Chirality of the mitotic #spindle: What is it good for and how is it produced? We have enjoyed this interdisciplinary collaboration with engineers and physicists! Thanks to Monika Trupinic, Barbara Kokanovic and @PonjavicIvana for the hot cell biology! https://t.co/LyvN1H4Oju pic.twitter.com/SX6oOsiT9S
— Iva Tolić (@Toliclab) August 13, 2021
Chirality is visible when you look at the spindle end-on. Microtubule bundles rotate clockwise (arrows) when moving towards the observer, meaning that they twist in a left-handed direction. This makes the spindle chiral. (HeLa cell, tubulin, colors show depth, LUT 16 colors). pic.twitter.com/W1Ny2TeGtM
— Iva Tolić (@Toliclab) August 13, 2021
What is the biological role of spindle chirality? It may help to maintain the robustness of the spindle under force! To test this idea, we compressed vertically oriented spindles along the axis. This resulted in spindle shortening (as expected) and an increase in twist! pic.twitter.com/tENa2nlVSE
— Iva Tolić (@Toliclab) August 13, 2021
Is the twist present at spindle birth or does it arise later? We measured the twist in different mitotic phases in HeLa cells expressing PRC1-GFP, which labels bridging fibers whose twist is then measured. You can see the bridging fibers here color-coded for depth. pic.twitter.com/x2smhh85Gd
— Iva Tolić (@Toliclab) August 13, 2021
We measured the twist also in RPE1 cells expressing CENP-A-GFP (color-coded for depth) and stained with SiR-tubulin (white). The twist was highest at the onset of anaphase in HeLa and RPE1 cells. HeLa cells have overall higher twist values. pic.twitter.com/VPzXmuxaFe
— Iva Tolić (@Toliclab) August 13, 2021
Here are slices through the spindle in the normal view (side view) and the end-on view. A strong left-handed twist is clearly visible at the bottom left, as the clockwise movement of the bridging fibers labeled with PRC1-GFP. pic.twitter.com/0Tj5CsG5CO
— Iva Tolić (@Toliclab) August 13, 2021
Spindle twist in prometaphase is close to 0, it is left-handed (negative) during metaphase, culminates at anaphase onset reaching a value of -2°/μm (in HeLa cells), and decreases towards 0 in late anaphase. pic.twitter.com/rB3zLqNBjx
— Iva Tolić (@Toliclab) August 13, 2021
How are the torques that make the spindle chiral generated? Motors that step along the microtubule with occasional side-steps in a certain direction may be responsible. Indeed, depletion of #Kif18A/kinesin-8 changes the twist in RPE1 cells from left-handed to right-handed! pic.twitter.com/ZWcCX1AYvG
— Iva Tolić (@Toliclab) August 13, 2021
Depletion of the #augmin complex (nucleator of new microtubules) or #PRC1 (crosslinker of antiparallel microtubules) also lead to right-handed twist! The switch from left-handed to right-handed twist means that competing mechanisms promote twisting in opposite directions. pic.twitter.com/mqSnmPM2s5
— Iva Tolić (@Toliclab) August 13, 2021
Round spindles have a stronger twist than elongated ones. Thus, the molecular mechanisms that generate larger bending moments, causing the spindles to be rounder, may also generate larger twisting moments, visible as stronger twist of the microtubule bundles. pic.twitter.com/0mTHclOpNP
— Iva Tolić (@Toliclab) August 13, 2021
In summary, #spindle #chirality depends on spindle shape, external forces, and on #motors (#Eg5, #Kif18A) that rotate #microtubules within the antiparallel overlaps. #PRC1 contributes to chirality by limiting microtubule rotation, and #augmin by nucleating bridging microtubules. pic.twitter.com/TDEuuHf0Si
— Iva Tolić (@Toliclab) August 13, 2021
A biological role of spindle chirality may be to allow for a passive mechanical response to forces, decreasing the risk of spindle breakage under high load. In contrast to metaphase, in late anaphase twist is absent, which promotes spindle rigidity and chromosome separation. pic.twitter.com/UySzGHmLT7
— Iva Tolić (@Toliclab) August 13, 2021
How to measure the twist of a mitotic spindle? This is not straightforward! Monika Trupinic and I are happy to have contributed to this work by @nenad_pavin and Arian Ivec, in which they designed a simple and robust “Oblique Circle Method”. https://t.co/5Zs7NBVonZ pic.twitter.com/SvTqDNhkyT
