Biology subjects

Tsai, F.

Publications and source records attributed to Tsai, F..

6 recordsLinked to original sources

bioRxiv2026

A Brugada-related KCNT1 mutation unveils its conductance-independent activation of store-operated Ca 2+ entry

BackgroundPotassium sodium-activated channel subfamily T member 1 (KCNT1) is a Na+-activated K+ channel, associated with epilepsy and cardiac diseases. Besides K+ conductance, KCNT1 is also involved in Ca2+ handling. KCNT1:R1106Q was identified in a Brugada Syndrome (BrS) patient; however, its pathological characteristics regarding either K+ or Ca2+ homeostasis remain to be fully elucidated. MethodsFura-2-loaded HEK293T cells were used for intracellular Ca2+ investigations, and whole-cell patch-clamp technique were used as complementary tests. Stromal interaction molecule 1 (STIM1) aggregation and endoplasmic reticulum (ER)-plasma membrane (PM) junctions in HeLa cells were visualized using spinning disc confocal microscope. Multielectrode array (MEA) assays were utilized to assess the field potential duration (FPD) and spontaneous beating rate of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). ResultsKCNT1 overexpression increased SOCE, which was further upregulated by the KCNT1R1106Q mutant. Furthermore, KCNT1 upregulated ER Ca2+ release and was found to localize at ER-PM junctions. The ER Ca2+ dynamics in KCNT1R1106-overexpressing cells was disrupted, while its SOCE remained intact. The KCNT1 cytoplasmic tail alone sufficiently modulated Ca2+ homeostasis. Given that KCNT1 possesses prominent charge-concentrated segments within its cytoplasmic domain, we substituted charged amino acids in either the 740-DDE-742 or 1114-RRLSR-1118 motifs with alanine; these substitutions abolished the KCNT1-mediated increases in ER Ca2+ release and ER-PM contact formation. MEA recordings of hiPSC-CMs revealed that the overexpression of KCNT1 shortened the FPD and accelerated the cardiomyocyte beating rate compared to baseline. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q was significantly attenuated compared to that of KCNT1WT. ConclusionsOur findings indicate that, independent of KCNT1-mediated ion conductance, the charged motifs located at both ends of the KCNT1 cytoplasmic tail serve as structural anchors to facilitate ER-PM contact formation. This non-conducting role endows KCNT1 with a K+ current-independent mechanism to modulate intracellular Ca2+ homeostasis and cardiomyocyte physiology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/731813v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@104e234org.highwire.dtl.DTLVardef@19eceb3org.highwire.dtl.DTLVardef@1148512org.highwire.dtl.DTLVardef@2c8118_HPS_FORMAT_FIGEXP M_FIG C_FIG Novelty and SignificanceO_ST_ABSWhat is known?C_ST_ABSO_LIKCNT1 is a Na+-activated K+ channel, and mutations within this gene are often associated with epilepsy and epilepsy-related cardiac diseases. C_LIO_LIBesides functioning as a rectifier, KCNT1 is implicated in cellular Ca2+ handling, such as regulating Ca2+ oscillations in rat cortical neurons. C_LIO_LIKCNT1:R1106Q missense mutation has been identified in a patient with Brugada Syndrome (BrS); however, its pathophysiological characteristics and underlying mechanisms remain uncharacterized. C_LI What New Information Does This Article Contribute?O_LIKCNT1 overexpression upregulates both ER Ca2+ release and store-operated Ca2+ entry (SOCE). KCNT1R1106Q further increased SOCE intensity, however, its increase in ER Ca2+ release and [Ca2+]ER were attenuated. C_LIO_LIThe cytoplasmic tail of KCNT1 alone is suNicient to modulate Ca2+ closely mimicking the eNects of full-length KCNT1. C_LIO_LIThe charged clusters distributed along the KCNT1 cytoplasmic tail facilitate ER-PM contact. Alanine substitutions abolish the enhancements in ER Ca2+ release and ER-PM contact formation, while SOCE modulation remains intact. C_LIO_LIThe KCNT1 cytoplasmic tail alone shortens the field potential duration (FPD) and accelerates the spontaneous beating rate in hiPSC-CMs. However, the magnitude of beating rate acceleration induced by KCNT1R1106Q is significantly attenuated compared to KCNT1WT. C_LI This study uncovers a novel, conductance-independent role for the KCNT1 channel in structural organization, demonstrating that the charged motifs on its cytoplasmic tail serve as physical anchors facilitating ER-PM junctions. This structural mechanism regulates intracellular Ca2+ homeostasis and subsequently alters cardiomyocyte electrophysiology. By decoupling KCNT1s structural impact from its ion-conducting function, these findings provide new mechanistic insights into how specific KCNT1 variants, such as the BrS-associated KCNT1:R1106Q mutation, may contribute to arrhythmogenesis through imbalanced Ca2+ handling rather than classical K+ current alterations.

