Biology subjects

Lee, J.-B.

Publications and source records attributed to Lee, J.-B..

9 recordsLinked to original sources

bioRxiv2025

PAF15 stabilizes PCNA on DNA and slows down its sliding dynamics

The PCNA-associated factor 15 (PAF15) protein is essential for human genome stability by regulating DNA replication and repair through its interaction with proliferating cell nuclear antigen (PCNA). Despite its significance, the mechanistic details of the PAF15-PCNA interaction remain insufficiently understood. Here, we reveal how PAF15 influences the sliding dynamics and function of PCNA. Using single-molecule diffusion analysis on a DNA skybridge platform, we show that PAF15 binding stabilizes PCNA in its DNA-bound state, reduces its diffusion rate along the duplex, restricts accessibility to PCNA PIP-box binding sites, and regulates PCNA loading and unloading on DNA via replication factor C (RFC). Our results are consistent with its coordinating role in both high-fidelity replication and lesion bypass. These findings establish PAF15 as a key regulator of PCNA function, serving as a mobile platform for DNA-editing enzymes and thereby influencing genome maintenance.

biophysics↗

bioRxiv2025

smDeepFLUOR: Single-Molecule Deep Learning Fluorescence Classification

Fluorescence intensity variation has long served as a primary readout for monitoring biological events. However, single-fluorophore signals arising from distinct molecular events often exhibit similar intensity profiles, making further classification challenging using conventional methods. In this study, we introduce smDeepFLUOR, a deep learning-based framework that resolves seemingly homogeneous spatiotemporal fluorescence signals by uncovering latent features imperceptible to conventional analyses. By leveraging a three-dimensional convolutional neural network trained on image sequences captured over 7 x 7 x 10 voxel windows, smDeepFLUOR reliably distinguishes specific from nonspecific protein binding, even across different experimental days, with an accuracy of up to 97%. Remarkably, smDeepFLUOR also captures real-time DNA synthesis kinetics by identifying subtle changes in the spatial distance between the fluorophore and the 3 end of nascent DNA, a feature undetectable by traditional methods. These classifications were achieved without incorporating explicit physical rules or engineered features, implying the presence of intrinsic, previously unrecognized differences in emission patterns. This approach significantly extends the analytical capabilities of single-molecule fluorescence imaging and opens new avenues for minimally labeled and label-free protein activities.

biophysics↗

bioRxiv2025

Inferring Spatially Varying Bending Stiffness of Biopolymers with Deep-Learning Approach

The bendability of biopolymers, including DNA, actin filaments, and microtubules, is crucial to diverse biological processes, such as cell motility and cytoskeletal organization. While conventional polymer models like the worm-like chain model assume uniform bending stiffness along the contour, biopolymers in reality often attain spatially varying bending stiffness as a result of complex cellular interactions. For instance, the bending stiffness of actin filaments and microtubules varies in response to associated binding proteins or chemical modifications. Despite its biological implications, measuring the position-dependent persistence length along polymer contours remains challenging. Here, we present a deep-learning-based method that quantitatively predicts spatially varying bending stiffness of biopolymers. Our framework segments a polymer chain into short over-lapping fragments, predicts local persistence lengths using a deep-learning model, and reconstructs full profiles. Using simulated data, we demonstrate that our approach achieves high accuracy and robustness even under data-scarce conditions. Applied to various biological systems, our method reveals a spatially varying stiffness from tip to base in filopodia as well as spatially heterogeneous stiffness in microtubules. Additionally, we investigate the internal mechanism of our deep-learning model and show that the model utilizes multiple physics-related features for persistence length estimation while adaptively adjusting its attention based on polymer stiffness.

biophysics↗

bioRxiv2024

DNA Hanger: Surface Free Single-Molecule Blotting Platform

Immunoblotting typically requires protein immobilization onto a membrane surface for quantification. Particularly, in real-time single-molecule blotting, stringent surface passivation is essential to prevent false-positive signals from nonspecific binding. In this study, we introduce a novel surface-free single-molecule blotting platform, termed the DNA Hanger. This platform employs a 3D structure of a quartz slide. Modified{lambda} -phage DNA molecules are randomly biotinylated along their bases and suspended between 4 m-high thin barriers, positioned away from the quartz slide surface. A light sheet, produced within the 3D structure, illuminates fluorophore-conjugated proteins bound to the biotinylated DNA, enabling single-molecule detection. The DNA Hanger assay significantly reduces nonspecific binding and enhances sensitivity to sub-picomolar concentrations, suggesting that this platform provides a novel, surface-condition-independent, and real-time approach in a wide range of single-molecule blotting.

biophysics↗

bioRxiv2024

Innovative Strategies for Enhancing Signal-to-Noise Ratio in Single-molecule Imaging: The Influence of Molecular Motion

Single-molecule fluorescence imaging has extensively revealed the dynamic and structural characteristics of biomolecules. However, its application is limited by the upper concentration of fluorophore-tagged biomolecules, which is in the sub-ten nanomolar range. We found that the signal-to-noise ratio (SNR) in single-molecule fluorescence imaging is strongly influenced by the size of fluorophore-labeled molecules in solution. Our computational simulations suggest that the faster diffusion of background fluorophores can enhance the SNR of target molecules. Moreover, we identified that the molecular motion through fluid flow can improve SNR. This study provides a novel perspective by emphasizing the importance of molecular motion in SNR and propose a rapid barrier-free method to increase the upper concentration limit in single-molecule imaging.

biophysics↗

bioRxiv2024

Tau condensation on DNA and localization on centromeres: A potential link to cell division

