Summary The kinetochore links chromosomes to dynamic spindle microtubules and drives both chromosome congression and segregation. To do so, the kinetochore must hold on to depolymerizing and polymerizing microtubules. At metaphase, one sister kinetochore couples to depolymerizing microtubules, pulling its sister along polymerizing microtubules [1,2]. Distinct kinetochore-microtubule interfaces mediate these behaviors: active interfaces transduce microtubule depolymerization into mechanical work, and passive interfaces generate friction as the kinetochore moves along microtubules [3,4]. Despite a growing understanding of the molecular components that mediate kinetochore binding [5–7], we do not know how kinetochores physically interact with polymerizing versus depolymerizing microtubule bundles, and whether they use the same mechanisms and regulation to do so. To address this question, we focus on the mechanical role of the essential load-bearing protein Hec1 [8–11]. Hec1’s affinity for microtubules is regulated by Aurora B phosphorylation on its N-terminal tail [12–15], but its role at the interface with polymerizing versus depolymerizing microtubules remains unclear. Here, we use laser ablation to trigger cellular pulling on mutant kinetochores and decouple sisters in vivo, and thereby separately probe Hec1’s role on polymerizing versus depolymerizing microtubules. We show that Hec1 tail phosphorylation tunes friction along polymerizing microtubules and yet does not compromise the kinetochore’s ability to grip depolymerizing microtubules. Together, the data suggest that kinetochore regulation has differential effects on engagement with growing and shrinking microtubules. Through this mechanism, the kinetochore can modulate its grip on microtubules over mitosis, and yet retain its ability to couple to microtubules powering chromosome movement.

11The spindle generates force to segregate chromosomes at cell division. In mammalian 12 cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle 13 anchors kinetochore-fibers in space and time to coordinate chromosome movement. 14 Yet, how it does so remains poorly understood as we lack tools to directly challenge this 15 anchorage. Here, we adapt microneedle manipulation to exert local forces on the 16 spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals that the 17 spindle retains local architecture in its center on the seconds timescale. Upon pulling, 18 sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting 19 chromosome stretching. Further, pulled kinetochore-fibers freely pivot around poles but 20 not around chromosomes, retaining their orientation within 3 µm of chromosomes. This 21 local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. 22Together, these observations indicate short-lived, specialized reinforcement of the 23 kinetochore-fiber in the spindle center. This could help the spindle protect local structure 24 near chromosomes from transient forces while allowing its remodeling over longer 25 timescales, thereby supporting robust chromosome attachments and movements. 26 27 48 spindle. K-fibers make contacts along their length with a dense network of non-49 kinetochore microtubules (non-kMTs) (Mastronarde et al., 1993;McDonald et al., 1992), 50 likely through both motor and non-motor microtubule binding proteins (Elting et al., 51 2017;Kajtez et al., 2016;Vladimirou et al., 2013). We know that the non-kMT network 52 bridges sister k-fibers together (Kajtez et al., 2016;Mastronarde et al., 1993; Witt, Ris & 53 Borisy, 1981), and that it can locally anchor k-fibers and bear load in the spindle's 54 longitudinal (pole-pole) axis (Elting et al., 2017). Yet, how the dynamic spindle 55 mechanically anchors k-fibers in space and time remains poorly mapped and 56 understood. Specifically, we do not know if k-fibers are anchored uniformly along their 57 length, to what structures they are anchored to, over what timescale this anchorage 58 persists before remodeling is allowed, or more broadly how local forces propagate 59 through the spindle's longitudinal and lateral axes. These questions are central to the 60spindle's ability to robustly maintain its structure, respond to force and ultimately move 61 chromosomes. 62We currently lack tools to apply forces with both spatial and temporal control to 63 mammalian spindles. For example, laser ablation, commonly used to alter forces in the 64 spindle, can locally perturb spindle structure, but lacks control over the duration and 65 direction of ensuing force changes. Further, mammalian spindles cannot yet be 66 reconstituted in vitro. To understand how the dynamic spindle robustly anchors k-fibers, 67 and to ultimately map mammalian spindle mechanics to function, we need approaches 68 to apply local and reproducible forces inside cells, with spatiotemp...