Molecular Biology↗

bioRxiv2024

Preclinical Proof of Concept for SNAPTM-TIL: a personalized, next-generation tumor-infiltrating lymphocyte therapy platform

Tumor-infiltrating lymphocyte (TIL) therapy, which involves extracting, expanding, and reinfusing immune cells to target cancer cells, has shown promise in melanoma treatment, but requires optimization for broader efficacy. The success of TIL therapy depends on the recognition of tumor-associated antigens, but neoantigen-reactive T-cells are often rare and exhausted in less immunogenic malignancies. Isolating T cells enriched in neoantigen reactivity prior to in vitro expansion and reinfusion may improve the response rates. To this end, our proprietary Specific Neo-Antigen Peptides (SNAP) technology platform improves the accuracy of neoantigen prediction and validation by combining advanced computational modeling and PepSeq, a high-throughput screen for the physical credentialing of putative neoantigens based on their affinity to bind patient-specific HLA class II proteins. This approach allows for the education and enrichment of TILs (SNAP-TILs) with personalized, predefined, highly immunogenic neoantigens prior to expansion. Using the SNAP platform, we consistently achieved, on average, an SNAP-TIL product comprising 96% CD3+ cells, with a mixture of 75% effector and 23% central memory cells. SNAP-TILs exhibited greater efficacy and selectivity in immune infiltration than TIL, which was expanded by the rapid expansion protocol alone using ex vivo models. SNAP-TIL was also reactive in highly and poorly immunogenic tumors, with 70% and 50% tumor growth inhibition in melanoma and pancreatic patient-derived xenograft models, respectively. This study demonstrates the novel benefit of our Personalized Neoantigen Pipeline approach, potentially providing a durable antitumor immune response for a larger proportion of cancer patients.

immunology↗

bioRxiv2024

Live-cell transcriptomics with engineered virus-like particles

The transcriptional state of a cell provides a multi-parameter representation of gene expression programs that reflect its identity and phenotype. However, current transcriptomic profiling technologies result in destruction of the biological sample, preventing direct analysis of transcriptional dynamics in the same living cells over time. Here, we developed a synthetic RNA export system called cellular self-reporting to address this fundamental technological limitation. We repurposed the murine leukemia virus retroviral protein Gag to enable diverse types of immortalized and primary mammalian cells to package cellular RNA molecules in virus-like particles (VLPs) for export into the extracellular environment. We applied self-reporting to interrogate the transcriptome-wide dynamics that occur during neuronal differentiation from induced pluripotent stem cells and detected gene expression profiles from individual live cells. Leveraging this genetically encodable approach, we expanded the capabilities of self-reporting through molecular engineering of VLP components. Pseudotyping VLPs with epitope-tagged envelope proteins enabled multiplexed selective live-cell readout of transcriptional states from heterogeneous co-cultures. Furthermore, structure-guided protein engineering of Gag fusions with human RNA binding domains improved the mRNA representation in self-reporting readouts and enabled the directed export of libraries of synthetic barcode transcripts. Taken together, this work establishes self-reporting as a facile and broadly enabling technology for live-cell, transcriptome-scale profiling of dynamic processes across diverse cell types and biological applications.

