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Open Access Publications from the University of California

Scholarly Works (9 results)

  • Thesis
  • Peer Reviewed
UC San Francisco Electronic Theses and Dissertations (2020)

For a cell to divide correctly, the spindle must connect to and align chromosomes and then generate force to move them into two daughter cells. The kinetochore is the macromolecular machine that connects chromosomes to a bundle of dynamic microtubules, the kinetochore-fiber (k-fiber). While we have a nearly complete parts list of kinetochore components and regulators, how they together give rise to the robust mechanics of the kinetochore-microtubule interface remains poorly understood. This is due to the fact that mammalian kinetochores and k-fibers cannot yet be reconstituted in vitro and there are few tools to perturb forces and measure the mechanics at this interface in vivo. In my thesis work I have addressed this gap using direct biophysical assays in mammalian cells to focus on two main questions about the kinetochore-microtubule interface. First, how do kinetochores hold on to microtubules that grow and shrink? Using live imaging to monitor spindle dynamics and laser ablation to challenge kinetochore grip, I show that regulation of the key microtubule binding protein Ndc80/Hec1 at the outer kinetochore by the kinase Aurora B specifically affects kinetochore movement on polymerizing microtubules without disrupting coupling to depolymerizing microtubules that generate force to move chromosomes. Second, at the other side of the interface, how do kinetochore-fibers remodel under force? I directly exert forces on individual mammalian k-fibers and find that even under high force for minutes they do not lose grip. Instead, k-fibers bend and elongate by polymerizing at normal rates at plus-ends and inhibiting depolymerization at minus-ends – thus ensuring robust connection to kinetochores. Altogether I find that robust kinetochore grip emerges from underlying properties of both the kinetochore and k-fiber microtubules – specialized regulation at the kinetochore allows the cell to adjust grip while still allowing force generation and dynamic mechanical feedback of k-fiber microtubules locally dissipates force, protecting spindle connections. These fundamental physical properties of the kinetochore-microtubule interface allow the spindle to faithfully segregate chromosomes and more broadly suggest a model for how force-generating cellular machines can also robustly maintain their structure.

    Cover page: Mechanical Robustness of the Mammalian Kinetochore-Microtubule Interface
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2020)
    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.
      Cover page: Microneedle manipulation of the mammalian spindle reveals specialized, short-lived reinforcement near chromosomes
      • Article
      • Peer Reviewed
      UC San Francisco Previously Published Works (2019)
      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.
        Cover page: The mammalian kinetochore–microtubule interface: robust mechanics and computation with many microtubules
        • Article
        • Peer Reviewed
        UC San Francisco Previously Published Works (2020)
        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.
          Cover page: Individual kinetochore-fibers locally dissipate force to maintain robust mammalian spindle structure
          • Thesis
          • Peer Reviewed
          UC San Diego Electronic Theses and Dissertations (2017)

          In my first ever art class, my teacher told me that ‘there are no mistakes in art’. At five years old, that statement struck me. No matter what I did, as an artist, I could always rectify my mistakes by making a new choice. In theatre, we can make many mistakes. I know that I have. But over my time here I have learned that those mistakes aren’t always lasting, and aren’t always a bad thing. Sometimes they can be ‘happy accidents’, sometimes discoveries, jumping off points, moments for introspection, or, in the very least, you can try again next time. In different moments in our lives, we can be

          distracted, deviate, and disabuse. There are so many issues we encounter, and much of our jobs and our lives are spent fixing these problems.

          Graduate school, in particular, was all about how many mistakes I could make in two and a half short years. Here I have had the time and space to strive, and fail, and learn. But in the end it was never really ‘failing’ – art is too subjective to ‘fail’. There is always a choice, a reason, or a ‘why’. Or a way to fix it. After it all, I can firmly say I have come out the other side better and stronger than before. I have been broken down and rebuilt many times over. And I hope it only continues from here.

          • 7 supplemental images
          Cover page: There Are No Mistakes in Art
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2015)
          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.
            Cover page: Kinesin-5: A Team Is Just the Sum of Its Parts
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2017)
            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.
              Cover page: Hec1 Tail Phosphorylation Differentially Regulates Mammalian Kinetochore Coupling to Polymerizing and Depolymerizing Microtubules
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2014)
              In Central America, the traditional temazcales or wood-fired steam baths, commonly used by many Native American populations, are often heated by wood fires with little ventilation, and this use results in high wood smoke exposure. Urinary mutagenicity has been previously employed as a non-invasive biomarker of human exposure to combustion emissions. This study examined the urinary mutagenicity in 19 indigenous Mayan families from the highlands of Guatemala who regularly use temazcales (N = 32), as well as control (unexposed) individuals from the same population (N = 9). Urine samples collected before and after temazcal exposure were enzymatically deconjugated and extracted using solid-phase extraction. The creatinine-adjusted mutagenic potency of urine extracts was assessed using the plate-incorporation version of the Salmonella mutagenicity assay with strain YG1041 in the presence of exogenous metabolic activation. The post-exposure mutagenic potency of urine extracts were, on average, 1.7-fold higher than pre-exposure samples (P < 0.005) and also significantly more mutagenic than the control samples (P < 0.05). Exhaled carbon monoxide (CO) was ~10 times higher following temazcal use (P < 0.0001), and both CO level and time spent in temazcal were positively associated with urinary mutagenic potency (i.e. P < 0.0001 and P = 0.01, respectively). Thus, the wood smoke exposure associated with temazcal use contributes to increased excretion of conjugated mutagenic metabolites. Moreover, urinary mutagenic potency is correlated with other metrics of exposure (i.e. exhaled CO, duration of exposure). Since urinary mutagenicity is a biomarker associated with genetic damage, temazcal use may therefore be expected to contribute to an increased risk of DNA damage and mutation, effects associated with the initiation of cancer.
                Cover page: Human urinary mutagenicity after wood smoke exposure during traditional temazcal use
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2022)
                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.
                  Cover page: ECM dimensionality tunes actin tension to modulate endoplasmic reticulum function and spheroid phenotypes of mammary epithelial cells.
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