Biology subjects

Low-Nam, S. T.

Publications and source records attributed to Low-Nam, S. T..

10 recordsLinked to original sources

bioRxiv2026

Single molecule Grb2 dynamics reveal spatiomechanical modulation of RTK signaling across length scales

Extracellular matrix (ECM)-mediated cell adhesion and tension regulate development and disease through crosstalk with Receptor Tyrosine Kinases (RTKs). RTKs stimulate proliferation, survival, and metabolic reprogramming via Ras/ERK and PI3K/Akt signaling. Emerging evidence identify the biophysical and biochemical integration of integrin-mediated adhesions with RTK signaling, however, the molecular mechanisms regulating this phenotype remain poorly understood. Here, we demonstrate that ECM stiffness potentiates ligand-mediated epidermal growth factor receptor (EGFR) signaling at scales ranging from the single adaptor protein, Grb2 upward to multicellular spheroids and tissues. We show that ECM stiffness-driven RTK signaling is mediated by integrin adhesions that reshape the membrane to induce the formation of RTK signaling hotspots distributed globally across the cell membrane. A parsimonious computational model and biochemical membrane perturbations causally implicate membrane curvature and topography in tension-dependent integrin-EGFR crosstalk. The results demonstrate how the mechanical properties of the ECM direct molecular recruitment and confinement of signaling molecules to regulate growth factor-dependent RTK signaling. Given that many RTK-driven solid tumors develop a stiff stroma, the findings could explain why many therapeutic tyrosine kinase inhibitors develop clinical resistance. The findings suggest clarifying how tension regulates transmembrane receptor signaling could identify unique force-dependent molecular regulators towards which new antitumor therapies could be applied.

biophysics↗

bioRxiv2026

T cell degranulation and payload delivery are modulated in space and time

Release of destructive payloads through degranulation is a hallmark of T cell cytotoxicity. Upon stimulation, tens to hundreds of granules are rapidly delivered to the interface with a target cell but only a small number of degranulation events occur. Two forms of degranulation have been observed and differ in the physical form of the payload released. The deposition of insoluble particles encapsulated in a proteinaceous shell may be more potent than release of purely soluble factors. The selection mechanisms and triggers for degranulation are still poorly understood, partly due to the lack of tools to dynamically map and distinguish each mode. Here we use high spatiotemporal resolution in vitro assays, coupled to a novel label-free strategy to characterize and track released particles, to show that degranulation is regulated not only by chemical but also physical aspects of the activation inputs. Furthermore, the released, insoluble particles are transported within the intermembrane junction with a preferred directionality. Immobilized stimulatory ligands favored the soluble degranulation mode suggesting that the particulate release is pertinent to mobile, physiological immune interfaces. Engineered chimeric antigen receptor (CAR) T cells have a defect in centralizing released particles, which may explain dampened toxicity and off-target tissue damage. Our results demonstrate how degranulation events are likely regulated by a combination of physical, chemical, and signaling mechanisms. We anticipate these findings will generate new strategies to bias degranulation outcomes and optimize the destructive capacity of single cytotoxic T cells. In the case of engineered T cells, efforts to restore the normal transport of insoluble particles could contribute to stronger therapeutics for cellular immunotherapy.

immunology↗

bioRxiv2025

CAR T cell cytotoxic responses are rapidly generated and sensitive to the unligated TCR

Chimeric antigen receptor (CAR) T cells expressing tumor-targeting engineered receptors can robustly eliminate cancer cells through secretion of cytotoxic factors. Durable remission in leukemia and lymphoma treatment has not been matched in solid tumors. Efforts to maximize tumor destruction and minimize toxicities have driven efforts to tune CAR signaling. However, the molecular mechanisms for CAR triggering and thresholds for activation are incompletely understood. Here, we measured the collection of CAR binding interactions that culminate in polarized delivery of lytic granules to the junction with the target. CAR T cells binarized cytotoxic activities in response to a few binding events and population outcomes were dominated by a subset of cells. Activation at the single molecule level matches the sensitivity of the native T cell receptor (TCR) and points to potent downstream signal propagation. Disruption of the unligated TCR with a transmembrane-targeting inhibitory peptide strongly dampened CAR T cell activation, indicating a critical crosstalk between the two receptors. Harnessing CAR T cell efficacy and reduction of toxicity will require new approaches to modify integration of the binding events, collected stochastically, that are rapidly digitized. These sensitive CAR T cell responses provide new insights into driving cytotoxic signaling through surface interaction engineering.

immunology↗

bioRxiv2025

PP2A activation drives aberrant macropinocytosis and cell death in pancreatic ductal adenocarcinoma

