Naegleria’s mitotic spindles are built from unique tubulins and highlight core spindle features

ABSTRACTNaegleria gruberi is a unicellular eukaryote whose evolutionary distance from animals and fungi has made it useful for developing hypotheses about the last common eukaryotic ancestor. Naegleria amoebae lack a cytoplasmic microtubule cytoskeleton and assemble microtubules only during mitosis, and thus provides a unique system to study the evolution and functional specificity of mitotic tubulins and the resulting spindle. Previous studies showed that Naegleria amoebae express a divergent α-tubulin during mitosis and we now show that Naegleria amoebae express a second mitotic α- and two mitotic β-tubulins. The mitotic tubulins are evolutionarily divergent relative to typical α- and β- tubulins, contain residues that suggest distinct microtubule properties, and may represent drug targets for the “brain-eating amoeba” Naegleria fowleri. Using quantitative light microscopy, we find that Naegleria’s mitotic spindle is a distinctive barrel-like structure built from a ring of microtubule bundles. Similar to those of other species, Naegleria’s spindle is twisted and its length increases during mitosis suggesting that these aspects of mitosis are ancestral features. Because bundle numbers change during metaphase, we hypothesize that the initial bundles represent kinetochore fibers, and secondary bundles function as bridging fibers.

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

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.

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

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.

Twist of the mitotic spindle culminates at anaphase onset and depends on microtubule-associated proteins along with external forces

ABSTRACTMechanical forces produced by motor proteins and microtubule dynamics within the mitotic spindle are crucial for the movement of chromosomes and their segregation into the emerging daughter cells. In addition to linear forces, rotational forces are present in the spindle, reflected in the left-handed twisted shapes of microtubule bundles that make the spindle chiral. However, the molecular origins of spindle chirality are unknown. Here we show that spindles are most twisted at the beginning of anaphase, and reveal multiple molecular players involved in spindle chirality. Inhibition of Eg5/kinesin-5 in a non-cancer cell line abolished spindle twist and depletion of Kif18A/kinesin-8 resulted in a right-handed twist, implying that these motors regulate twist likely by rotating the microtubules around one another within the antiparallel overlaps of bridging fibers. Depletion of the crosslinker PRC1 resulted in a right-handed twist, indicating that PRC1 may contribute to the twist by constraining free rotation of microtubules. Overexpression of PRC1 abolished twist, possibly due to increased torsional rigidity of the bundles. Depletion of augmin led to a right-handed twist, suggesting that twist depends on the geometry of microtubule nucleation. Round spindles were more twisted than elongated ones, a notion that we directly tested by compressing the spindle along its axis, which resulted in stronger left-handed twist, indicating a correlation between bending moments and twist. We conclude that spindle twist is controlled by multiple molecular mechanisms acting at different locations within the spindle as well as forces, and propose a potential physiological role of twist in promoting passive mechanical response of the spindle to forces during metaphase.

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

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.

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

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 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.

Microtubule organization within mitotic spindles revealed by serial block face scanning EM and image analysis

Serial block face scanning electron microscopy (SBF-SEM) is a powerful method to analyze cells in 3D. Here, working at the resolution limit of the method, we describe a correlative light-SBF-SEM workflow to resolve microtubules of the mitotic spindle in human cells. We present three examples of uses for this workflow which are not practical by light microscopy and/or TEM. First, distinguishing closely associated microtubules within K-fibers; second, resolving bridging fibers in the mitotic spindle; third, visualizing membranes in mitotic cells, relative to the spindle apparatus. Our workflow also includes new computational tools for exploring the spatial arrangement of MTs within the mitotic spindle. We use these tools to show that microtubule order in mitotic spindles is sensitive to the level of TACC3 on the spindle.