bioengineering↗

bioRxiv2022

Functional antibodies exhibit light chain coherence

The vertebrate adaptive immune system modifies the genome of individual B cells to encode antibodies binding particular antigens1. In most mammals, antibodies are composed of a heavy and a light chain which are sequentially generated by recombination of V, D (for heavy chains), J, and C gene segments. Each chain contains three complementarity-determining regions (CDR1-3), contributing to antigen specificity. Certain heavy and light chains are preferred for particular antigens2-21. We considered pairs of B cells sharing the same heavy chain V gene and CDRH3 amino acid sequence and isolated from different donors, also known as public clonotypes22,23. We show that for naive antibodies (not yet adapted to antigens), the probability that they use the same light chain V gene is [~]10%, whereas for memory (functional) antibodies it is [~]80%. This property of functional antibodies is a phenomenon we call light chain coherence. We also observe it when similar heavy chains recur within a donor. Thus, though naive antibodies appear to recur by chance, the recurrence of functional antibodies reveals surprising constraint and determinism in the processes of V(D)J recombination and immune selection. For most functional antibodies, the heavy chain determines the light chain.

immunology↗

bioRxiv2022

enclone: precision clonotyping and analysis of immune receptors

Half a billion years of evolutionary battle forged the vertebrate adaptive immune system, an astonishingly versatile factory for molecules that can adapt to arbitrary attacks. The history of an individual encounter is chronicled within a clonotype: the descendants of a single fully rearranged adaptive immune cell. For B cells, reading this immune history for an individual remains a fundamental challenge of modern immunology. Identification of such clonotypes is a magnificently challenging problem for three reasons: O_LIThe cell history is inferred rather than directly observed: the only available data are the sequences of V(D)J molecules occurring in a sample of cells. C_LIO_LIEach immune receptor is a pair of V(D)J molecules. Identifying these pairs at scale is a technological challenge and cannot be done with perfect accuracy--real samples are mixtures of cells and fragments thereof. C_LIO_LIThese molecules can be intensely mutated during the optimization of the response to particular antigens, blurring distinctions between kindred molecules. C_LI It is thus impossible to determine clonotypes exactly. All solutions to this problem make a trade-off between sensitivity and specificity; useful solutions must address actual artifacts found in real data. We present enclone1, a system for computing approximate clonotypes from single cell data, and demonstrate its use and value with the 10x Genomics Immune Profiling Solution. To test it, we generate data for 1.6 million individual B cells, from four humans, including deliberately enriched memory cells, to tax the algorithm and provide a resource for the community. We analytically determine the specificity of enclones clonotyping algorithm, showing that on this dataset the probability of co-clonotyping two unrelated B cells is around 10-9. We prove that using only heavy chains increases the error rate by two orders of magnitude. enclone comprises a comprehensive toolkit for the analysis and display of immune receptor data. It is ultra-fast, easy to install, has public source code, comes with public data, and is documented at bit.ly/enclone. It has three "flavors" of use: (1) as a command-line tool run from a terminal window, that yields visual output; (2) as a command-line tool that yields parseable output that can be fed to other programs; and (3) as a graphical version (GUI).

immunology↗

bioRxiv2019

CloneRetriever: retrieval of rare clones from heterogeneous cell populations

BackgroundMany biological processes, such as cancer metastasis, organismal development, and development of resistance to cytotoxic therapy, rely on the emergence of rare sub-clones from a larger population. Understanding how the genetic and epigenetic features of diverse clones affect clonal fitness provides insight into molecular mechanisms underlying selective processes. However, identifying causal drivers of clonal fitness remains challenging. Population-level analysis has limited resolution to characterize clones prior to selection, while high-resolution single-cell methods are destructive and challenging to scale across large populations, limiting further functional analysis of relevant clones.\n\nResultsHere, we develop CloneRetriever, a methodology for tracking and retrieving rare clones throughout their response to selection. CloneRetriever utilizes a CRISPR sgRNA-barcode library that allows isolation of viable cells from specific clones within the barcoded population using a sequence-specific retrieval reporter. We demonstrated that CloneRetriever can measure clonal fitness of cancer cell models in vitro and retrieve targeted clones at abundance as low as 1 in 1,883 in a heterogeneous cell population.\n\nConclusionsCloneRetriever provides a means to track and access specific and rare clones of interest across dynamic changes in population structure to comprehensively explore the basis of these changes.

genomics↗