RESEARCH

Membrane immunobiophysics: The immune cell membrane serves as a central hub for dynamic communication with the extracellular environment and contains the vast majority of therapeutic receptor targets in immunotherapy. Immune cells also exhibit dynamic, complex membrane shapes (Image reveals the 3D shape of a human T cell, Nat Commun, 2025). What role does membrane topology play in guiding the decision-making of T cells? How can these insights inform the optimization of therapeutic agent design to maximize T cell function and therapeutic efficacy? This research utilizes single-molecule and super-resolution microscopy techniques to investigate membrane-mediated intercellular and intracellular communication. Our group has developed a versatile pan-membrane-protein labeling technique optimized for live-cell super-resolution imaging. This approach is rapid, highly efficient, resistant to photobleaching, and compatible with multiplexed detection, making it uniquely applicable to primary immune cells. Using this approach, we have successfully visualized the dynamic membrane interactions between T cells and cancer cells in the context of immunotherapy. Our research has also revealed the involvement of specialized membrane structures in signaling regulation and membrane-crossing activities of engineered nanomaterials into immune cells. Our work has demonstrated microvilli-localized transmembrane proteins that regulate T-cell receptor signaling, as well as super-resolved extended membrane fibers involved in antigen uptake and storage by dendritic cells (Nano Lett, 2022). Furthermore, we have revealed the membrane-penetrating activity of engineered nanoparticles at the single-particle level (Nanoscale, 2021).

Representative publications:

• Gunasekara H, Cheng Y-S, Perez-Silos V, Zevallos-Morales A, Abegg D, Burgess A, Gong L-W, Minshall RD, Adibekian A, Murga-Zamalloa C, Ondrus AE, Hu YS*, Unveiling cellular communications through rapid pan-membrane-protein labeling. Nat Commun. 2025;16(1):3584.PMID: 40234465; PMCID: PMC12000395. Link.
• Jing H, Saed B, Pálmai M, Gunasekara H, Snee PT, Hu YS*, Fluorescent artificial antigens revealed extended membrane networks utilized by live dendritic cells for antigen uptake, Nano Lett. 2022. PMID: 35499493 pdf Link
• Jing H, Pálmai M, Saed B, George A, Snee PT, Hu YS^*, Cytosolic delivery of membrane-penetrating QDs into T cell lymphocytes: implications in immunotherapy and drug delivery, Nanoscale, 2021. PMID: 33688882 pdf Link

Counting molecules in situ with single-molecule labeling: Precise molecular quantification establishes baseline levels of molecular components within the cellular machinery, which is essential for maintaining proper physiological functions. These baseline levels also serve as references to identify disease-associated dysregulation, enabling the identification of novel mechanisms masked by ensemble-averaged measurements. This research is based on the premise that biological functions are encoded in the transient, non-covalent interactions among biomolecules. As such, the dynamic behaviors of biomolecules can be harnessed to improve super-resolution imaging toward precise molecular quantification. Our group has pioneered the development of a versatile single-molecule labeling technique using commercially available reagents. The technique diverges from standard immunofluorescence staining by directly capturing antibody binding and unbinding events to achieve super-resolution imaging (Image reveals the single-molecule labeling principle, ACS Nano, 2022). The advantage of this labeling technique is that the interaction frequency directly correlates with the substrate concentration, enabling precise molecular quantification in situ. By employing two orthogonal approaches—scaling down the association (on) rate via probe concentration or scaling up the dissociation (off) rate via chemical perturbation—we have successfully demonstrated single-molecule localization microscopy using this concept (Biophys J, 2024; Bioconjug Chem, 2023).

Representative publications:

• Gunasekara H, Perera T, Chao CJ, Bruno J, Saed B, Anderson J^#, Zhao Z, Hu YS*. Phalloidin-PAINT: Enhanced quantitative nanoscale imaging of F-actin. Biophys J. 2024;123(18):3051-3064. PMID: 38961624. Link.
• Gunasekara H^⫽, Perera T^⫽, Anderson J^#, Saed B, Ramseier N, Neama K^#, Hu YS^*. Superresolution imaging with single-antibody labeling. Bioconjug. Chem. 2023;34(5):825-833. PMID: 37145839; PMCID: PMC10859171. pdf Link
• Gunasekara H, Munaweera R, Hu YS^*, Chaotropic perturbation of noncovalent interactions of the hemagglutinin tag monoclonal antibody fragment enables superresolution molecular census, ACS Nano. 2022. PMID: 34797055 pdf Link Correction: Gunasekara H, Munaweera R, Novotná L, Lillemeier BF*, Hu YS^*, ACS Nano. 2022. PMID: 36417787 pdf Link

