Skip to main content
eScholarship
Open Access Publications from the University of California

Scholarly Works (11 results)

  • Article
  • Peer Reviewed
UC Davis Previously Published Works (2018)
The physicist Edward Purcell wrote in 1977 about mechanisms that cells could use to propel themselves in a low Reynolds number environment. Reporting in Developmental Cell, O'Neill et al. (2018) provide direct evidence for one of these mechanisms by optogenetically driving the migration of cells suspended in liquid through RhoA activation.
    Cover page: “Rho”ing a Cellular Boat with Rearward Membrane Flow
    • Thesis
    • Peer Reviewed
    UC Davis Electronic Theses and Dissertations (2021)

    Neutrophils are early responders of the innate immune system that use chemotaxis, the directed migration of cells along a chemical gradient, to reach sites of infection and inflammation. Deciphering the spatial patterns of chemoattractant signals is a fundamental challenge at the cellular level due to diffusion and inherent stochasticity of signaling molecules. Despite these sources of noise, neutrophils respond to changing chemoattractant gradients with high spatial and temporal precision by integrating information from receptors with a cell-autonomous polarity and motility signaling program1–4. The wiring of the chemotaxis signaling network is complex with multiple layers of feedbacks and potentially feedforward connections. Understanding how cell polarity programs interpret and respond to chemotaxis receptor signals remains a key challenge in the field, as deciphering the interconnections of the network are not trivial. To better understand how cell polarity regulators in the Rho GTPase family signal within chemotaxing cells, I developed a high-magnification chemotaxis assay that enables measurement of Rho GTPase activity biosensors downstream of photo-activation of chemoattractants (Chapter 2). Chemoattractant gradients are inherently spatial, and it remains unclear how cells encode this spatially relevant information from the receptors in the downstream chemotaxis signaling programs. Experiments using diffusible chemoattractants are limiting, as achieving well-defined and highly local receptor stimulation is unfeasible. To overcome this challenge, I developed a new molecular tool kit that uses light-driven chemotaxis signaling to improve the spatial and temporal control of receptor activation. I paired this receptor with a red-shifted biosensor for a cell front polarity regulator, enabling stimulation and response measurements in the same cells. Using this system, I explored how cell polarity signals are propagated in space and time downstream of receptor stimulation. Additionally, I investigated how negative signals shape the polarity response (Chapter 3). Through collaboration, the light-activated chemotaxis receptor was also used to unlock another experimental question that was limited by chemoattractant diffusion. Specifically, we investigated whether existing cell front/back polarity is resistant to receptor activation at the cell rear (Chapter 4). Collectively, the methodologies developed, and information gleaned from this work lay the foundation for systematic interrogation of spatial signal transduction that occurs during neutrophil chemotaxis.

