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Abstract
High-fidelity transmission of the genome through cell division requires that all sister kinetochores bind to dynamic microtubules (MTs) from opposite spindle poles. The application of opposing forces to this bioriented configuration produces tension that stabilizes kinetochore–microtubule (kt–MT) attachments. Defining the magnitude of force that is applied to kinetochores is central to understanding the mechano-molecular underpinnings of chromosome segregation; however, existing kinetochore force measurements span orders of magnitude. Here we measure kinetochore forces by engineering two calibrated force sensors into the Drosophila kinetochore protein centromere protein (CENP)-C. Measurements of both reporters indicate that they are, on average, under ∼1–2 piconewtons (pNs) of force at metaphase. Based on estimates of the number of CENP-C molecules and MTs per Drosophila kinetochore and envisioning kinetochore linkages arranged such that they distribute forces across them, we propose that kinetochore fibres (k-fibres) exert hundreds of pNs of poleward-directed force to bioriented kinetochores.
Highlights
High-fidelity transmission of the genome through cell division requires that all sister kinetochores bind to dynamic microtubules (MTs) from opposite spindle poles
The behaviour of force reporters inserted into the central unstructured region of centromere protein (CENP)-C were compared with negative controls in which the sensors were placed at the C terminus so that they would not be subjected to force (Fig. 1c)
Insertion of the tension sensor module (TSMod) reporter into CENP-C did not disrupt its function as chromosome alignment was indistinguishable between control cells and cells treated with Doublestranded RNAs (dsRNAs) targeting the 30-untranslated region (30-UTR) of the CENP-C transcript to knockdown the endogenous CENP-C (Fig. 2b)
Summary
High-fidelity transmission of the genome through cell division requires that all sister kinetochores bind to dynamic microtubules (MTs) from opposite spindle poles. While motor-mediated forces produced during lateral kt–MT interactions facilitate proper chromosome congression[5,6], they are neither sufficient to satisfy the spindle assembly checkpoint nor to support accurate chromosome segregation during mitosis[7] Rather, these outcomes require the formation of stable end-on kt– MT attachments that are mediated by a conserved MT-binding complex in the outer kinetochore called the KMN (KNL-1, Mis[12] complex, Ndc[80] complex) network[8]. When biorientation is established, opposing poleward forces produce tension across sister kinetochores that stabilizes kt–MT attachments and contributes to spindle assembly checkpoint satisfaction[16,17,18]. In contrast to earlier measurements of 700 pN per kinetochore (50 pN per MT)[23], a more recent optical trapping study in meiotic insect cells and a
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