Self-organization of kinetochore-fibers in human mitotic spindles
- Department of Physics, Harvard University, United States
- Experimental Center, Faculty of Medicine Carl Gustav Carus, Technische Universität Dresden, Germany
- Department of Molecular and Cellular Biology, Harvard University, United States
- John A Paulson School of Engineering and Applied Sciences, Harvard University, United States
- Center for Computational Biology, Flatiron Institute, United States
Figures
Figure 1
Many KMT minus ends are not in the vicinity of the pole.
(A) A sample half spindle showing the KMTs from the EM ultrastructure. KMTs are shown in red while minus ends are marked in black. The spindle pole lies at 0µm on the spindle axis while the …
Figure 2 with 2 supplements
Photoactivation of spindle tubulin in live HeLa cells.
(A) Photoactivation experiment showing PA-GFP:alpha-tubulin and SNAP-SIR:centrin immediately preceding photoactivation, 0 s, 30 s, and 60 s after photoactivation with a 750nm femtosecond pulsed …
Figure 2—figure supplement 1
Z Perpendicular point spread function calibration.
(A) Sample images from the z-scan of PA-GFP tubulin photoactivated with 750nm two-photon photoactivation. The line profile below the image is generated by averaging 15 pixels on either side of the …
Figure 2—figure supplement 2
PA-GFP:Alpha Tubulin Bleaching Calibration.
(A) Time series of activated tubulin in spindles. The whole spindle was photoactivated with a 750nm femtosecond pulsed laser and left to equilibrate for 5 minutes before imaging (B) Mean integrated …
Figure 3 with 2 supplements
Spatial dependence of photoconversion parameters.
(A) Sample photoactivated frames (488 nm, 500ms exposure, 5s frame rate) and line profiles from a line drawn near the kinetochore. (B) Sample photoconverted frames and line profiles from a line …
Figure 3—figure supplement 1
Position dependence of one photon photoactivation in the spindle.
(A) Sample photoconverted frames (561 nm, 500ms exposure, 5 s frame rate) and line profiles from a line drawn near the kinetochore (B) Line speed vs. initial position of the line drawn on the …
Figure 3—figure supplement 2
Comparison of speed of tubulin in near the spindle axis and across the entire spindle width.
(A) Sample photoactivation images and line profile taken by averaging pixels across the entire spindle width displayed by the dotted box superimposed over the images. (B) Sample photoactivation …
Figure 4 with 2 supplements
Measuring nematic alignment of non-KMTs and KMTs (3D reconstructed cell #1).
(A) Sample from a 3D reconstruction of non-KMTs (yellow) and KMTS (red) from electron tomography (Kiewisz et al., 2022). (B) Mean local orientation of non-KMTs projected into a 2D XY plane averaged …
Figure 4—figure supplement 1
Measuring nematic alignment of non-KMTs and KMTs (3D reconstructed cell #2).
(A) Sample from a 3D reconstruction of non-KMTs (yellow) and KMTS (red) from electron tomography (Kiewisz et al., 2022). (B) Mean local orientation of non-KMTs projected into a 2D XY plane averaged …
Figure 4—figure supplement 2
Measuring nematic alignment of non-KMTs and KMTs (3D reconstructed cell #3).
(A) Sample from a 3D reconstruction of non-KMTs (yellow) and KMTS (red) from electron tomography (Kiewisz et al., 2022). (B) Mean local orientation of non-KMTs projected into a 2D XY plane averaged …
Figure 5 with 4 supplements
Experiment and theory of the orientation field of MTs in HeLa spindles.
(A) Orientation field of MTs from averaging three spindle reconstructions from electron tomography. (B) Orientation field of MTs from averaging polarized light microscopy (LC-PolScope) data from 11 …
Figure 5—figure supplement 1
Comparison of PolScope and EM predicted measurements of the retardance area in HeLa spindles.
(A) Sample PolScope retardance image with ellipse fit to the boundary of the spindle. The retardance area is calculated by multiplying the mean retardance in the spindle by the fit minor axis of the …
Figure 5—figure supplement 2
Boundary conditions for the active liquid crystal models.