Seven new homoleptic complexes of the form A2[M(pin(F))2] have been synthesized with the dodecafluoropinacolate (pin(F))(2-) ligand, namely (Me4N)2[Fe(pin(F))2], 1; (Me4N)2[Co(pin(F))2], 2; ((n)Bu4N)2[Co(pin(F))2], 3; {K(DME)2}2[Ni(pin(F))2], 4; (Me4N)2[Ni(pin(F))2], 5; {K(DME)2}2[Cu(pin(F))2], 7; and (Me4N)2[Cu(pin(F))2], 8. In addition, the previously reported complexes K2[Cu(pin(F))2], 6, and K2[Zn(pin(F))2], 9, are characterized in much greater detail in this work. These nine compounds have been characterized by UV-vis spectroscopy, cyclic voltammetry, elemental analysis, and for paramagnetic compounds, Evans method magnetic susceptibility. Single-crystal X-ray crystallographic data were obtained for all complexes except 5. The crystallographic data show a square-planar geometry about the metal center in all Fe (1), Ni (4), and Cu (6, 7, 8) complexes independent of countercation. The Co species exhibit square-planar (3) or distorted square-planar geometries (2), and the Zn species (9) is tetrahedral. No evidence for solvent binding to any Cu or Zn complex was observed. Solvent binding in Ni can be tuned by the countercation, whereas in Co only strongly donating Lewis solvents bind independent of the countercation. Indirect evidence (diffuse reflectance spectra and conductivity data) suggest that 5 is not a square-planar compound, unlike 4 or the literature K2[Ni(pin(F))2]. Cyclic voltammetry studies reveal reversible redox couples for Ni(III)/Ni(II) in 5 and for Cu(III)/Cu(II) in 8 but quasi-reversible couples for the Fe(III)/Fe(II) couple in 1 and the Co(III)/Co(II) couple in 2. Perfluorination of the pinacolate ligand results in an increase in the central C-C bond length due to steric clashes between CF3 groups, relative to perhydropinacolate complexes. Both types of pinacolate complexes exhibit O-C-C-O torsion angles around 40°. Together, these data demonstrate that perfluorination of the pinacolate ligand makes possible highly unusual and coordinatively unsaturated high-spin metal centers with ready thermodynamic access to rare oxidation states such as Ni(III) and Cu(III).

The spindle generates force to segregate chromosomes at cell division. In mammalian cells, kinetochore-fibers connect chromosomes to the spindle. The dynamic spindle anchors kinetochore-fibers in space and time to move chromosomes. Yet, how it does so remains poorly understood as we lack tools to directly challenge this anchorage. Here, we adapt microneedle manipulation to exert local forces on the spindle with spatiotemporal control. Pulling on kinetochore-fibers reveals the preservation of local architecture in the spindle-center over seconds. Sister, but not neighbor, kinetochore-fibers remain tightly coupled, restricting chromosome stretching. Further, pulled kinetochore-fibers pivot around poles but not chromosomes, retaining their orientation within 3 μm of chromosomes. This local reinforcement has a 20 s lifetime, and requires the microtubule crosslinker PRC1. Together, these observations indicate short-lived, specialized reinforcement in the spindle center. This could help protect chromosome attachments from transient forces while allowing spindle remodeling, and chromosome movements, over longer timescales.

Mechanical characteristics of single biological cells are used to identify and possibly leverage interesting differences among cells or cell populations. Fluidity-hysteresivity normalized to the extremes of an elastic solid or a viscous liquid-can be extracted from, and compared among, multiple rheological measurements of cells: creep compliance versus time, complex modulus versus frequency, and phase lag versus frequency. With multiple strategies available for acquisition of this nondimensional property, fluidity may serve as a useful and robust parameter for distinguishing cell populations, and for understanding the physical origins of deformability in soft matter. Here, for three disparate eukaryotic cell types deformed in the suspended state via optical stretching, we examine the dependence of fluidity on chemical and environmental influences at a timescale of ∼1 s. We find that fluidity estimates are consistent in the time and frequency domains under a structural damping (power-law or fractional-derivative) model, but not under an equivalent-complexity, lumped-component (spring-dashpot) model; the latter predicts spurious time constants. Although fluidity is suppressed by chemical cross-linking, we find that ATP depletion in the cell does not measurably alter the parameter, and we thus conclude that active ATP-driven events are not a crucial enabler of fluidity during linear viscoelastic deformation of a suspended cell. Finally, by using the capacity of optical stretching to produce near-instantaneous increases in cell temperature, we establish that fluidity increases with temperature-now measured in a fully suspended, sortable cell without the complicating factor of cell-substratum adhesion.