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Open Biol
2022 Mar 01;123:210389. doi: 10.1098/rsob.210389.
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Reconstitution of an active human CENP-E motor.
Craske B
,
Legal T
,
Welburn JPI
.
Abstract
CENP-E is a large kinesin motor protein which plays pivotal roles in mitosis by facilitating chromosome capture and alignment, and promoting microtubule flux in the spindle. So far, it has not been possible to obtain active human CENP-E to study its molecular properties. Xenopus CENP-E motor has been characterized in vitro and is used as a model motor; however, its protein sequence differs significantly from human CENP-E. Here, we characterize human CENP-E motility in vitro. Full-length CENP-E exhibits an increase in run length and longer residency times on microtubules when compared to CENP-E motor truncations, indicating that the C-terminal microtubule-binding site enhances the processivity when the full-length motor is active. In contrast with constitutively active human CENP-E truncations, full-length human CENP-E has a reduced microtubule landing rate in vitro, suggesting that the non-motor coiled-coil regions self-regulate motor activity. Together, we demonstrate that human CENP-E is a processive motor, providing a useful tool to study the mechanistic basis for how human CENP-E drives chromosome congression and spindle organization during human cell division.
Figure 1. The first predicted coiled-coil of human CENP-E weakly facilitates dimerization of motor domains. (a) Coiled-coil prediction of full-length CENP-E by Paircoil2. Dashed vertical lines represent truncations. (b) Constructs used in this study. KT = kinetochore-binding domain, MT = second microtubule-binding site, GCN4 = GCN4 leucine zipper domain, His = hexahistidine tag, mNeon = mNeonGreen fluorescent protein. (c) Schematic representation of a single-molecule motility assay. (d) Kymographs of CENP-E483-2mNeon and K560-GFP at indicated nanomolar concentrations for motility assays. (e) Native PAGE analysis of purified CENP-E483-2mNeon oligomeric status. M = monomer, D = dimer. (f) Histogram representation of velocities for CENP-E483-2mNeon (n = 346) at 50 nM fit to a single Gaussian distribution (r2 = 0.978), mean of the Gaussian fit ± s.e.m. are reported in the graph, median ± s.e = 131.6 ± 4.1 nm s−1. (g) 1 − cumulative frequency distribution of run lengths for CENP-E483-2mNeon at 50 nM (n = 346) fit to a single-exponential decay (r2 = 0.982). (h) 1 − cumulative frequency distribution of residency times for CENP-E483-2mNeon at 50 nM (n = 346) fit to a single-exponential decay (r2 = 0.990).
Figure 2. (Opposite.) Stable CENP-E dimers are robustly processive in vitro. (a) Coomassie stained gel of purified CENP-E483LZ-2mNeon and CENP-E754-2mNeon after SDS-PAGE. Arrowheads indicate purified protein. (b) Kymographs of 5 nM CENP-E483LZ-2mNeon and 3.5 nM CENP-E754-2mNeon moving along single microtubules. (c) Schematic representation of photobleaching and intensity analysis assay. (d) Histogram distribution of CENP-E483LZ-2mNeon velocities (n = 774) fitted to a single Gaussian distribution (r2 = 0.992), mean of the Gaussian fit ± s.e.m. are reported in the graph, median ± s.e. = 160.3 ± 2.7 nm s−1. (e) 1 − cumulative frequency of run lengths measured for CENP-E483LZ-2mNeon (n = 774) and fitted to a single-exponential distribution (r2 = 0.986). (f) 1 − cumulative frequency of residency times measured for CENP-E483LZ-2mNeon (n = 774) and fit to a single-exponential distribution (r2 = 0.969). (g) Histogram distribution of CENP-E754-2mNeon velocities (n = 289) fit to a single Gaussian distribution (r2 = 0.996), mean of the Gaussian fit ± s.e.m. are reported in the graph, median ± s.e. = 154.3 ± 4.0 nm s−1. (h) 1 − cumulative frequency of run lengths measured for CENP-E754-2mNeon (n = 289) and fit to a single-exponential distribution (r2 = 0.993). (i) 1 − cumulative frequency of residency times measured for CENP-E754 (n = 289) and fit to a single-exponential distribution (r2 = 0.966). (j) Example four-step photobleaching trace of CENP-E754-2mNeon. (k) Histogram distribution of CENP-E754-2mNeon bleaching steps (n = 187). (l) Initial fluorescence intensity distribution of CENP-E754-2mNeon (n = 88) and K560-2mNeon (n = 117).
Figure 3. Full-length human CENP-E is a processive motor. (a) Coomassie stained gel of purified CENP-EFL-mNeon after SDS-PAGE. (b) Histogram distribution for microtubule gliding velocities of CENP-EFL-mNeon (n = 93), mean of the Gaussian fit ± s.e.m. are reported. (c) Example of a kymograph showing a single-CENP-EFL-mNeon dimer moving along a microtubule. CENP-EFL-mNeon was imaged at 12.5 nM. (d) Histogram distribution of CENP-EFL-mNeon velocities (n = 61) fitted to a double Gaussian distribution (r2 = 0.958), mean of the Gaussian fit ± s.e.m. are reported. (e) 1 − cumulative frequency of run lengths measured for CENP-EFL-mNeon (n = 61) and fitted to a single-exponential distribution (r2 = 0.951). (f) 1 − cumulative frequency of residency times measured for CENP-EFL-mNeon (n = 61) and fitted to a single-exponential distribution (r2 = 0.995). (g) Example two-step photobleaching trace of CENP-EFL-mNeon. (h) Histogram distribution of CENP-EFL-mNeon bleaching steps (n = 102). (i) Initial fluorescence intensity distribution of CENP-EFL-mNeon (n = 102).
Figure 4. The non-motor regions of human CENP-E regulate processive motility. (a) Quantification of total landing rates for CENP-EFL-mNeon (n = 98, n = number of microtubules) and CENP-E754-2mNeon (n = 100, n = number of microtubules). Welch's t-test, p < 0.0001. (b) Quantification of processive landing rates for CENP-EFL-mNeon (n = 98, n = number of microtubules) and CENP-E754-2mNeon (n = 100, n = number of microtubules). Welch's t-test, p < 0.0001.
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