— Iva Tolić (@Toliclab) August 4, 2021
Because the spindle shape reflects the forces within it, measurement of the shapes of microtubule bundles is important for the understanding of forces that act on chromosomes during mitosis. The spindle is a chiral object, as microtubule bundles follow a left-handed helical path. pic.twitter.com/M0elnmoeMj
— Iva Tolić (@Toliclab) August 4, 2021
In the Oblique Circle Method, microtubule bundles are tracked in 3D and their coordinates are then translated and rotated. pic.twitter.com/8HjUqwiggs
— Iva Tolić (@Toliclab) August 4, 2021
To fit a circular arc in a reproducible way, we first fit a plane, and then fit a circle that lies in this plane. The radius of the circle and the position of the circle center, together with the normal vector of the bundle plane, determine the geometry of the traced bundle. pic.twitter.com/guw0gcTzsB
— Iva Tolić (@Toliclab) August 4, 2021
The curvature of the bundle can be directly calculated as 1 over the radius of the fitted circle. The twist, however, cannot be calculated in a straightforward manner. We introduce the twist value as a change of the azimuthal angle with respect to the axial position. pic.twitter.com/24X8LW8LQU
— Iva Tolić (@Toliclab) August 4, 2021
Overall, the recent discovery of spindle chirality suggests that there are rotational forces in the spindle in addition to linear ones. We hope that the Oblique Circle Method for measuring curvature and twist will help to understand the forces that act on chromosomes in mitosis. pic.twitter.com/4jwMjpyHa3
— Iva Tolić (@Toliclab) August 4, 2021
How do chromosomes get aligned on the mitotic spindle? By asymmetric poleward flux of kinetochore fibers! This story started with a physical model by @nenad_pavin & @BozanDomagoj, motivating @risteski_ and @Michaela_Jag to develop methodology to test it. https://t.co/NpWWr89YRR pic.twitter.com/M0fp7wXVfC
— Iva Tolić (@Toliclab) July 27, 2021
Chromosome alignment at the spindle equator promotes proper chromosome segregation and depends on pulling forces by kinetochore fiber tips and polar ejection forces. But kinetochore fibers are also subjected to forces driving their poleward flux! pic.twitter.com/MakIeNz8f4
— Iva Tolić (@Toliclab) July 27, 2021
In the Flux-Driven Centering Model, motor proteins accumulate in the antiparallel overlaps between kinetochore fibers and bridging microtubules, where they slide the microtubules apart and drive their flux. pic.twitter.com/W9fAUhlZO6
— Iva Tolić (@Toliclab) July 27, 2021
This centering mechanism works so that the longer kinetochore fiber fluxes faster than the shorter one, moving the kinetochores towards the center. The reason for the faster flux of the longer kinetochore fiber is its longer overlap with bridging microtubules. pic.twitter.com/5rh9criNOn
— Iva Tolić (@Toliclab) July 27, 2021
The difference in the flux of sister kinetochore fibers is the core of the centering mechanism. You can see this in the animation based on the Flux-Driven Centering Model. pic.twitter.com/eWVnazGb8Q
— Iva Tolić (@Toliclab) July 27, 2021
To test the model, we need to measure the poleward flux of different classes of microtubules (kinetochore and bridging). So we developed a speckle microscopy assay by using a very low concentration (1 nM) of SiR-tubulin. Each speckle marks an individual microtubule. pic.twitter.com/5uH1sdKONn
— Iva Tolić (@Toliclab) July 27, 2021
Measurements of speckle velocities in human spindles reveal that longer kinetochore fibers flux faster than shorter ones, which is a key feature of the Flux-Driven Centering mechanism. pic.twitter.com/qS02hGooWe
— Iva Tolić (@Toliclab) July 27, 2021
In the model, the tension between sister kinetochores opposes the flux of kinetochore fibers, making it slower than the flux of the bridging fiber. Speckle microscopy experiments confirm this prediction. pic.twitter.com/LwG1A1OWKX
— Iva Tolić (@Toliclab) July 27, 2021