At the spindle boundary, a tangential anchoring condition enforces that the director field lies tangent to the boundary. At the +1 point defects near the centrosomes, a radial anchoring condition …
Figure 5—figure supplement 3
Comparison of the predicted angles from the active liquid crystal theory.
(A) Orientation field of MTs from a 2D approximate active liquid crystal theory. (B) Orientation field of MTs from the central slice of a 3D active liquid theory (C) Orientation field of MTs from a …
Figure 5—figure supplement 4
Experimentally measured orientation field of MTs in HeLa spindles compared to theoretical predictions with point defects localized on the spindle periphery.
(A) Orientation field of MTs from averaging EM reconstructions from three spindles. (B) Orientation field of MTs from averaging polarized light microscopy (LC-PolScope) data from eleven spindles. (C)…
Figure 6 with 3 supplements
Predicting the KMT minus end speeds from the steady state distribution of minus ends along streamlines.
(A) Eight representative KMTs from spindle reconstructions by electron tomography (red), with their minus ends (black dots) and the streamlines (thin black lines) these minus ends are located on. …
Figure 6—figure supplement 1
Comparison of EM and fit liquid crystal theory for individual reconstructed spindles.
(A) Average MT orientation from reconstructed spindle #1. (B) Theoretical model of the spindle geometry with tangential anchoring at the elliptical spindle boundary conditions and point defects at …
Figure 6—figure supplement 2
Density distribution of non-KMT minus ends along streamlines.
For both ends of each non-KMT, the streamline trajectory from the non-KMT end was calculated by integrating along the nematic director field for that spindle. The distance from each end to the …
Figure 6—figure supplement 3
Simulated distribution of minus ends along streamlines using either a nucleate at kinetochore model (blue) or a capture from spindle recruitment model (green), compared to the experimentally measured minus distribution from electron microscopy reconstructions (black).
KMTs were nucleated and plus ends were placed at positions drawn from the distribution of kinetochores along streamlines. For the capture from spindle model, the KMT minus ends were initially placed …
Figure 7 with 5 supplements
Model predicted tubulin flux compared to observed values.
(A) Sample simulated images and line profiles from a photoconversion simulation using KMT minus end speeds in the nucleate at kinetochore model. (B) Comparison of the predicted spatial dependence …
Figure 7—figure supplement 1
Density distribution of kinetochores along streamlines.
The position of kinetochores in each sample cell was projected onto the streamline trajectories computed in Figure 6—figure supplement 3 (black dots) and binned from all three cells. The …
Figure 7—figure supplement 2
Sample experimental line profile from a photoconversion experiment and a fit modified Cauchy profile (), The fit profile was generated by drawing a photoconverted line on the simulated spindle (Figure 7A) and projecting the calculated tubulin intensity onto the spindle axis with the modified Cauchy profile with various central positions l0, widths w, and Cauchy exponent a.
Best fit was determined from a χ2 minimization algorithm.
Figure 7—figure supplement 3
Sample simulated images from photoconversion in the capture from spindle model.
For each streamline, a photoconverted line was drawn on the simulated, idealized spindle using the fit modified Caucy profile from Figure 7—figure supplement 2. The photoconverted tubulin intensity …
Figure 7—figure supplement 4
Model predicted tubulin flux compared to observed values without minus end depolymerization at the pole.
(A) Sample simulated images and line profiles from a photoconversion simulation using KMT minus end speeds in the nucleate at kinetochore model. (B) Sample simulated images and line profiles from a …
Figure 7—figure supplement 5
Model predicted tubulin flux compared to observed values with constant KMT minus end speed along streamlines in a narrow region near the spindle axis.
(A) Sample photoactivated cell and associated line profile generated by averaging the intensity in 5 pixels on either side of the spindle axis in the dotted box (B) Sample simulated images and line …
Figure 8 with 1 supplement
Summary of a nucleate at kinetochore model of KMT dynamics and structure in HeLa cells.
(A) Summary of the steps of the model: 1. KMTs nucleate at kinetochores 2. KMTs grow along streamlines 3. KMTs slow down as they grow 4. KMTs treadmill near the pole; and 5. KMTs detach. (B) KMT …
Figure 8—figure supplement 1
Line spreading after photoactivation.