      Cover page: Analysis of neutrophil chemotaxis spatial signal processing using optogenetic receptor control
      • Article
      • Peer Reviewed
      UC Davis Previously Published Works (2021)
      During chemotaxis, neutrophils use cell surface G Protein Coupled Receptors to detect chemoattractant gradients. The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities. Positive feedback could promote rapid signal spreading, yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell. How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing. Here we directly measure information flow from receptors to Cdc42 by pairing zebrafish parapinopsina, an optogenetic G Protein Coupled Receptor with reversible ON/OFF control, with a spectrally compatible red/far red Cdc42 Fluorescence Resonance Energy Transfer biosensor. Using this toolkit, we show that positive and negative signals downstream of G proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.
        Cover page: Optogenetic control of receptors reveals distinct roles for actin- and Cdc42-dependent negative signals in chemotactic signal processing
        • Article
        • Peer Reviewed
        UC Davis Previously Published Works (2021)
        During chemotaxis, neutrophils use cell surface G-Protein Coupled Receptors (GPCRs) to detect chemoattractant gradients 1–4 . The downstream signaling system is wired with multiple feedback loops that amplify weak inputs and promote spatial separation of cell front and rear activities 1, 5–8 . Positive feedback could promote rapid signal spreading 9 , yet information from the receptors is transmitted with high spatial fidelity, enabling detection of small differences in chemoattractant concentration across the cell 1 . How the signal transduction network achieves signal amplification while preserving spatial information remains unclear. The GTPase Cdc42 is a cell-front polarity coordinator that is predictive of cell turning, suggesting an important role in spatial processing 10 . To directly measure information flow from receptors to Cdc42, we paired zebrafish parapinopsina, an optogenetic GPCR that allows reversible ON/OFF receptor control with a spectrally compatible red/far red Cdc42 FRET biosensor. Using this new toolkit, we show that positive and negative signals downstream of G-proteins shape a rapid, dose-dependent Cdc42 response. Furthermore, F-actin and Cdc42 itself provide two distinct negative signals that limit the duration and spatial spread of Cdc42 activation, maintaining output signals local to the originating receptors.
          Creative Commons 'BY-NC-ND' version 4.0 license
          • Article
          • Peer Reviewed
          UC Davis Previously Published Works (2023)
          Maintaining persistent migration in complex environments is critical for neutrophils to reach infection sites. Neutrophils avoid getting trapped, even when obstacles split their front into multiple leading edges. How they re-establish polarity to move productively while incorporating receptor inputs under such conditions remains unclear. Here, we challenge chemotaxing HL60 neutrophil-like cells with symmetric bifurcating microfluidic channels to probe cell-intrinsic processes during the resolution of competing fronts. Using supervised statistical learning, we demonstrate that cells commit to one leading edge late in the process, rather than amplifying structural asymmetries or early fluctuations. Using optogenetic tools, we show that receptor inputs only bias the decision similarly late, once mechanical stretching begins to weaken each front. Finally, a retracting edge commits to retraction, with ROCK limiting sensitivity to receptor inputs until the retraction completes. Collectively, our results suggest that cell edges locally adopt highly stable protrusion/retraction programs that are modulated by mechanical feedback.
            Creative Commons 'BY' version 4.0 license
            • Article
            • Peer Reviewed
            UC Davis Previously Published Works (2015)
            In both physiological and cell culture systems, EGF-stimulated ERK activity occurs in discrete pulses within individual cells. Many feedback loops are present in the EGF receptor (EGFR)-ERK network, but the mechanisms driving pulsatile ERK kinetics are unknown. Here, we find that in cells that respond to EGF with frequency-modulated pulsatile ERK activity, stimulation through a heterologous TrkA receptor system results in non-pulsatile, amplitude-modulated activation of ERK. We further dissect the kinetics of pulse activity using a combination of FRET- and translocation-based reporters and find that EGFR activity is required to maintain ERK activity throughout the 10-20-minute lifetime of pulses. Together, these data indicate that feedbacks operating within the core Ras-Raf-MEK-ERK cascade are insufficient to drive discrete pulses of ERK activity and instead implicate mechanisms acting at the level of EGFR.
              Cover page: Receptor Level Mechanisms Are Required for Epidermal Growth Factor (EGF)-stimulated Extracellular Signal-regulated Kinase (ERK) Activity Pulses*
              • Article
              • Peer Reviewed
              UC Davis Previously Published Works (2021)
              To migrate efficiently to target locations, cells must integrate receptor inputs while maintaining polarity: a distinct front that leads and a rear that follows. Here we investigate what is necessary to overwrite pre-existing front-rear polarity in neutrophil-like HL60 cells migrating inside straight microfluidic channels. Using subcellular optogenetic receptor activation, we show that receptor inputs can reorient weakly polarized cells, but the rear of strongly polarized cells is refractory to new inputs. Transient stimulation reveals a multi-step repolarization process, confirming that cell rear sensitivity to receptor input is the primary determinant of large-scale directional reversal. We demonstrate that the RhoA/ROCK/myosin II pathway limits the ability of receptor inputs to signal to Cdc42 and reorient migrating neutrophils. We discover that by tuning the phosphorylation of myosin regulatory light chain we can modulate the activity and localization of myosin II and thus the amenability of the cell rear to 'listen' to receptor inputs and respond to directional reprogramming.
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2022)
                To control their movement, cells need to coordinate actin assembly with the geometric features of their substrate. Here, we uncover a role for the actin regulator WASP in the 3D migration of neutrophils. We show that WASP responds to substrate topology by enriching to sites of inward, substrate-induced membrane deformation. Superresolution imaging reveals that WASP preferentially enriches to the necks of these substrate-induced invaginations, a distribution that could support substrate pinching. WASP facilitates recruitment of the Arp2/3 complex to these sites, stimulating local actin assembly that couples substrate features with the cytoskeleton. Surprisingly, WASP only enriches to membrane deformations in the front half of the cell, within a permissive zone set by WASP's front-biased regulator Cdc42. While WASP KO cells exhibit relatively normal migration on flat substrates, they are defective at topology-directed migration. Our data suggest that WASP integrates substrate topology with cell polarity by selectively polymerizing actin around substrate-induced membrane deformations in the front half of the cell.
                  Cover page: WASP integrates substrate topology and cell polarity to guide neutrophil migration
                  • Article
                  • Peer Reviewed
                  UC Davis Previously Published Works (2023)

