Alexandra Long

All Publications

• MAP9/MAPH-9 supports axonemal microtubule doublets and modulates motor movement. Developmental cell Tran, M. V., Khuntsariya, D., Fetter, R. D., Ferguson, J. W., Wang, J. T., Long, A. F., Cote, L. E., Wellard, S. R., Vazquez-Martinez, N., Sallee, M. D., Genova, M., Magiera, M. M., Eskinazi, S., Lee, J. D., Peel, N., Janke, C., Stearns, T., Shen, K., Lansky, Z., Magescas, J., Feldman, J. L. 2023

  Abstract

  Microtubule doublets (MTDs) comprise an incomplete microtubule (B-tubule) attached to the side of a complete cylindrical microtubule. These compound microtubules are conserved in cilia across the tree of life; however, the mechanisms by which MTDs form and are maintained invivo remain poorly understood. Here, we identify microtubule-associated protein 9 (MAP9) as an MTD-associated protein. We demonstrate that C.elegans MAPH-9, a MAP9 homolog, is present during MTD assembly and localizes exclusively to MTDs, a preference that is in part mediated by tubulin polyglutamylation. We find that loss of MAPH-9 causes ultrastructural MTD defects, including shortened and/or squashed B-tubules with reduced numbers of protofilaments, dysregulated axonemal motor velocity, and perturbed cilia function. Because we find that the mammalian ortholog MAP9 localizes to axonemes in cultured mammalian cells and mouse tissues, we propose that MAP9/MAPH-9 plays a conserved role in regulating ciliary motors and supporting the structure of axonemal MTDs.

  View details for DOI 10.1016/j.devcel.2023.12.001

  View details for PubMedID 38159567

• ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells. The EMBO journal Kai, F., Ou, G., Tourdot, R. W., Stashko, C., Gaietta, G., Swift, M. F., Volkmann, N., Long, A. F., Han, Y., Huang, H. H., Northey, J. J., Leidal, A. M., Viasnoff, V., Bryant, D. M., Guo, W., Wiita, A. P., Guo, M., Dumont, S., Hanein, D., Radhakrishnan, R., Weaver, V. M. 2022: e109205

  Abstract

  Patient-derived organoids and cellular spheroids recapitulate tissue physiology with remarkable fidelity. We investigated how engagement with a reconstituted basement membrane in three dimensions (3D) supports the polarized, stress resilient tissue phenotype of mammary epithelial spheroids. Cells interacting with reconstituted basement membrane in 3D had reduced levels of total and actin-associated filamin and decreased cortical actin tension that increased plasma membrane protrusions to promote negative plasma membrane curvature and plasma membrane protein associations linked to protein secretion. By contrast, cells engaging a reconstituted basement membrane in 2D had high cortical actin tension that forced filamin unfolding and endoplasmic reticulum (ER) associations. Enhanced filamin-ER interactions increased levels of PKR-like ER kinase effectors and ER-plasma membrane contact sites that compromised calcium homeostasis and diminished cell viability. Consequently, cells with decreased cortical actin tension had reduced ER stress and survived better. Consistently, cortical actin tension in cellular spheroids regulated polarized basement membrane membrane deposition and sensitivity to exogenous stress. The findings implicate cortical actin tension-mediated filamin unfolding in ER function and underscore the importance of tissue mechanics in organoid homeostasis.

  View details for DOI 10.15252/embj.2021109205

  View details for PubMedID 35880301

• A not-so-simple twist of fate. Developmental cell Long, A. F., Stearns, T. 2021; 56 (4): 402–4

  Abstract

  Multiciliated cells are considered terminally differentiated, yet tissues bearing them are remodeled during development and after injury. In this issue of Developmental Cell, Tasca etal. (2021) show that multiciliated epithelial cells are lost via two different Notch-dependent processes, apoptosis and transdifferentiation, during developmental remodeling of the Xenopus epidermis.