(A) Photoactivation experiment showing PA-GFP:alpha-tubulin preceding photoactivation, 0min, 1min, and 2min after photoactivation with a 750nm femtosecond pulsed laser (B) Line profile generated by …
Appendix 1—figure 1
Sample geometry of spindle streamlines used in the simulation.
Geometry of the spindle streamlines used in the simulations. The thin lines show the trajectories of nematic streamlines in the spindle bulk. The thick black line shows the elliptical boundary of …
Appendix 1—figure 2
Length distribution of non-KMTs in the spindle.
Binned histogram of the lengths of non-KMTs in three reconstructed mitotic HeLa spindle. Black dots: electron microscopy data; black line: exponential fit. Mean MT length is 2.0±0.05 µm.
Appendix 1—figure 3
Predicted photoconverted line speed for various uniform non-KMT motion speeds.
The speed of the non-KMTs was varied (assorted colors) in 0.5 µm/min increments in a 2D confocal imaging spindle simulation.
Appendix 1—figure 4
pindle background profile.
(A) Sample representative spindle image (Green: mCherry:tubulin). (B) The intensity of the tubulin marker projected onto the spindle axis and then averaged for n=72 half spindles. The spindle axis …
Appendix 1—figure 5
Height of the opposite pole over time.
The peak height averaged from n=5 spindles displaying a clear opposite peak (black dots) is fit to an exponential (black line).
Videos
Animation 1
Simulated tubulin photoconversion in a 3D model spindle.
Model simulation of the motion of KMTs in a nucleate at kinetochore model. KMTs are shown in red, KMT minus ends are shown in black, photoconverted tubulin is shown in yellow. The model runs for 5 …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (Homo sapiens) | HeLa Kyoto | Gerlich Lab, IMBA, Vienna Austria | - | - |
| Transfected construct (Homo sapiens) | pBABE-puro CENP-A:GFP | Yu et al., 2019 | - | CENP-A C-terminally labeled with sfGFP; in retroviral vector with puromycin selection marker |
| Transfected construct (Homo sapiens) | pBABE-hygro SNAP:Centrin | This paper (Needleman Lab, Harvard) | - | Centrin N-terminally labeled with a SNAP tag; in retroviral vector with hygromycin selection marker |
| Transfected construct (Homo sapiens) | pJAG98(pBABE-blast) mEOS3.2:alpha tubulin | Yu et al., 2019 | - | Alpha tubulin N-terminally labeled with mEOS3.2; in retroviral vector with blastcidin selection marker |
| Transfected construct (Homo sapiens) | pIRESneo-PA-GFP-alpha Tubulin | Tulu et al., 2003 | Alpha tubulin N-terminally labeled with PA-GFP in a vector with a neomycin marker | |
| Commercial assay or kit | SNAP-Cell 647-SiR | New England Biolabs | - | Catalog number S9102S |
| Software algorithm | Interactive spindle photoconversion analysis GUI (MATLAB 2020b) | This paper (Dryad) | - | - |
| Software algorithm | Photoconversion simulation package | This paper (Dryad) | - | - |
| Software algorithm | Photoconversion control and imaging | Wu et al., 2016 | - | Controls custom confocal photoconversion for arbitrary geometry |
| Software algorithm | Polarized light microscopy control software | https://openpolscope.org/ | - | - |
Appendix 1—table 1
Parameters values and sources.
| Simulation Parameter | Value | Source |
|---|---|---|
| KMT Trajectories, | - | Nematic Theory (Figure 5 and. 6 A) |
| KMT Velocity | Varies | Mass Conservation Analysis (Figure 6E) |
| KMT Stability, | 0.4 min–1 | Photoconversion (Figure 3G) |
| Non-KMT Mean Length, | 2 µm | Electron Microscopy (Appendix 1—figure 2) |
| Photoconverted Line Width, | 150 nm | Converted Line Profile (Figure 7—figure supplement 2) |
| Background Height, | 0.06 | Opposite Peak Height (Appendix 1—figure 5) |
| Background Rise Time, | 60 s | Opposite Peak Height (Appendix 1—figure 5) |
