scholarly journals Coordinated Poleward Flux of Sister Kinetochore Fibers Drives Chromosome Alignment

2021 ◽  
Author(s):  
Patrik Risteski ◽  
Domagoj Božan ◽  
Mihaela Jagrić ◽  
Agneza Bosilj ◽  
Nenad Pavin ◽  
...  
Keyword(s):  
Sister Kinetochore ◽  
Chromosome Alignment ◽  
Poleward Flux

scholarly journals The coupling between sister kinetochore directional instability and oscillations in centromere stretch in metaphase PtK1 cells

2012 ◽  
Vol 23 (6) ◽  
pp. 1035-1046 ◽  
Author(s):  
Xiaohu Wan ◽  
Daniela Cimini ◽  
Lisa A. Cameron ◽  
E. D. Salmon
Keyword(s):  
High Resolution ◽  
Dynamic Instability ◽  
Sister Kinetochore ◽  
Kinetics Of ◽  
Poleward Flux

Kinetochores bound to kinetochore microtubules (kMTs) exhibit directional instability in mammalian and other mitotic vertebrate cells, oscillating between poleward (P) and away-from-the-pole (AP) movements. These oscillations are coupled to changes in length of kMTs in a way that maintains a net stretch of the centromere. To understand how sister kinetochore directional instability and kMT plus-end dynamic instability are coupled to oscillations in centromere stretch, we tracked at high resolution the positions of fluorescent kinetochores and their poles for oscillating chromosomes within spindles of metaphase PtK1 cells. We found that the kinetics of P and AP movement are nonlinear and different. By subtracting contributions from the poleward flux of kMTs, we found that maximum centromere stretch occurred when the leading kinetochore switched from depolymerization to polymerization, whereas minimum centromere stretch occurred on average 7 s after the initially trailing kinetochore switched from polymerization to depolymerization. These differences produce oscillations in centromere stretch at about twice the frequency of kinetochore directional instability and at about twice the frequency of centromere oscillations back and forth across the spindle equator.

scholarly journals Tension-dependent Regulation of Microtubule Dynamics at Kinetochores Can Explain Metaphase Congression in Yeast

2005 ◽  
Vol 16 (8) ◽  
pp. 3764-3775 ◽  
Author(s):  
Melissa K. Gardner ◽  
Chad G. Pearson ◽  
Brian L. Sprague ◽  
Ted R. Zarzar ◽  
Kerry Bloom ◽  
...  
Keyword(s):  
Fluorescence Recovery After Photobleaching ◽  
Attachment Site ◽  
Spatial Gradient ◽  
Attachment Sites ◽  
Sister Kinetochore ◽  
Chromosome Alignment ◽  
Pulling Forces ◽  
Best Fit ◽  
Theory And Experiment ◽  
Rule Out

During metaphase in budding yeast mitosis, sister kinetochores are tethered to opposite poles and separated, stretching their intervening chromatin, by singly attached kinetochore microtubules (kMTs). Kinetochore movements are coupled to single microtubule plus-end polymerization/depolymerization at kinetochore attachment sites. Here, we use computer modeling to test possible mechanisms controlling chromosome alignment during yeast metaphase by simulating experiments that determine the 1) mean positions of kinetochore Cse4-GFP, 2) extent of oscillation of kinetochores during metaphase as measured by fluorescence recovery after photobleaching (FRAP) of kinetochore Cse4-GFP, 3) dynamics of kMTs as measured by FRAP of GFP-tubulin, and 4) mean positions of unreplicated chromosome kinetochores that lack pulling forces from a sister kinetochore. We rule out a number of possible models and find the best fit between theory and experiment when it is assumed that kinetochores sense both a spatial gradient that suppresses kMT catastrophe near the poles and attachment site tension that promotes kMT rescue at higher amounts of chromatin stretch.

scholarly journals Sliding of kinetochore fibers along bridging fibers helps center the chromosomes on the spindle

2021 ◽  
Author(s):  
Patrik Risteski ◽  
Mihaela Jagrić ◽  
Iva M. Tolić
Keyword(s):  
Chromosome Segregation ◽  
Human Cells ◽  
Remarkable Feature ◽  
Mechanical Coupling ◽  
Chromosome Alignment ◽  
The Difference ◽  
Bridging Fibers ◽  
Lateral Length ◽  
Poleward Flux

