<i>Naegleria’s</i>mitotic spindles are built from unique tubulins and highlight core spindle features

Trupinić, Monika; Ivec, Arian; Swafford, Austin D.; Nolton, Emily; Rice, Luke M.; Tolić, Iva M.; Fritz‐Laylin, Lillian K.; Wadsworth, Patricia

doi:10.1101/2021.02.23.432318

Cited by 3 publications

(4 citation statements)

References 71 publications

(102 reference statements)

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“…In contrast to the left-handed twist of human spindles, spindles in the amoeba Naegleria gruberi are twisted in a right-handed fashion (Velle et al, 2021), which may be due to the differences in kinesins and other microtubule-associated proteins between Naegleria and humans. What determines the direction and amount of twist in spindles of different organisms remains an intriguing topic for future studies.…”

Section: Mechanisms That Generate Spindle Twistmentioning

confidence: 87%

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The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Trupinić

Kokanović

Ponjavić

et al. 2020

Preprint

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Mechanical 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.

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Section: Mechanisms That Generate Spindle Twistmentioning

confidence: 87%

“…Another organism whose spindles are prominently twisted is a unicellular eukaryote, amoeba Naegleria gruberi. Interestingly, amoeba's spindles are predominantly twisted in a right-handed fashion (Velle et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Trupinić

Kokanović

Ponjavić

et al. 2020

Preprint

Self Cite

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“…Because the synthetic spindle is similar to spindles found in HeLa cells, this phenomenological function can be used for all bundles in HeLa spindles and also for spindles that have shapes similar to HeLa cells. For the majority of bundles, this correction will be less than 5% ( 18 , 19 ).…”

Section: Resultsmentioning

confidence: 99%

“…We have recently shown that the spindle in human cells is a chiral object, as bundles follow a left-handed helical path (17,18). Chirality is also present in the spindles of amoeba Naegleria gruberi, though the twist is right-handed (19). The chirality of the spindle is best visualized by looking at the spindle end on, i.e., along the pole-to-pole axis, to observe the three-dimensional shapes with a helical twist (Fig.…”

Section: Introductionmentioning

confidence: 99%

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

Ivec

Trupinić

Tolić

et al. 2021

Biophysical Journal

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The highly ordered spatial organization of microtubule bundles in the mitotic spindle is crucial for its proper functioning. The recent discovery of twisted shapes of microtubule bundles and spindle chirality suggests that the bundles extend along curved paths in three dimensions, rather than being confined to a plane. This, in turn, implies that rotational forces, i.e., torques, exist in the spindle in addition to the widely studied linear forces. However, studies of spindle architecture and forces are impeded by a lack of a robust method for the geometric quantification of microtubule bundles in the spindle. In this work, we describe a simple method for measuring and evaluating the shapes of microtubule bundles by characterizing them in terms of their curvature and twist. By using confocal microscopy, we obtain three-dimensional images of spindles, which allows us to trace the entire microtubule bundle. For each traced bundle, we first fit a plane and then fit a circle lying in that plane. With this robust method, we extract the curvature and twist, which represent the geometric information characteristic for each bundle. As the bundle shapes reflect the forces within them, this method is valuable for the understanding of forces that act on chromosomes during mitosis.

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A conserved pressure-driven mechanism for regulating cytosolic osmolarity

Garner

Beckford

Weeda

et al. 2023

Preprint

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Controlling intracellular osmolarity is essential to all cellular life. Cells that live in hypo-osmotic environments like freshwater must constantly battle water influx to avoid swelling until they burst. Many eukaryotic cells use contractile vacuoles to collect excess water from the cytosol and pump it out of the cell. Although contractile vacuoles are essential to many species, including important pathogens, the mechanisms that control their dynamics remain unclear. To identify basic principles governing contractile vacuole function, we here investigate the molecular mechanisms of two species with distinct vacuolar morphologies from different eukaryotic lineages—the discobanNaegleria gruberi, and the amoebozoan slime moldDictyostelium discoideum. Using quantitative cell biology we find that, although these species respond differently to osmotic challenges, they both use actin for osmoregulation, as well as vacuolar-type proton pumps for filling contractile vacuoles. We also use analytical modeling to show that cytoplasmic pressure is sufficient to drive water out of contractile vacuoles in these species, similar to findings from the alveolateParamecium multimicronucleatum. Because these three lineages diverged well over a billion years ago, we propose that this represents an ancient eukaryotic mechanism of osmoregulation.

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

Cited by 3 publications

References 71 publications

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

The chirality of the mitotic spindle provides a mechanical response to forces and depends on microtubule motors and augmin

Oblique circle method for measuring the curvature and twist of mitotic spindle microtubule bundles

A conserved pressure-driven mechanism for regulating cytosolic osmolarity

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