  View details for DOI 10.1016/j.devcel.2021.02.003

  View details for PubMedID 33621489

• Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure. The Journal of cell biology Long, A. F., Suresh, P., Dumont, S. 2020; 219 (8)

  Abstract

  At cell division, the mammalian kinetochore binds many spindle microtubules that make up the kinetochore-fiber. To segregate chromosomes, the kinetochore-fiber must be dynamic and generate and respond to force. Yet, how it remodels under force remains poorly understood. Kinetochore-fibers cannot be reconstituted in vitro, and exerting controlled forces in vivo remains challenging. Here, we use microneedles to pull on mammalian kinetochore-fibers and probe how sustained force regulates their dynamics and structure. We show that force lengthens kinetochore-fibers by persistently favoring plus-end polymerization, not by increasing polymerization rate. We demonstrate that force suppresses depolymerization at both plus and minus ends, rather than sliding microtubules within the kinetochore-fiber. Finally, we observe that kinetochore-fibers break but do not detach from kinetochores or poles. Together, this work suggests an engineering principle for spindle structural homeostasis: different physical mechanisms of local force dissipation by the k-fiber limit force transmission to preserve robust spindle structure. These findings may inform how other dynamic, force-generating cellular machines achieve mechanical robustness.

  View details for DOI 10.1083/jcb.201911090

  View details for PubMedID 32435797

• Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes ELIFE Suresh, P., Long, A. F., Dumont, S. 2020; 9

  Abstract

  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.

  View details for DOI 10.7554/eLife.53807

  View details for Web of Science ID 000524007500001

  View details for PubMedID 32191206

  View details for PubMedCentralID PMC7117910

• The mammalian kinetochore-microtubule interface: robust mechanics and computation with many microtubules CURRENT OPINION IN CELL BIOLOGY Long, A. F., Kuhn, J., Dumont, S. 2019; 60: 60–67

  Abstract

  The kinetochore drives chromosome segregation at cell division. It acts as a physical link between chromosomes and dynamic microtubules, and as a signaling hub detecting and processing microtubule attachments to control anaphase onset. The mammalian kinetochore is a large macromolecular machine that forms a dynamic interface with the many microtubules that it binds. While we know most of the kinetochore's component parts, how they work together to give rise to its robust functions remains poorly understood. Here we highlight recent findings that shed light on this question, driven by an expanding physical and molecular toolkit. We present emerging principles that underlie the kinetochore's robust microtubule grip, such as redundancy, specialization, and dynamicity, and present signal processing principles that connect this microtubule grip to robust computation. Throughout, we identify open questions, and define simple engineering concepts that provide insight into kinetochore function.

  View details for DOI 10.1016/j.ceb.2019.04.004

  View details for Web of Science ID 000486545800009

  View details for PubMedID 31132675

  View details for PubMedCentralID PMC6756937

• Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules CURRENT BIOLOGY Long, A. F., Udy, D. B., Dumont, S. 2017; 27 (11): 1692-+

  Abstract

  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] in mammalian cells. 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.

  View details for DOI 10.1016/j.cub.2017.04.058

  View details for Web of Science ID 000402814600057

  View details for PubMedID 28552353

  View details for PubMedCentralID PMC5502739

• Kinesin-5: A Team Is Just the Sum of Its Parts DEVELOPMENTAL CELL Hueschen, C. L., Long, A. F., Dumont, S. 2015; 34 (6): 609–10

  Abstract

  How the cell builds a spindle remains an open question. In this issue of Developmental Cell, Shimamoto, Forth, and Kapoor (2015) show that kinesin-5 motor ensembles can exert sliding forces that scale with microtubule overlap length. This behavior could allow microtubule architecture-dependent modulation of force and contribute to spindle self-organization.

  View details for DOI 10.1016/j.devcel.2015.09.010

  View details for Web of Science ID 000361941500001

  View details for PubMedID 26418291

• Structural and Electronic Properties of Old and New A(2)[M(pin(F))(2)] Complexes INORGANIC CHEMISTRY Tahsini, L., Specht, S. E., Lum, J. S., Nelson, J. M., Long, A. F., Golen, J. A., Rheingold, A. L., Doerrer, L. H. 2013; 52 (24): 14050–63

  Abstract

  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).

  View details for DOI 10.1021/ic401837y

  View details for Web of Science ID 000328668400034

  View details for PubMedID 24261319

• Mechanical Fluidity of Fully Suspended Biological Cells BIOPHYSICAL JOURNAL Maloney, J. M., Lehnhardt, E., Long, A. F., Van Vliet, K. J. 2013; 105 (8): 1767–77

  Abstract

  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.

  View details for DOI 10.1016/j.bpj.2013.08.040

  View details for Web of Science ID 000325838500006

  View details for PubMedID 24138852

  View details for PubMedCentralID PMC3797573