ABSTRACTChromosome alignment at the spindle equator during metaphase is the most remarkable feature of mitosis, which promotes proper chromosome segregation and depends on the forces exerted at the plus end of kinetochore microtubules and polar ejection forces. However, forces arising from lateral mechanical coupling of kinetochore fibers with non-kinetochore microtubules play a role in chromosome alignment, but the mechanism is unclear. Here we develop a speckle microscopy assay to measure the poleward flux of individual microtubules in spindles of human cells and show that bridging microtubules slide apart and undergo poleward flux at a higher rate than kinetochore microtubules. Depletion of the microtubule coupler NuMa increased the difference in the flux velocity of kinetochore and bridging microtubules, suggesting that sliding forces from the bridging fiber are transmitted largely through NuMa onto the associated kinetochore fibers. Depletions of Kif18A/kinesin-8, Kif4A/kinesin-4, as well as double depletions of Kif18A together with Kif4A or Kif18A together with the crosslinker of antiparallel microtubules PRC1 increased the flux velocity of kinetochore fibers up to the velocity of bridging fibers, due to the increased coupling resulting from the extended antiparallel overlap regions. We found severe kinetochore misalignment after double Kif18A and Kif4A as well as Kif18A and PRC1 depletions compared to a single Kif18A depletion, suggesting that forces within the bridging fiber have a centering effect on the kinetochores. We propose that lateral length-dependent sliding forces that the bridging fiber exerts onto kinetochore fibers drive the movement of kinetochores towards the spindle center, thereby promoting chromosome alignment.

scholarly journals Optogenetic control of PRC1 reveals its role in chromosome alignment on the spindle by overlap length-dependent forces

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Mihaela Jagrić ◽  
Patrik Risteski ◽  
Jelena Martinčić ◽  
Ana Milas ◽  
Iva M Tolić
Keyword(s):  
Metaphase Chromosome ◽  
Mechanical Coupling ◽  
Chromosome Position ◽  
Sister Kinetochore ◽  
Chromosome Alignment ◽  
Bridging Fibers ◽  
Optogenetic Control

During metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to partially disassemble bridging fibers and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and largely lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochore fibers.

scholarly journals Augmin regulates kinetochore tension and spatial arrangement of spindle microtubules by nucleating bridging fibers

2020 ◽  
Author(s):  
Martina Manenica ◽  
Valentina Štimac ◽  
Isabella Koprivec ◽  
Juraj Simunić ◽  
Iva M. Tolić
Keyword(s):  
Critical Role ◽  
Spatial Arrangement ◽  
Knock Out ◽  
Daughter Cells ◽  
Sister Kinetochore ◽  
Division Spindle ◽  
Spindle Microtubules ◽  
Bridging Fibers ◽  
Poleward Flux

ABSTRACTThe mitotic spindle functions as a molecular micromachine that evenly distributes chromosomes into two daughter cells during cell division. Spindle microtubules in human cells are mainly nucleated at the centrosome and on the lateral surface of existing microtubules by the augmin complex. However, it is unknown how the augmin-mediated nucleation affects functionally distinct microtubule bundles and consequently the forces within the spindle. Here we show, by using siRNA depletion and CRISPR knock-out of the augmin complex subunits HAUS6 or HAUS8, that augmin is crucial for the nucleation of bridging microtubules, which laterally link sister kinetochore fibers. Augmin depletion resulted in a reduction in the number of microtubules within bridging fibers by around 80% and in kinetochore fibers by 40%, suggesting that the bridging microtubules are mainly nucleated at the surface of present microtubules. In augmin-depleted cells, the interkinetochore distance decreased preferentially for kinetochores that lack a bridging fiber, independently of the thickness of their k-fibers, implying that augmin affects forces on kinetochores largely via bridging fibers. Without augmin the number of bridging fibers decreased, with the remaining ones mostly confined to the spindle periphery with an increased overlap length. A slower poleward flux of microtubules after augmin depletion is indicative of slower sliding within the bridging fiber. Our results demonstrate a critical role of augmin in the formation of bridging microtubules and proper architecture of the metaphase spindle, suggesting a model where sliding of augmin-nucleated bridging microtubules promotes poleward flux of k-fibers and thus tension on kinetochores.

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

2019 ◽  
Author(s):  
Mihaela Jagrić ◽  
Patrik Risteski ◽  
Jelena Martinčić ◽  
Ana Milas ◽  
Iva M. Tolić
Keyword(s):  
Metaphase Chromosome ◽  
Mechanical Coupling ◽  
Chromosome Position ◽  
Sister Kinetochore ◽  
Chromosome Alignment ◽  
Bridging Fibers ◽  
Optogenetic Control

AbstractDuring metaphase, chromosome position at the spindle equator is regulated by the forces exerted by kinetochore microtubules and polar ejection forces. However, the role of forces arising from mechanical coupling of sister kinetochore fibers with bridging fibers in chromosome alignment is unknown. Here we develop an optogenetic approach for acute removal of PRC1 to disassemble bridging fibers, and show that they promote chromosome alignment. Tracking of the plus-end protein EB3 revealed longer antiparallel overlaps of bridging microtubules upon PRC1 removal, which was accompanied by misaligned and lagging kinetochores. Kif4A/kinesin-4 and Kif18A/kinesin-8 were found within the bridging fiber and lost upon PRC1 removal, suggesting that these proteins regulate the overlap length of bridging microtubules. We propose that PRC1-mediated crosslinking of bridging microtubules and recruitment of kinesins to the bridging fiber promotes chromosome alignment by overlap length-dependent forces transmitted to the associated kinetochores fibers.

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