSET has also been implicated in minusend- directed polewards movement, however the mechanism remains unknown. These migratory events contribute to distinct steps of congression, with chromosomes first being moved polewards by dynein (steps 1 and 2), before being ejected towards the equator by CENP-E (steps 3 and 4). Motor driven congression often fails to maintain chromosomes at the spindle equator, and as such, these lateral attachments are converted into the default end-on attachment state during the formation of the

alongside the mature K-fibre of an aligned bi-oriented chromosome, a movement that was facilitated by a force-generating lateral interaction between the P-kinetochore and K-fibre (Kapoor et al., 2006) (Fig. 5, Chr 2). This behaviour was abolished by CENP-E depletion, establishing that CENP-E drives a bi-orientation independent congression mechanism (Kapoor et al., 2006). Further validation of this model came from studies that specifically enriched for the lateral sliding pathway. Here, DCP was prevented by abrogating end-on attachment with depletion of the Ndc80 complex component Nuf2 (Cai et al., 2009). Remarkably, CENP-E

mediated lateral sliding could align all chromosomes in 50% of cells when the minus-end directed motor HSET was also depleted (Cai et al., 2009). Similar observations were made when the major end-on attachment factor Ndc80 was codepleted with MCAK (Iemura and Tanaka, 2015).

Interestingly, these studies highlighted that MCAK and HSET depletion could suppress an unknown P force, and allow for CENP-E mediated congression in the absence of end-on attachment, but how? The current idea is that this effect is indirect, as HSET and MCAK both negatively regulate the stability of microtubules required for CENP-E sliding toward the equator (Iemura and Tanaka, 2015).

Somewhat unexpectedly, CENP-E has been implicated in the DCP mechanism in vivo. In an experiment designed to simulate DCP, where end-on attached sisters are transported by depolymerising astral

inhibition was found to perturb microtubule tracking (Gudimchuk et al., 2013). Moreover, CENP-E was shown to track with polymerising and depolymerising microtubules in vitro, and can transduce the force from filament disassembly to beads (Gudimchuk et al., 2013). Together, these data provided compelling evidence that the motor contributes to DCP (Fig 3c). However, 80% of bi-oriented kinetochore pairs in a bipolar spindle successfully congressed when depleted of CENP-E (Bancroft et al., 2015), and CENP-E is not required for the oscillation of aligned sister-pairs

(Jaqaman et al., 2010). Nevertheless, microtubule dynamics at aligned kinetochore-pairs are perturbed when CENP-E is inhibited or depleted (Maffini et al., 2009). It must be noted that CENP-E is significantly reduced at aligned sisters when compared to polar kinetochores. As such, the primary function of CENP-E appears to be as a lateral sliding mediator that congresses peripheral chromosomes (Barisic et al., 2014).

1.5.2 The polar ejection force

The existence of a polar ejection force (PEF) was proposed by Darlington in 1937. This idea formed the basis of his ‘polar repulsion’ model of

congression (Darlington, 1937), which stated that centromeres are repelled from the poles with a force inversely proportional to their proximity

(Darlington, 1937). While failing to account for congression (Levesque and Compton, 2001), the existence of the PEF was later demonstrated by laser

cutting experiments (Rieder et al., 1986). Here, an acentric chromosome fragment was shown to be actively transported away from its proximal pole (Rieder et al., 1986). This behaviour was abolished by the addition of nocodazole, suggesting that microtubules were critical for PEF generation (Ault et al., 1991). Importantly, these studies also showed that the PEF was exerted along chromosomes arms, as oppose to the centromere as

originally predicted (Darlington, 1937). Consistent with this, both in vivo and in vitro suites later showed that plus-end directed chromosome arm

associated kinesins (chromokinesins) Kif4a and Kid create the PEF (Antonio et al., 2000; Bieling et al., 2010; Brouhard and Hunt, 2005; Funabiki and Murray, 2000; Levesque and Compton, 2001; Yajima et al., 2003). Thus, the PEF is an AP force generated by the plus-end directed walking of chromatin-tethered kinesin along microtubules. As microtubule concentration is proportional to proximity to the pole, the strength of the PEF is inversely proportional to distance from the pole (Cane et al., 2013). In terms of congression, the individual depletion of Kif4a or Kid has little effect. In contrast, their co-depletion results in significant alignment defects, suggesting these motors may have independent and/or coordinated

functions (Wandke et al., 2012). In this regard, Kid, the principal PEF generator, is dispensable for chromosome alignment, but has a role in the control of aligned kinetochore dynamics and kinetochore position sensing (Levesque and Compton, 2001; Stumpff et al., 2012; Wandke et al., 2012). With respect to the former, Kid depletion decreases the velocity of, and

increases the rate of directional switching at, aligned kinetochore-pairs. In contrast, the function of Kif4a is less well characterised. It has been suggested to antagonise Kid by reducing microtubule dynamicity in early mitosis via the suppression of polymerisation, a finding that suggests it may have a PEF independent function (Wandke et al., 2012). Indeed, this may involve the regulation of antiparallel microtubule overlaps, a well

documented function of Kif4a in cytokinesis (Nunes Bastos et al., 2013). Nevertheless, several studies have implicated Kif4a and kinesin-4 motors in PEF generation, although more work is necessary to tease out the specific contribution (Antonio et al., 2000; Bieling et al., 2010; Brouhard and Hunt, 2005; Funabiki and Murray, 2000; Levesque and Compton, 2001; Yajima et al., 2003).