Does the relationship between the bridging and kinetochore fiber flux hold also in spindles with changed flux velocities? We depleted a set of spindle proteins and found that the bridging fiber flux was always faster than or equal to the kinetochore fiber flux. pic.twitter.com/7MP9AZyNyZ
— Iva Tolić (@Toliclab) July 27, 2021
In spindles depleted of Ndc80, which do not have kinetochore fibers, the bridging fiber flux was unchanged, suggesting that the flux is generated within this fiber. In contrast, CENP-E depletion slowed down the bridging fiber flux, so this motor may slide bridging microtubules. pic.twitter.com/Pr0nMe3cc3
— Iva Tolić (@Toliclab) July 27, 2021
Centering is more efficient when kinetochore fiber flux is slower than the bridging fiber flux, allowing for sliding of kinetochore fibers along bridging fibers. In agreement with this prediction, extreme kinetochore misalignment was accompanied by fast kinetochore fiber flux. pic.twitter.com/M6BkyXKpw8
— Iva Tolić (@Toliclab) July 27, 2021
Overall, the treatments with the kinetochore fiber flux velocity similar to that of untreated cells show efficient centering, whereas treatments with faster kinetochore fiber flux show worse centering. pic.twitter.com/zDXW7UVS4Z
— Iva Tolić (@Toliclab) July 27, 2021
What caused this speeding up of the kinetochore fiber flux in the treatments with misaligned kinetochores? The model suggests that changes in the overlap length can change flux velocities. Indeed, PRC1-labeled overlaps were longer after Kif18A and Kif4A depletion. pic.twitter.com/fYp3xFvDbS
— Iva Tolić (@Toliclab) July 27, 2021
Overall, PRC1-labeled overlaps were longer in treatments with faster kinetochore fiber flux, suggesting that the sliding forces generated within the bridging fiber are transferred to the kinetochore fibers through the antiparallel overlaps between these two types of fibers. pic.twitter.com/bYdZAvy1S1
— Iva Tolić (@Toliclab) July 27, 2021
Depletion of the microtubule crosslinker NuMa slowed down the kinetochore fiber flux without affecting the bridging fiber flux. This supports the idea that NuMA acts as a crosslinker transmitting the sliding forces from the bridging fiber onto the associated kinetochore fibers. pic.twitter.com/YUugYblDol
— Iva Tolić (@Toliclab) July 27, 2021
This is a summary of what we learned by combining theoretical physics with cell biology: Length-dependent sliding forces exerted by the bridging fiber onto kinetochore fibers promote chromosome alignment. Great working with @risteski_ @Michaela_Jag @BozanDomagoj @nenad_pavin! pic.twitter.com/EQgokRNFFe
— Iva Tolić (@Toliclab) July 27, 2021
It’s a Tweetorial Thursday, time for a #DBIOtweetorial brought to you by the #engageDBIO crew! This week’s host is Iva Tolic @Toliclab from @institutrb, tweeting about the mitotic #spindle and its mechanical curiosities. pic.twitter.com/jztzRB67fQ
— APS-DBIO (@ApsDbio) July 1, 2021
The mitotic spindle is a biological micro-machine that never fails to fascinate! A quick recap: The spindle segregates the chromosomes into two equal parts destined to the future daughter cells. It is made of microtubules and hundreds of other proteins. pic.twitter.com/bjFk3uE2TR — APS-DBIO (@ApsDbio) July 1, 2021
The spindle is a tiny mechanical device, so mechanical perturbations have been especially useful in uncovering how it works. Laser ablation of a kinetochore (the chromosome-microtubule connection site) demonstrated that microtubules pull on kinetochores.https://t.co/fZcLd4NrV2 pic.twitter.com/DpKiiFV1Ks — APS-DBIO (@ApsDbio) July 1, 2021
Laser ablation of a kinetochore fiber identified a dynein-powered force that pulls severed microtubules back into the spindle, repairing spindle architecture. https://t.co/69Q9LVZzJfhttps://t.co/6MbT0j1uNqhttps://t.co/zSoJ4qDCod pic.twitter.com/HkXlk1f0Vw
— APS-DBIO (@ApsDbio) July 1, 2021
Before its reincorporation in the spindle, the kinetochore fiber stub rotates with its tip moving away from the spindle. This revealed that the kinetochore fibers are linked by a bridging fiber, which is bent due to rotational forces.https://t.co/uP2eGEbUmE pic.twitter.com/YCgcddb6Tr — APS-DBIO (@ApsDbio) July 1, 2021
