Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells

Milas, Ana; Jagrić, Mihaela; Martinčić, Jelena; Tolić, Iva Marija

doi:10.1016/bs.mcb.2018.03.024

Cited by 14 publications

(12 citation statements)

References 68 publications

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“…Unlike previous optogenetic approaches (Fielmich et al, 2018;Okumura et al, 2018;van Haren et al, 2018;Yang et al, 2013;Zhang et al, 2017), this method allows for global lossof-function of full-length spindle proteins, relying on simple protein tagging rather than domain splitting, with no need of chromophore addition. Moreover, this method may be implemented with other optical perturbations (Milas et al, 2018) and used as "in vivo pulldown" for probing protein-protein interactions in different phases of the cell cycle. However, this approach depends on high turnover of the protein in comparison with the time scale of interest.…”

Section: An Optogenetic System For Acute Selective and Reversible Rmentioning

confidence: 99%

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

Jagrić

Risteski

Martinčić

et al. 2019

Preprint

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During metaphase, chromosome position at the spindle equator is mainly regulated by the forces exerted by kinetochore microtubules. However, the role of forces arising from mechanical coupling between sister kinetochore fibers and bridging fibers, whose antiparallel microtubules are crosslinked by protein regulator of cytokinesis 1 (PRC1), in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 and show that PRC1 promotes kinetochore alignment. PRC1 removal resulted in reduction of bridging fibers and straightening of outermost kinetochore fibers. The inter-kinetochore distance decreased, the metaphase plate widened, and lagging kinetochores appeared, suggesting a role of PRC1 in regulating forces on kinetochores. MKLP1/kinesin-6 was lost from the spindle together with PRC1, whereas Kif4A/kinesin-4 remained on chromosomes and CLASP1, Kif18A/kinesin-8, and CENP-E/kinesin-7 on kinetochore fiber tips. We conclude that in metaphase PRC1, by mechanically coupling bridging and kinetochore fibers, regulates spindle mechanics and buffers kinetochore movements, promoting chromosome alignment.

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Section: An Optogenetic System For Acute Selective and Reversible Rmentioning

confidence: 99%

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

Jagrić

Risteski

Martinčić

et al. 2019

Preprint

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“…Indeed, MT and k-fibers minus-ends dynamics in mitosis still remain largely less understood than plus-ends dynamics due to the fact that they are embedded into the main spindle and thus difficult to isolate and image. Advanced imaging methods and perturbation tools, including laser ablation, now allow to isolate and track MT minus-ends in space and time 8,[27][28][29] . In this context, our work provides new insights on the regulation of k-fibers dynamics by involving a new player, IFT88, in the control of NuMA local enrichment at minus-ends.…”

Section: Discussionmentioning

confidence: 99%

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their reincorporation into mitotic spindles

Taulet

Douanier

Vitre

et al. 2019

Preprint

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To build and maintain mitotic spindle architecture, molecular motors exert spatially regulated forces on microtubules (MT) minus-ends. This spatial regulation is required to allow proper chromosomes alignment through the organization of kinetochore fibers (k-fibers). NuMA was recently shown to target dynactin to MT minus-ends and thus to spatially regulate dynein activity. However, given that k-fibers are embedded in the spindle, our understanding of the machinery involved in the targeting of proteins to their minus-ends remains limited. Intraflagellar transport (IFT) proteins were primarily studied for their ciliary roles but they also emerged as key regulators of cell division. Taking advantage of MT laser ablation, we show here that IFT88 concentrates at k-fibers minus-ends and is required for their re-anchoring into spindles by controlling NuMA accumulation. Indeed, IFT88 interacts with NuMA and is required for its enrichment at newly generated k-fibers minus-ends. Combining nocodazole washout experiments and IFT88 depletion, we further show that IFT88 is required for the reorganization of k-fibers into spindles and thus for efficient chromosomes alignment in mitosis. Overall, we propose that IFT88 could serve as a mitotic MT minus-end adaptor to concentrate NuMA at minus-ends thus facilitating kfibers incorporation into the main spindle.can be considered as a platform for such a regulation. Indeed, NuMA was recently shown to target dynactin to minus-ends and thus spatially regulate dynein activity 8,9 . If proteins recruitment at MT plus-end has been well-documented, the specific targeting of proteins to MTs and k-fiber minusends has yet to be fully understood 10 . Indeed, continuous reintegration of k-fibers happens inside an established spindle and is essential to preserve its integrity and to ensure proper chromosomes alignment 6-8 . However, given that k-fibers are embedded in the main spindle, our understanding of the machinery involved in the targeting of proteins to their minus-ends remains limited.The intraflagellar transport (IFT) machinery is a well conserved intra-cellular transport system that has been studied for a long time for its role in cilia formation and function in non-dividing cells 11 .During IFT-mediated transport, kinesin and dynein motors drive the bidirectional transport of IFT trains from the base to the tip of the cilium in an anterograde movement and from the tip to the base in a retrograde movement 12 . IFTs were therefore accepted to function as cargos, for example, of axoneme precursors such as tubulins as well as molecules of the signal transduction machinery inside the cilium 13,14 . More recently, IFTs were shown to contribute to ciliary motor activation 15 indicating that a lot remains to be done to fully understand the roles of this complex machinery. Interestingly, in addition to their ciliary roles, IFTs were also shown to function in interphase in the regulation of MT dynamics in the cytoplasm 16 . Moreover, they were shown to contribute to intracellular transport in n...

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“…One of the most successful applications of LS-OT has been the trapping and/or damaging of (mitotic) chromosomes. This kind of studies will keep providing critical information on mitosis kinetics, regulation and consequences of chromosomal dysfunctionality ( Kong et al, 2017 ; Milas et al, 2018 ; Odell et al, 2019 ). For example, a very recent publication reports on a very fine level of LS disruption of particular mitotic machinery elements in order to better understand mitotic chromosome migration ( Forer and Berns, 2020 ).…”

Section: Perspectivesmentioning

confidence: 99%

“…Given the fine control that OT offer to trap and manipulate either whole chromosomes or chromosome fragments ( Berns et al, 1989 ; Harsono et al, 2013 ; Milas et al, 2018 ), plus the possibility to directly produce chromosome fragments with LS ( Berns et al, 2006 ), it is clear that these tools should be promptly employed to discern the mechanisms of chromothripsis. For example, less subtle procedures, like exposure of cell cultures to mild concentrations of ROS, has been proved to be an effective method to induce large amounts of micronuclei ( Blázquez-Castro and Stockert, 2015 ).…”

Section: Perspectivesmentioning

confidence: 99%

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

Blázquez‐Castro

Fernández‐Piqueras

Santos

2020

Front. Bioeng. Biotechnol.

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Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers, in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presented.

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Optogenetic reversible knocksideways, laser ablation, and photoactivation on the mitotic spindle in human cells

Cited by 14 publications

References 68 publications

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

Optogenetic control of PRC1 reveals that bridging fibers promote chromosome alignment by overlap length-dependent forces

IFT88 controls NuMA enrichment at k-fibers minus-ends to facilitate their reincorporation into mitotic spindles

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

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