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