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