Laser ablation also revealed that a single mechanical unit consisting of two sister kinetochore fibers and their bridge is able to separate the kinetochores, via motors that slide the antiparallel bridging microtubules apart. https://t.co/GlMtuatbgyhttps://t.co/HCGUi4ml3b pic.twitter.com/B06xcjDZE2
— APS-DBIO (@ApsDbio) July 1, 2021
Sophisticated experiments (a kinetochore fiber’s pulled by a microneedle) –> sister kinetochore fibers are strongly linked and don’t pivot around kinetochores, but around the pole, suggesting reinforcements near kinetochores. https://t.co/WOdkAvc6tmhttps://t.co/es0ROksU1d pic.twitter.com/LmCQtbKsl8 — APS-DBIO (@ApsDbio) July 1, 2021
Optogenetic removal of PRC1 (the protein that provides this reinforcement by bundling bridging microtubules) from the spindle to the cell membrane led to the conclusion that bridging fibers help to align the chromosomes at the spindle midplane.https://t.co/7J9kN8rNYn pic.twitter.com/5YONsbwSyl
— APS-DBIO (@ApsDbio) July 1, 2021
How does this work? Kinetochore fibers are under forces that drive their poleward flux, creating a tug-of-war between sister kinetochore fibers, which promotes kinetochore centering (https://t.co/8m3okwiVza). This flux is powered by four kinesins (https://t.co/FgrGXI8hlb). pic.twitter.com/8s59cI464w — APS-DBIO (@ApsDbio) July 1, 2021
The spindle is so cool that it even shows chirality! The microtubule bundles making up the spindle have a left-handed helix-like shape, visible as the rotation of bridging fibers around the spindle axis when the spindle is observed along the axis. https://t.co/5KMg4p4x6B pic.twitter.com/PoKdbHS2Ow
— APS-DBIO (@ApsDbio) July 1, 2021
That’s it! I hope I’ve convinced you that spindles are exciting to explore! Big thanks to @ApsDbio + #EngageDBIO team for arranging the #DBIOtweetorial series! Read more about #spindle #mechanobiology in https://t.co/vHArPhlVUz Enjoy! — Your host today, Iva Tolic @Toliclab pic.twitter.com/tZKE3A4OWQ — APS-DBIO (@ApsDbio) July 1, 2021
How do #chromosomes know where the #spindle center is? @risteski_ @Michaela_Jag @nenad_pavin and I discuss intricacies of chromosome alignment and flux-driven centering in the #Cytoskeleton Special Issue of @CurrentBiology! Movie by @isabella__k @Stimac_V.https://t.co/oQep4XRKYD pic.twitter.com/2UZXYkhsxm
— Tolić lab (@Toliclab) May 25, 2021
Here comes a little thread. Chromosomes align in a fascinating lineup at the equatorial plane in metaphase to ensure synchronous poleward movement of chromatids in anaphase and proper nuclear reformation in telophase. This alignment relies on microtubules & associated proteins. pic.twitter.com/Uyn0Wfokej — Tolić lab (@Toliclab) May 25, 2021
Molecular players of chromosome alignment⁰ regulate the dynamics of kinetochore microtubules that pull on kinetochores, generate polar ejection forces that push on chromosome arms, and slide apart bridging microtubules and thus also the attached kinetochore microtubules. pic.twitter.com/HjDrsczhEp
— Tolić lab (@Toliclab) May 25, 2021
Pushing forces exerted by microtubules can center chromosomes because they decrease with an increasing distance from the pole. Due to the higher number of microtubules coming from the nearer pole and their higher force, the chromosome will be pushed towards the spindle center. pic.twitter.com/yYG0Hhqp1O — Tolić lab (@Toliclab) May 25, 2021
Some motor proteins can ‘measure’ microtubule length and preferentially suppress the dynamics of longer microtubules, thereby centering the chromosome. These motors must walk all the way to the microtubule tip to gather there in a microtubule length-dependent manner. pic.twitter.com/a5jx0PMMMj
— Tolić lab (@Toliclab) May 25, 2021
But there is a third possibility: Chromosomes may be centered by microtubule length-dependent pulling forces! We recently proposed a model based on pulling forces generated along the length of the microtubule.https://t.co/obuwb2ysXg pic.twitter.com/7TmhENtlSI — Tolić lab (@Toliclab) May 25, 2021
How does this work? First, a displaced chromosome has a longer overlap between the kinetochore fiber from the distal pole and the bridging microtubules from the same pole. This leads to larger friction as the bridging microtubules slide apart, which centers the chromosome. pic.twitter.com/xN798sin9n