Tau protein, traditionally recognized for stabilizing microtubules and forming pathological aggregates, has recently been observed to form condensates in various contexts. While its condensation with RNA has been well studied, the interaction between tau and DNA, along with its biological significance, remains less explored. Here, using single-molecule experiments, we found that tau binds stably to naked DNA at nanomolar concentrations, leading to the local co- condensation of tau and DNA. These tau condensates on DNA can also interface with microtubules, leveraging taus known role in promoting microtubule growth and organization. The dynamic nature of these condensates facilitates the remodeling of the DNA-microtubule assembly. Interestingly, two phosphomimetic tau mutants, T231D/S235D and S262D, retained their affinity for DNA but differed in their ability to link microtubules to DNA. Furthermore, imaging of HEK-293 and SH-SY5Y cells in early mitosis revealed that tau localizes on centromeres, poised to capture nascent mitotic spindles. Building on these observations, we speculate that tau may play a novel role in mitosis, where tau clusters facilitate the early registration of mitotic spindles to chromosomes before kinetochore-mediated attachment. We also discuss the potential implications of this model in conditions where abnormal cell cycle re-entry and tau activity may disrupt cell division.

biophysics↗

bioRxiv2022

In vivo CRISPR screens reveal Serpinb9 and Adam2 as regulators of immune therapy response in lung cancer

How the genetic landscape of a tumor governs the tumors response to immunotherapy remains largely elusive. Here, we established a direct in vivo CRISPR/Cas9 gene editing methodology to assess the immune-modulatory capabilities of 573 putative cancer genes associated with altered cytotoxic activity in human cancers. Using KrasG12D- and BrafV600E-driven mouse lung cancer models, we identify Serpinb9 and Adam2 as our top immune suppressive and immune enhancing genes, respectively. Mechanistically, we show that Serpinb9 ablation in KrasG12D- and BrafV600E-mutant lung tumor cells greatly enhances the efficacy of cytotoxic T-cells in vitro and in vivo. ADAM2 is a cancer testis antigen broadly expressed in human cancers such as lung adenocarcinoma (13.9%), renal (74.7%), prostate (72.4%), uterine (28.6%) and invasive breast (9.5%) cancer. In our mouse models, we show that Adam2 expression is induced in KrasG12D- but not BrafV600E-driven murine lung tumors and that its expression is further enhanced by immunotherapy. We show that loss of Adam2 significantly decreases KrasG12D-lung tumor burden but blocks the efficacy of cytotoxic T-cells. Consistently, Adam2 overexpression dramatically increases tumor growth and enhances immunotherapy efficacy. Mechanistically, we find that Adam2s oncogenic function depends on modulating the tumor immune microenvironment by restraining productive type I and type II interferon responses as well as cytokine signaling, reducing the presentation of tumor-associated antigen, and modulating surface expression of several immunoregulatory receptors within Kras-driven lung tumors. Adam2 expression also leads to reduced levels of immune checkpoint inhibitors such as Pd-l1, Lag3, Tigit and Tim3. This reduced exhaustion within the tumor microenvironment may explain why ex vivo expanded and adoptively transferred cytotoxic T-cells show enhanced cytotoxic efficacy against Adam2 overexpressing lung tumors. Together, our study highlights the power of integrating cancer genomic with in vivo CRISPR/Cas9 screens to uncover how cancer-associated genetic alterations control responses to immunotherapies.

cancer biology↗

bioRxiv2021

The Formation of a Stable Sliding Clamp Discriminates MSH2-MSH3 and MSH2-MSH6 Mismatch Interaction

MutS homologs (MSH) are highly conserved core components of DNA mismatch repair (MMR). Mismatch recognition provokes ATP-binding by MSH proteins that drives a conformational transition from a short-lived lesion-searching clamp to an extremely stable sliding clamp on the DNA. Once on DNA the MSH sliding clamps provide a platform for the assembly of MMR strand-specific excision components beginning with the highly conserved MutL homologs (MLH/PMS). Previous studies with short mismatch-containing oligonucleotides revealed an MSH ATP hydrolysis (ATPase) cycle that included mismatch recognition, the formation of an ATP-bound sliding clamp and dissociation from the end of a mismatched DNA that ultimately recovers the mismatch binding conformation. We found that ATP-bound MSH complexes on blocked-end or very long DNA are extremely stable under a range of ionic conditions. These observations underpinned the development of a high-throughput fluorescence resonance energy transfer (FRET) system capable of clearly distinguishing between HsMSH2-HsMSH3 and HsMSH2-HsMSH6 activities that is suitable for chemical inhibitor screens.

biochemistry↗

bioRxiv2018

F-actin dynamics transform filopodial bridges into intercellular nanotubes capable of distant cell communication

A novel actin-based bridge connecting cells has been recognized as a new pathway for the distant transport of cytoplasmic components, viruses, or pathogenic substances between cells. However, it is not yet known how such a fine structure extends over several hundred micrometres and remains robust for several hours. Using optical fluorescence imaging methods, we found that random contact promotes the formation of filopodial bridges through N-cadherin interactions between filopodia, which are slender actin-rich plasma membrane protrusions. These filopodial bridges eventually evolve into a single actin-based bridge (intercellular nanotube) that connects two cells via an intermediate state that involves a helical structure. Surprisingly, the twisting of two filopodia is likely to result from the rotational motion of actin filaments inside the filopodia by myosin V. The accumulated torsion of the filopodia triggers the release of one of the paired filopodia, whose end is attached to the other cell body by an N-cadherin cluster. The resulting retraction of the filopodium by retrograde F-actin flow leaves a single bridge. The N-cadherin/catenin cluster is likely to form a synapse between the intercellular nanotube and the cell body. This study sheds light on the formation mechanism of the filopodial bridge-based intercellular nanotubes for long-distance communication between cells.

biophysics↗