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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2013)

Cellular membranes organize signal transduction, serving as platforms for protein interactions, as well as direct modulators of enzyme function. Since many signaling reactions converge at the plasma membrane, understanding the coupling between protein

function and membrane localization of signaling complex is of fundamental importance. However, a quantitative description of many of such interactions has been lacking, largely due to the difficulty in performing measurements in the complex cellular environment. Here, we reconstitute and quantitatively probe the kinetics between small GTPase Ras and its activators: Son of Sevenless (SOS) and Ras guanyl nucleotide-releasing protein

(RasGRP) using supported membranes.

The activation of the membrane-associated Ras by cytosolic guanine nucleotide exchange factors, SOS and RasGRP, is a key step in a plethora of receptor-mediated cell signaling pathways. Both SOS and RasGRP contain multiple domains and several of which

are known to regulate the activation of SOS/RasGRP. In this dissertation, we study in parallel the kinetics of SOS and RasGRP-catalyzed Ras activation as well as the mechanisms that release the autoinhibition of SOS/RasGRP at single molecule level.

We develop an assay with supported bilayers functionalized with H-Ras to monitor the enzymatic activities from hundreds of individual SOS proteins. The data reveals that SOS is dynamically heterogeneous, sampling a broad distribution of turnover rates via

stochastic fluctuations between distinct states. The regulatory domains allosterically inhibit SOS by suppressing fluctuations to high activity states with sensitivity to both Ras nucleotide state and lipids. On the subject of Ras activation by RasGRP, the functional role of lipid interacting C1 domain of RasGRP1 has been studied. The preliminary results suggest that diacylglycerol (DAG), through the interaction with the C1 domain, regulates the function of RasGRP1 primarily by increasing its membrane localization.

    Cover page: Study of signal transduction in reconstituted membranes: Ras activation by SOS and RasGRP1
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2024)
    Nuclear factor kappa B (NF-κB) is a key regulator in immune signaling and is known to exhibit a digital activation pattern. Yet the molecular basis underlying the heterogeneity in NF-κB activation at single-cell level is not entirely understood. Here, we show that NF-κB activation in single cells is largely regulated by intrinsic differences at the receptor level. Using the genome editing and time-lapse imaging, we directly characterize endogenous TNFR1 dynamics and NF-κB activation from the same single cells. Total internal reflection fluorescence (TIRF) microscopy shows that endogenous TNFR1 forms pre-ligand clusters in the resting cells. Upon tumor necrosis factor (TNF) stimulation, the diffusion coefficient of membrane TNFR1 was significantly decreased and a substantial level of TNFR1 undergoes oligomerization to form trimers and hexamers. Moreover, multi-color cell imaging reveals that both digital and graded information processing regulate NF-κB activation across different TNFR1 expression levels. Our results indicate that single-cell NF-κB activation potential strongly correlates with its TNFR1 characteristics.
      Cover page: TNFR1 mediates heterogeneity in single-cell NF-κB activation.
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2021)
        Cover page: NF-kappa B responds to absolute differences in cytokine concentrations
        • Article
        • Peer Reviewed
        UC Irvine Previously Published Works (2021)
        Cells receive a wide range of dynamic signaling inputs during immune regulation, but how gene regulatory networks measure such dynamic inputs is not well understood. Here, we used microfluidic single-cell analysis and mathematical modeling to study how the NF-κB pathway responds to immune inputs that vary over time such as increasing, decreasing, or fluctuating cytokine signals. We found that NF-κB activity responded to the absolute difference in cytokine concentration and not to the concentration itself. Our analyses revealed that negative feedback by the regulatory proteins A20 and IκBα enabled differential responses to changes in cytokine dose by providing a short-term memory of previous cytokine concentrations and by continuously resetting kinase cycling and receptor abundance. Investigation of NF-κB target gene expression showed that cells exhibited distinct transcriptional responses under different dynamic cytokine profiles. Our results demonstrate how cells use simple network motifs and transcription factor dynamics to efficiently extract information from complex signaling environments.
          Cover page: NF-κB responds to absolute differences in cytokine concentrations.
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2016)
          The assembly of cell surface receptors with downstream signaling molecules is a commonly occurring theme in multiple signaling systems. However, little is known about how these assemblies modulate reaction kinetics and the ultimate propagation of signals. Here, we reconstitute phosphotyrosine-mediated assembly of extended linker for the activation of T cells (LAT):growth factor receptor-bound protein 2 (Grb2):Son of Sevenless (SOS) networks, derived from the T-cell receptor signaling system, on supported membranes. Single-molecule dwell time distributions reveal two, well-differentiated kinetic species for both Grb2 and SOS on the LAT assemblies. The majority fraction of membrane-recruited Grb2 and SOS both exhibit fast kinetics and single exponential dwell time distributions, with average dwell times of hundreds of milliseconds. The minor fraction exhibits much slower kinetics, extending the dwell times to tens of seconds. Considering this result in the context of the multistep process by which the Ras GEF (guanine nucleotide exchange factor) activity of SOS is activated indicates that kinetic stabilization from the LAT assembly may be important. This kinetic proofreading effect would additionally serve as a stochastic noise filter by reducing the relative probability of spontaneous SOS activation in the absence of receptor triggering. The generality of receptor-mediated assembly suggests that such effects may play a role in multiple receptor proximal signaling processes.
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2014)
            Activation of the small guanosine triphosphatase H-Ras by the exchange factor Son of Sevenless (SOS) is an important hub for signal transduction. Multiple layers of regulation, through protein and membrane interactions, govern activity of SOS. We characterized the specific activity of individual SOS molecules catalyzing nucleotide exchange in H-Ras. Single-molecule kinetic traces revealed that SOS samples a broad distribution of turnover rates through stochastic fluctuations between distinct, long-lived (more than 100 seconds), functional states. The expected allosteric activation of SOS by Ras-guanosine triphosphate (GTP) was conspicuously absent in the mean rate. However, fluctuations into highly active states were modulated by Ras-GTP. This reveals a mechanism in which functional output may be determined by the dynamical spectrum of rates sampled by a small number of enzymes, rather than the ensemble average.
              Cover page: Ras activation by SOS: Allosteric regulation by altered fluctuation dynamics
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