— Tolić lab (@Toliclab) May 25, 2021
Second, a displaced chromosome has a longer antiparallel overlap between the kinetochore fiber extending towards the distal pole and the bridging microtubules coming from the opposite pole, leading to a larger motor-driven sliding force within this overlap. pic.twitter.com/lRBFULX7og — Tolić lab (@Toliclab) May 25, 2021
Finally, we compare centering mechanisms in yeast and human cells. In yeast, when kinetochores move towards a pole, the longer kinetochore microtubules collect more kinesin-8 motors, which induce their catastrophe, thereby bringing the kinetochores back to the spindle center. pic.twitter.com/ywBJwX0EBr
— Tolić lab (@Toliclab) May 25, 2021
In human spindles, motors slide overlapping microtubules and drive their poleward flux. Because the longer kinetochore fiber has a longer overlap with the bridging fiber, its flux is faster, pulling the kinetochores towards the spindle center. We call it “flux-driven centering”. pic.twitter.com/VhIBDYHDyY — Tolić lab (@Toliclab) May 25, 2021
Anaphase
Spindle elongation in human cells is powered by joint activity of kinesin-5 and kinesin-4. Without spindle elongation, chromosome segregation often fails, demonstrating its role in successful cell division. Here comes a tweetorial! (1/10)https://t.co/WUSTfn0Z43 pic.twitter.com/25BhTDkWc9
— Tolić lab (@Toliclab) May 4, 2021
Is spindle elongation in anaphase driven by motor proteins that slide antiparallel microtubules apart, which is known as the “sliding filament mechanism”, or by pushing forces generated by microtubules as they grow against barriers? (2/10) pic.twitter.com/RnIUaag800 — Tolić lab (@Toliclab) May 4, 2021
Spindle elongation depends on the joint activity of the motor EG5/kinesin-5 and the microtubule crosslinker PRC1. Without them, elongation is completely blocked, which is visible from this movie with chromosomes stained by SiR-DNA and color-coded for depth. (3/10) pic.twitter.com/WWtRoIO1OY
— Tolić lab (@Toliclab) May 4, 2021
Spindle elongation block induced by combined disruption of PRC1 and EG5 leads to massive chromosome missegregation! The whole chromosome mass often moves to one daughter cell at the time when the cleavage furrow starts forming. (4/10) pic.twitter.com/dSoh7rZ2zx — Tolić lab (@Toliclab) May 4, 2021
EG5 is a microtubule slider, but how does PRC1 contribute to spindle elongation? By depleting midzone proteins one by one combined with EG5 inactivation, we found that PRC1 promotes spindle elongation by recruiting KIF4A/kinesin-4 to the midzone. (5/10) pic.twitter.com/L4XQSTeKWv
— Tolić lab (@Toliclab) May 4, 2021
Combined disruption of KIF4A and EG5 mimics combined disruption of PRC1 and EG5. Thus, cells without KIF4A and EG5 also have total failure of chromosome segregation due to blocked spindle elongation. (6/10) pic.twitter.com/9R8Ybl3sSd — Tolić lab (@Toliclab) May 4, 2021
Is the spindle elongation block due to weak midzone microtubules? This is unlikely because expansion microscopy shows normal midzone microtubule bundles. (7/10) pic.twitter.com/L1li90vANH
— Tolić lab (@Toliclab) May 4, 2021
Or is it something related to astral microtubules? This is also unlikely because combined disruption of KIF4A and EG5 does not change the number or dynamics of EB3 comets. (8/10) pic.twitter.com/F35UuSEbnu — Tolić lab (@Toliclab) May 4, 2021
Or is the elongation block due to perturbed microtubule sliding? Yes, as photoactivation of tubulin shows abolished sliding after joint disruption of KIF4A and EG5. So, KIF4A and EG5 drive spindle elongation together by sliding apart the midzone microtubules. (9/10) pic.twitter.com/Y32qO6znIW
— Tolić lab (@Toliclab) May 4, 2021
We propose that two mechanistically distinct sliding modules, one based on a self-sustained and the other on a crosslinker-assisted motor, power the mechanism of spindle elongation in human cells. All done by incredible @Kruno14683771 @PonjavicIvana Renata Buda @risteski_ (10/10) pic.twitter.com/uOFqR1osSd — Tolić lab (@Toliclab) May 4, 2021