Pancreatic cancer is highly aggressive with a five-year survival rate of just 13%. Metabolic rewiring in response to oncogenic signals plays a critical role in pancreatic ductal adenocarcinoma (PDAC) survival, tumor growth, and metastasis. These alterations make PDAC tumors dependent on anabolic metabolism for survival, highlighting a unique vulnerability that can be therapeutically exploited. However, during nutrient deprivation, PDAC cells can circumvent this vulnerability by engulfing extracellular fluids to replenish amino acids in a process called, macropinocytosis. This process can be induced downstream of oncogenic KRAS expression, a small GTPase that is almost universally mutated in PDAC patients. The inhibition of macropinocytosis in vivo reduces PDAC tumor growth, emphasizing the importance of this pathway to cancer cell survival. However, the signaling mechanisms that regulate this process remain poorly understood. Protein phosphatase 2A (PP2A) is a heterotrimeric complex that regulates a wide variety of cell signaling pathways, including KRAS, and is commonly dysregulated in human PDAC tumors. Here, we show that acute PP2A activation prevents macropinosome processing leading to cell death. Furthermore, we demonstrate that PP2A posttranslationally regulates the lipid kinase, PIKfyve, a key regulator of macropinosome-lysosome fusion. Finally, we determine that PP2A activating compounds can function synergistically with metabolic inhibitors, supporting a new therapeutic strategy in this aggressive and deadly cancer. Together, our results implicate PP2A as a critical suppressor of PDAC metabolic plasticity and highlight the use of PP2A activating compounds to prevent PDAC nutrient scavenging.

cancer biology↗

bioRxiv2025

A mechanistic model for dynamics between CAR T cells and target cells captures features that determine killing profiles

Chimeric antigen receptor (CAR) T cells represent a potent, programmable therapeutic that repurposes T cell cytotoxicity toward target cell elimination. Direct killing of tumor cells has been demonstrated in several cancer contexts but deficits in destroying solid tumors have been attributed to molecular and mechanical complexity. Understanding and predicting the efficacy of CAR T cell therapy requires a rigorous framework to capture the mechanistic interactions between immune cells and tumor targets. Toward enumerating such features and to quantitatively describe tumor cell survival in response to CAR T cell visitation, we propose a parametric hazard rate model for the right-censored conditional lifetime of a tumor cell, having the form of a time-discounted integral of cell:cell engagement. The model, conditioned using data on the numbers, durations, and modes of contacts made during the stochastic encounters between the two cell types, extracts mechanistic details from the experiments. We suggest two types of parameters to encapsulate features of CAR T cell killing potency ({kappa}) and the resilience of the tumor ({gamma}), respectively. These parsimonious parameters, nevertheless, generate substantial insights. Firstly, phenotypic heterogeneity from interactions mediated by substrate-engaged CAR T cells dominate killing outcomes, while those initiated in suspension contribute minimally. This emphasizes that a subset of the CAR T cell population is dominating the population outcome. Secondly, the model can be used to predict the killing as a function of time upon perturbation of the system. In this case, biasing the arrival process, toward more engagement in the adherent mode, can tune the rate of killing. Collectively, insights from the model framework imply that CAR T cell accumulation and killing efficiency depend not on the sum of individual T cell interactions, but rather on the weighted, time-dependent sum of heterogeneous interactions that are environmentally modulated. The model facilitates predictive insights into how contact sequences modulate CAR T cell responses and may provide strategies to alter therapeutic regimens.

biophysics↗

bioRxiv2025

Simultaneous particle tracking, phase retrieval and point spread function reconstruction

3D tracking and localization of particles, typically fluorescently labeled biomolecules, provides a direct means of monitoring cellular transport and communication. However, sample-induced wavefront distortions of emitted fluorescent light as it passes through the sample and onto the detector often yield point spread function (PSF) aberrations, presenting an important challenge to 3D particle tracking using pre-calibrated PSFs. PSF calibration is typically performed outside cellular samples, ignoring sample-induced aberrations, which can result in localization errors on the order of tens to hundreds of nanometers, ultimately compromising sub-diffraction limited tracking. In practice, correcting sample-induced aberrations currently requires sample-specific hardware adjustments, such as adaptive optics. Yet, information on sample-induced aberrations and PSF shape can be directly decoded from data collected using a 3D imaging setup (e.g., bi-focal). To this end, we propose a framework for simultaneous particle tracking, phase retrieval, and PSF reconstruction (SPT-PR) directly from the input data themselves. We apply it to sub-diffraction tracking of lytic granules released at the immunological synapse of T cells revealing slower motions in proximity of the plasma cell membrane, consistent with assembly of the fusion machinery and, ultimately, degranulation and release of toxic payloads. To accomplish this, we operate within a Bayesian paradigm, placing continuous priors on all possible pupil phase and amplitudes warranted by the data without limiting ourselves to a finite Zernike set-thereby allowing capture of intricate pupil phase details. We benchmark our framework using a wide range of synthetic and experimental data from static to diffusing particles, and generalize to multiple diffusing particles with overlapping PSFs. Further, as a result of simultaneous particle tracking, phase retrieval, and PSF reconstruction, we retrieve the pupil phase with errors smaller than 10% under a range of realistic scenarios while demonstrating that for tracking lytic granules under an idealized Gaussian PSF assumption, we recover discrepancies as large as hundreds of nanometers.