                  Background

                  In light of the growing appreciation for the role of collective cell motility in metastasis, a deeper understanding of the underlying signaling pathways will be critical to translating these observations to the treatment of advanced cancers. Here, we examine the contribution of Wnt/planar cell polarity (Wnt/PCP), one of the non-canonical Wnt signaling pathways and defined by the involvement of the tetraspanin-like proteins Vangl1 and Vangl2, to breast tumor cell motility, collective cell invasiveness and mammary tumor metastasis.

                  Methods

                  Vangl1 and Vangl2 knockdown and overexpression and Wnt5a stimulation were employed to manipulate Wnt/PCP signaling in a battery of breast cancer cell lines representing all breast cancer subtypes, and in tumor organoids from MMTV-PyMT mice. Cell migration was assessed by scratch and organoid invasion assays, Vangl protein subcellular localization was assessed by confocal fluorescence microscopy, and RhoA activation was assessed in real time by fluorescence imaging with an advanced FRET biosensor. The impact of Wnt/PCP suppression on mammary tumor growth and metastasis was assessed by determining the effect of conditional Vangl2 knockout on the MMTV-NDL mouse mammary tumor model.

                  Results

                  We observed that Vangl2 knockdown suppresses the motility of all breast cancer cell lines examined, and overexpression drives the invasiveness of collectively migrating MMTV-PyMT organoids. Vangl2-dependent RhoA activity is localized in real time to a subpopulation of motile leader cells displaying a hyper-protrusive leading edge, Vangl protein is localized to leader cell protrusions within leader cells, and actin cytoskeletal regulator RhoA is preferentially activated in the leader cells of a migrating collective. Mammary gland-specific knockout of Vangl2 results in a striking decrease in lung metastases in MMTV-NDL mice, but does not impact primary tumor growth characteristics.

                  Conclusions

                  We conclude that Vangl-dependent Wnt/PCP signaling promotes breast cancer collective cell migration independent of breast tumor subtype and facilitates distant metastasis in a genetically engineered mouse model of breast cancer. Our observations are consistent with a model whereby Vangl proteins localized at the leading edge of leader cells in a migrating collective act through RhoA to mediate the cytoskeletal rearrangements required for pro-migratory protrusion formation.
                    Cover page: Vangl-dependent Wnt/planar cell polarity signaling mediates collective breast carcinoma motility and distant metastasis
                    • Article
                    • Peer Reviewed
                    UC Davis Previously Published Works (2023)
                    Targeting common oncogenic drivers of glioblastoma multiforme (GBM) in patients has remained largely ineffective, raising the possibility that alternative pathways may contribute to tumor aggressiveness. Here we demonstrate that Vangl1 and Fzd7, components of the non-canonical Wnt planar cell polarity (Wnt/PCP) signaling pathway, promote GBM malignancy by driving cellular proliferation, migration, and invasiveness, and engage Rho GTPases to promote cytoskeletal rearrangements and actin dynamics in migrating GBM cells. Mechanistically, we uncover the existence of a novel Vangl1/Fzd7 complex at the leading edge of migrating GBM cells and propose that this complex is critical for the recruitment of downstream effectors to promote tumor progression. Moreover, we observe that depletion of FZD7 results in a striking suppression of tumor growth and latency and extends overall survival in an intracranial mouse xenograft model. Our observations support a novel mechanism by which Wnt/PCP components Vangl1 and Fzd7 form a complex at the leading edge of migratory GBM cells to engage downstream effectors that promote actin cytoskeletal rearrangements dynamics. Our findings suggest that interference with Wnt/PCP pathway function may offer a novel therapeutic strategy for patients diagnosed with GBM.
                      Cover page: A complex of Wnt/planar cell polarity signaling components Vangl1 and Fzd7 drives glioblastoma multiforme malignant properties
                      Top