We are happy and honored to be in such good company among the authors of the book on Mitosis in Seminars in Cell and Developmental Biology. Kruno Vukusic did an impressive job of synthesizing a large body of literature and giving new ideas on anaphase B. https://t.co/IuWIpLiZib
— Tolić lab (@Toliclab) April 12, 2021
Thanks to Helder Maiato @mitosisrocks for the invitation and Ivana Saric for the neat drawings! pic.twitter.com/8mhO6ij8kz — Tolić lab (@Toliclab) April 12, 2021
Anaphase mitotic spindle is a highly complex structure made up of different microtubule subpopulations, all contributing to various extents to the active poleward movement of chromosomes and spindle elongation. pic.twitter.com/1dT30b7KZ1
— Tolić lab (@Toliclab) April 12, 2021
Spindle elongation and chromosome segregation occur at precisely regulated velocities during different stages of anaphase B, driven by different force-generating mechanisms. pic.twitter.com/OSHhbe2Z1S — Tolić lab (@Toliclab) April 13, 2021
The origin of the forces required for spindle elongation during anaphase B is still debated between two views depending on where the force is generated: by pushing from the inside of the spindle or pulling from the outside of the spindle. pic.twitter.com/SiGVovgo5V
— Tolić lab (@Toliclab) April 13, 2021
How and where are the pushing and pulling forces that drive spindle elongation generated? Here is a summary of the proposed mechanisms. pic.twitter.com/VvFN94c9iv — Tolić lab (@Toliclab) April 13, 2021
In human spindles, the forces that drive anaphase B are mainly generated by sliding apart of antiparallel microtubules in the spindle midzone. The experimental results that support this “internal” force generation are summarized in this drawing. pic.twitter.com/tPLHunyc4e
— Tolić lab (@Toliclab) April 13, 2021
During the transition from metaphase to anaphase, the localization of major motor proteins and non-motor regulators changes, with many of them moving to the antiparallel microtubules of the spindle midzone. pic.twitter.com/LmA8WYEEH3 — Tolić lab (@Toliclab) April 13, 2021
If you want to know more, check also other papers on anaphase by Kruno Vukusic: https://t.co/6117sW2NHqhttps://t.co/QIQGkJClUx A new paper showing that microtubule sliding modules based on EG5 and PRC1-dependent KIF4A drive spindle elongation is coming out in Dev Cell in May.
— Tolić lab (@Toliclab) April 13, 2021
How does the #spindle align the #chromosomes? We found that bridging fibers lead the chromosomes towards the spindle middle by overlap length-dependent forces. Congrats @Michaela_Jag, @risteski_, Jelena Martincic and Ana Milas! @eLife https://t.co/QZmCjUK8dH (1/13) — Tolić lab (@Toliclab) March 9, 2021
We developed a fast and reversible optogenetic tool to remove the microtubule crosslinker PRC1 (shown in pink) from the bridging fibers in the spindle to the cell membrane. This led to displacement of kinetochores (cyan) from the spindle equator. (2/13) pic.twitter.com/ISpgTI6vUs — Tolić lab (@Toliclab) March 9, 2021
Optogenetic PRC1 removal increased the frequency of lagging kinetochores. Cells with lagging kinetochores in anaphase had a smaller inter-kinetochore distance before anaphase, suggesting that imperfect kinetochore segregation is related to a decrease in tension. (3/13) pic.twitter.com/yz8ExaaImC — Tolić lab (@Toliclab) March 9, 2021
What is the reason for kinetochore misalignment when PRC1 is removed? It could be related to 1) microtubules in the bridging fibers, 2) polar ejection forces, 3) proteins that modulate the dynamics of kinetochore microtubules. (4/13) pic.twitter.com/ANPtnkEqvD — Tolić lab (@Toliclab) March 9, 2021
Bridging fibers were thinner after optogenetic PRC1 removal, and tracking of EB3 on growing tips of bridging microtubules revealed that their overlap regions were longer. (5/13) pic.twitter.com/1g9thFmBLA — Tolić lab (@Toliclab) March 9, 2021
How is the length of the bridging microtubules and their overlaps controlled? We found that Kif4A and Kif18A localize in the bridging fibers during metaphase, which you can see here in cross-sections of vertically oriented spindles. (6/13) pic.twitter.com/Mdl0lDPRHh — Tolić lab (@Toliclab) March 9, 2021