biophysics↗

bioRxiv2025

SPTnet: a deep learning framework for end-to-end single-particle tracking and motion dynamics analysis

Single-particle tracking (SPT) provides high-resolution spatial-temporal information on biomolecule dynamics. However, localization inaccuracies, limited track lengths, heterogeneous fluorescence backgrounds, and potential molecular motion blur pose significant challenges that hinder the accurate extraction of movement trajectories and their underlying motion behavior. The conventional SPT pipeline struggles to comprehensively address detection, localization, linkage, and motion parameter inference simultaneously, resulting in information loss during sequential processing. To overcome these challenges, we propose SPTnet, an end-to-end deep learning framework that leverages a Transformer-based architecture to optimize trajectory and motion parameter estimations in parallel through a global loss. SPTnet bypasses traditional SPT processes, directly inferring molecular trajectories and motion parameters from fluorescence microscopy videos with a precision approaching the statistical information limit. Our results demonstrate that SPTnet outperforms conventional methods under commonly encountered but challenging conditions such as short trajectories, low signal-to-noise ratio (SNR), heterogeneous backgrounds, motion blur, and especially when molecules exhibit non-Brownian behaviors.

biophysics↗

bioRxiv2024

Direct measurement of PIP2 densities in biological membranes using a peptide-based sensor

Organization and composition of the plasma membrane are important modulators of many cellular programs. Phosphatidylinositol phosphate (PIP) lipids are low abundance membrane constituents with different arrangements of phosphate groups around an inositol head group that regulate a large number of signaling pathways. Many strategies have been developed to detect and track PIP species to monitor their clustering, mobility, and interaction with binding partners. We implement a peptide-based ratiometric sensor for the detection of PI(4,5)P2 lipids in reconstituted membrane systems that permit absolute quantification of PI(4,5)P2 densities down to physiological levels. The sensor is membrane permeable and easily applicable to measurements in living cells. Application of calibrated sensors to cells expressing common mutations in the small GTPase, Ras, showed a reshaping of surface PI(4,5)P2 levels and distributions in a mutation-specific manner. The rapid implementation of this quantitative sensing strategy to cellular studies of cellular signaling, membrane organization and dynamics should be broadly applicable.

biochemistry↗

bioRxiv2021

Discrete LAT condensates encode antigen information from single pMHC:TCR binding events

LAT assembly into a two-dimensional protein condensate is a prominent feature of antigen discrimination by T cells. Here, we use single-molecule imaging techniques to resolve the spatial position and temporal duration of each pMHC:TCR molecular binding event while simultaneously monitoring LAT condensation at the membrane. An individual binding event is sufficient to trigger a LAT condensate, which is self-limiting, and neither its size nor lifetime is correlated with the duration of the originating pMHC:TCR binding event. Only the probability of the LAT condensate forming is related to the pMHC:TCR binding dwell time. LAT condenses abruptly, but after an extended delay from the originating binding event. A LAT mutation that facilitates phosphorylation at the PLC-{gamma}1 recruitment site shortens the delay time to LAT condensation and alters T cell antigen specificity. These results identify a role for the LAT protein condensation phase transition in setting antigen discrimination thresholds in T cells.

biophysics↗

bioRxiv2017

Membrane bending begins at any stage of clathrin-coat assembly and defines endocytic dynamics

Summary ParagraphClathrin-mediated endocytosis internalizes membrane from the cell surface by reshaping flat regions of membrane into spherical vesicles(1, 2). The relationship between membrane bending and clathrin coatomer assembly has been inferred from electron microscopy and structural biology, without directly visualization of membrane bending dynamics (3-6). This has resulted in two distinct and opposing models for how clathrin bends membrane (7-10). Here, polarized Total Internal Reflection Fluorescence microscopy was improved and combined with electron microscopy, atomic force microscopy, and super-resolution imaging to measure membrane bending during endogenous clathrin and dynamin assembly in living cells. Surprisingly, and not predicted by either model, the timing of membrane bending was variable relative to clathrin assembly. Approximately half of the time, membrane bending occurs at the start of clathrin assembly, in the other half, the onset of membrane bending lags clathrin arrival, and occasionally completely assembled flat clathrin transitions into a pit. Importantly, once the membrane bends, the process proceeds to scission with similar timing. We conclude that the pathway of coatomer formation is versatile and can bend the membrane during or after the assembly of the clathrin lattice. These results highlight the heterogeneity in this fundamental biological process, and provide a more complete nanoscale view of membrane bending dynamics during endocytosis.

cell biology↗