Spindle scaling is governed by cell boundary regulation of microtubule nucleation

Rieckhoff, Elisa Maria; Berndt, Frederic; Golfier, Stefan; Decker, Franziska; Elsner, Maria; Ishihara, Keisuke; Brugués, Jan

doi:10.1101/2020.06.15.136937

Cited by 8 publications

(8 citation statements)

References 64 publications

(92 reference statements)

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“…, 2013 ; Rieckhoff et al. , 2019 , 2020 ). Second, while most MAPs that slow down depolymerization have been reported to accelerate polymerization in minimal conditions ( Drechsel et al.…”

Section: Discussionmentioning

confidence: 99%

Spatial variation of microtubule depolymerization in large asters

Ishihara

Decker

Caldas

et al. 2021

MBoC

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Microtubule plus end depolymerization rate is a potentially important target of physiological regulation, but it has been challenging to measure, so its role in spatial organization is poorly understood. Here we apply a method for tracking plus ends based on time difference imaging to measure depolymerization rates in large interphase asters growing in Xenopus egg extract. We observed strong spatial regulation of depolymerization rates, which were higher in the aster interior compared to the periphery, and much less regulation of polymerization or catastrophe rates. We interpret these data in terms of a limiting component model, where aster growth results in lower levels of soluble tubulin and MAPs in the interior cytosol compared to that at the periphery. The steady-state polymer fraction of tubulin was ∼30%, so tubulin is not strongly depleted in the aster interior. We propose that the limiting component for microtubule assembly is a MAP that inhibits depolymerization, and that egg asters are tuned to low microtubule density. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text]

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“…, 2013 ; Rieckhoff et al. , 2019 , 2020 ). Second, while most MAPs that slow down depolymerization have been reported to accelerate polymerization in minimal conditions ( Drechsel et al.…”

Section: Discussionmentioning

confidence: 99%

Spatial variation of microtubule depolymerization in large asters

Ishihara

Decker

Caldas

et al. 2021

MBoC

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“…It was suggested that these flows can arise from the high degree of connectivity introduced by molecular motors. However, in contrast to stabilized mixtures, microtubules in the spindle are constantly nucleated throughout the structure ( Oh et al, 2016, Decker et al, 2018, Rieckhoff et al, 2020 ) and turnover rapidly ( Brugués et al, 2012, Redemann et al, 2017, Rieckhoff et al, 2020 ). In particular, microtubule nucleation is an auto-catalytic process ( Ishihara et al, 2014, Oh et al, 2016, Decker et al, 2018 ), which leads to microtubule structures that are genuinely different from the ones obtained in other simplified in vitro systems ( Sanchez et al, 2012, Roostalu et al, 2018, Fürthauer et al, 2019 ).…”

Section: Introductionmentioning

confidence: 99%

A gelation transition enables the self-organization of bipolar metaphase spindles

Dalton

Oriola

Decker

et al. 2021

Preprint

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The mitotic spindle is a highly dynamic bipolar structure that emerges from the self-organization of microtubules, molecular motors, and other proteins. Sustained motor-driven poleward flows of short dynamic microtubules play a key role in the bipolar organization of spindles. However, it is not understood how the local activity of motor proteins generates these large-scale coherent poleward flows. Here, we combine experiments and simulations to show that a gelation transition enables long-ranged microtubule transport causing spindles to self-organize into two oppositely polarized microtubule gels. Laser ablation experiments reveal that local active stresses generated at the spindle midplane propagate through the structure thereby driving global coherent microtubule flows. Simulations show that microtubule gels undergoing rapid turnover can exhibit long stress relaxation times, in agreement with the long-ranged flows observed in experiments. Finally, we show that either disrupting such flows or decreasing the network connectivity can lead to a microtubule polarity reversal in spindles both in the simulations and in the experiments. Thus, we uncover an unexpected connection between spindle rheology and architecture in spindle self-organization.

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“…For example, the EB1 C-terminal half could interfere with cortical MT capture, although mCherry-Zdk1-EB1C does not localize to the cortex in blue light. Notably, although MT growth has been linked to spindle length [29,30], π-EB1 photoinactivation-mediated spindle shortening was not accompanied by a MT growth rate decrease. Instead, we think that acute EB1 inhibition causes an imbalance of forces acting on the spindle poles that actively drives spindle shortening.…”

Section: Resultsmentioning

confidence: 99%

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

Dema

Wittmann

2021

Preprint

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Chromosome segregation is accomplished by the mitotic spindle, a bipolar micromachine built primarily from microtubules. Different microtubule populations contribute to spindle function: Kinetochore microtubules attach and transmit forces to chromosomes, antiparallel interpolar microtubules support spindle structure, and astral microtubules connect spindle poles to the cell cortex [1,2]. In mammalian cells, End Binding (EB) proteins associate with all growing microtubule plus ends throughout the cell cycle and serve as adaptors for a diverse group of +TIPs that control microtubule dynamics and interactions with other intracellular structures [3]. Because binding of many +TIPs to EB1 and thus microtubule-end association is switched off by mitotic phosphorylation [4-6] the mitotic function of EBs remains poorly understood. To analyze how EB1 and associated +TIPs on different spindle microtubule populations contribute to mitotic spindle dynamics, we use a light sensitive EB1 variant, π-EB1, that allows local, acute and reversible inactivation of +TIP association with growing microtubule ends in live cells [7]. We find that acute π-EB1 photoinactivation results in rapid and reversible metaphase spindle shortening and transient relaxation of tension across the central spindle. However, in contrast to interphase, π-EB1 photoinactivation does not inhibit microtubule growth in metaphase, but instead increases astral microtubule length and number. Yet, in the absence of EB1 activity astral microtubules fail to engage the cortical dynein/dynactin machinery and spindle poles move away from regions of π-EB1 photoinactivation. In conclusion, our optogenetic approach reveals mitotic EB1 functions that remain hidden in genetic experiments likely due to compensatory molecular systems regulating vertebrate spindle dynamics.

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Spindle scaling is governed by cell boundary regulation of microtubule nucleation

Cited by 8 publications

References 64 publications

Spatial variation of microtubule depolymerization in large asters

Spatial variation of microtubule depolymerization in large asters

A gelation transition enables the self-organization of bipolar metaphase spindles

Optogenetic EB1 inactivation shortens metaphase spindles by disrupting cortical force-producing interactions with astral microtubules

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