Organelle morphology and dynamics: This research investigates the spatio-temporal coordination of intracellular organelles in regulating the transport and secretion of signaling molecules that mediate and regulate immunity and inflammation. The research contributes to science in two areas. 1) Cytokine biology (Image shows intracellular cytokines within a single T cell). Our imaging work has advanced cytokine biology by uncovering enhanced vesicular trafficking as a mechanism underlying increased interleukin-2 production following T-cell activation (Biophys J, 2024). In parallel, we are developing labeling and imaging techniques to profile cytokines in their non-soluble forms, including membrane-bound cytokines and cytokines associated with extracellular vesicles, and to understand their novel roles in cancer biology and inflammation. 2) Mitochondrial dynamics and motility. We have developed super-resolution and live-imaging techniques to quantitatively characterize mitochondrial morphology and dynamics, and utilize these biophysical readouts to develop novel biomarkers of immune cell function. The research aims to identify molecular mechanisms masked by ensemble-level biochemical and omics measurements.

Representative publications:

• Saed B, Ramseier NT, Perera T, Anderson J^#, Burnett J, Gunasekara H, Burgess A, Jing H, Hu YS*, Increased vesicular dynamics and nanoscale clustering of IL-2 after T cell activation. Biophys J. 2024;123(15):2343-2353. PMID: 38532626; PMCID: PMC11331045. Link.
• Jiang Y, Krantz S, Qin X, Li S, Gunasekara H, Kim Y-M, Zimnicka A, Bae M, Ma K, Toth PT,  Hu Y, Shajahan-Haq AN, Patel HH, Gentile S, Bonini MG, Rehman J, Liu Y, Minshall RD*, Caveolin-1 controls mitochondrial damage and ROS production by regulating fission-fusion dynamics and mitophagy, Redox Biol. 2022. PMID: 35413643 PMCID: PMC9018165 pdf Link
• Waye AA , Ticiani E, Sharmin Z,  Perez SV, Perera T, Tu A, Buhimschi IA, Murga-Zamalloa CA, Hu YS, Veiga-Lopez A*. Reduced bioenergetics and mitochondrial fragmentation in human primary cytotrophoblasts induced by an EGFR-targeting chemical mixture. Chemosphere. 2024;364:143301. doi: 10.1016/j.chemosphere.2024.143301. Epub ahead of print. PMID: 39251161. Link.

From single molecules to translational impact: Single-molecule methods offer unique clinical insights that are not revealed at bulk levels. Our work has visualized oncogenic transcriptional programming in T-cell lymphoma at the single-molecular level within the native cellular environment. We have visualized the anti-cancer effects of clinically relevant small-molecule compounds, including histone deacetylase inhibitors and ribosome biogenesis inhibitors. In addition, we are leveraging the single-molecule sensitivity to understand therapeutic antibodies in immune checkpoint blockade through their interactions with membrane targets. Unlike standard assays, these studies develop ultrasensitive platforms to evaluate the performance of therapeutic agents using patient-derived materials, thereby advancing precision medicine.

Representative publications:

• Ramseier NT, Matsuda A, Lin AY*, Hu YS*, Uncovering bispecific antibody mechanisms via 3D superresolution imaging of T cell-tumor cell membrane contacts, Blood, Volume 146, Supplement 1, 2025, Page 4338, ISSN 0006-4971. Link.
• Kady N, Abdelrahman S, Rauf A, Burgess A, Weiss J, Gunasekara H, Ramseier N, Maine I, Zevallos-Morales A, Perez-Silos V, Wolfe A, Hristov A, Brown N, Inamdar K, Sverdlov M, Hu Y, Murga-Zamalloa C, Wang C*, and Wilcox R*. The GATA-3 dependent transcriptome and tumor microenvironment are regulated by eIF4E and XPO1 in T-cell lymphomas. Blood. 2025;145(6):597-611.PMID: 39652777. Link.
• Geng X, Wang C, Abdelrahman S, Perera T, Saed B, Hu YS, Wolfe A, Reneau J, Murga-Zamalloa C, Wilcox RA*. GATA-3 dependent gene transcription is impaired upon HDAC inhibition. Clin Cancer Res. 2024;30(5):1054–1066. PMID: 38165708; PMCID: PMC10922852 Link.