Depletion of Kif4A or Kif18A resulted in longer PRC1-labeled overlaps, suggesting that they regulate the overlap length by suppressing the dynamics of bridging microtubules. (7/13) pic.twitter.com/JbjtljP5Jo — Tolić lab (@Toliclab) March 9, 2021
Optogenetic PRC1 removal resulted in removal of Kif4A from the bridging fiber. This can explain the elongation of overlaps: Kif4A removal led to excessive microtubule growth and thus longer overlaps in metaphase, similar to the long overlaps in anaphase after Kif4A siRNA. (8/13) pic.twitter.com/4Yw6fkPX58 — Tolić lab (@Toliclab) March 9, 2021
We propose that Kif4A and Kif18A at the plus ends of bridging microtubules regulate the overlap length. Kif4A and Eg5 within the bridging fiber possibly slide the microtubules apart, whereas PRC1 stabilizes the overlaps together with MKLP1, Eg5, and other crosslinkers. (9/13) pic.twitter.com/uLOxt8b1cK — Tolić lab (@Toliclab) March 9, 2021
After PRC1 removal, Kif4A remained on chromosome arms, suggesting that polar ejection forces were not perturbed. Kif18A, CLASP1, and CENP-E remained on kinetochore fiber tips, arguing against a change in k-microtubule dynamics as the cause of kinetochore misalignment. (10/13) pic.twitter.com/jQ8Nf5aFui — Tolić lab (@Toliclab) March 9, 2021
In contrast to the acute optogenetic PRC1 removal, long-term PRC1 depletion by siRNA does not lead to chromosome misalignment. This difference suggests the existence of compensatory mechanisms that regulate alignment during long-term depletion. (11/13) pic.twitter.com/mE5YDhBNVv — Tolić lab (@Toliclab) March 9, 2021
In summary, crosslinking of bridging microtubules by PRC1 promotes chromosome alignment by overlap length-dependent forces transmitted to the kinetochore fibers. The overlap facing the farther pole is longer and thus makes more force, pulling the chromosome to the center. (12/13) pic.twitter.com/YGeSxuesoH — Tolić lab (@Toliclab) March 9, 2021
Centering efficiency depends on the relative asymmetry in the overlap length on either side, so short overlaps lead to better centering. Thus, PRC1 helps chromosome alignment through the control of the overlap length between bridging and kinetochore fibers. (13/13) pic.twitter.com/dPNZIeK2VW — Tolić lab (@Toliclab) March 9, 2021
Thanks to @eLife for showcasing our paper! Shining a light on cell division https://t.co/x82t1Fv2Jm — Tolić lab (@Toliclab) March 21, 2021
Researchers at @Toliclab optogenetically controlled the important cell-division protein PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces https://t.co/hO8WkjT3le pic.twitter.com/ov11n2VNU3 — eLife – the journal (@eLife) March 15, 2021
Interested in how organelles, cells, and tissues respond, generate, and coordinate mechanical inputs and outputs? @Dev_Cell recently had a special issue Mechanics behind Cell and Developmental Biology. @nenad_pavin and I wrote about the #spindle.https://t.co/PDzFl6VZXo — Tolić lab (@Toliclab) February 15, 2021
The complexity of the mitotic #spindle motivates the development of a variety of approaches complementary to genetics and biochemistry. Because the spindle is a mechanical machine, the understanding of its functioning requires approaches based on mechanical perturbations. pic.twitter.com/RiLd70Enx5 — Tolić lab (@Toliclab) February 15, 2021
One of the key questions about the #spindle is what forces act on #chromosomes. Mechanobiology experiments led to a picture where pulling forces exerted by #kinetochore #microtubules are opposed by polar ejection forces that push on chromosome arms. pic.twitter.com/xIeG8rYSdO — Tolić lab (@Toliclab) February 15, 2021
Laser cutting of #kinetochore fibers led to the discovery of the mechanism of their reincorporation in the #spindle and of bridging fibers that link sister kinetochore fibers and regulate the forces acting on #chromosomes. pic.twitter.com/KITtoPqGPl — Tolić lab (@Toliclab) February 15, 2021
Motivated by an unusual prediction from a theoretical model, experiments showed that the #microtubule bundles show a left-handed twisted shape, making the whole #spindle a #chiral structure. pic.twitter.com/brbrp6N1mT — Tolić lab (@Toliclab) February 15, 2021