Biology subjects

Lee, K.-Y.

Publications and source records attributed to Lee, K.-Y..

9 recordsLinked to original sources

bioRxiv2026

Structures of Cas9-Bound Double-Stranded DNA Mini-Circle Reveal Impacts of DNA Shape on Cas9 Target Interrogation

CRISPR-Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA at specific sites and has been adapted as a powerful tool for genome manipulation. Cas9 recognizes its target through multiple conformational transitions coordinated between the Cas9 ribonucleoprotein and the DNA duplex. Such transitions, and consequently Cas9 targeting specificity, are expected to be significantly influenced by the collective duplex physical properties referred to as DNA shape. To advance our currently limited understanding of the interplay between DNA shape and Cas9 target interrogation, we solved two cryo-EM structures of SpyCas9 bound to a cognate target embedded in a relaxed 95-base-pair DNA double-stranded minicircle. The Cas9-bound DNA segment engages in similar interactions involved in PAM-binding and R-loop initiation as those observed in Cas9-bound linear DNA. However, R-loop is limited to less than three base-pairs, thus interfering with Cas9 cleavage. The minicircle DNA, which is fully resolved, retains its global shape. As Cas9 locally unwinds the protospacer, the closed-ring topology constrains the movement of the paired PAM-distal DNA duplex, thus interfering with R-loop propagation. These data provide detailed insight into the interplay between DNA shape and Cas9 structure and function, and may shed light on genome-editing and manipulation in environments with varied DNA topologies.

biophysics↗

bioRxiv2026

Cryo-EM Structure of a 95-Basepair Double-Stranded DNA Minicircle at 5.3 A Resolution

Double-stranded DNA minicircles have been observed in a variety of biological settings and are also widely employed in biotechnology, therapeutic applications, and basic research. Here, we report a cryo-EM structure of a 95-basepair minicircle (dsMC95) at a 5.3 [A] resolution. dsMC95 forms a closed ring as designed and no local deformation is observed. The two DNA strands are fully resolved, with the major and minor grooves clearly distinguishable. Analysis reveals a nine-fold periodicity in the helical twist, which corresponds to approximately 10.56 base pairs per turn. Together with groove width analysis, the data indicate that dsMC95 maintains a B-DNA configuration. The dsMC95 ring exhibits an in-plane ellipticity of 1.13 and an out-of-plane displacement of 15{degrees}, with differences in out-of-plane displacements observed between the two half-segments. The dsMC95 structure, which is the only free DNA cryo-EM structure with a resolution better than 6 [A] to date, allows comparison to other structures to better understand DNA physical features such as bending. The findings advance our understanding of DNA structure under topological constraints and may inform studies of naturally occurring small circular DNA as well as the manipulation of DNA in nanotechnology applications.

biophysics↗

bioRxiv2025

The respiratory phase causally modulates the readiness potential amplitude

Previous research has shown that the respiratory phase correlates with both voluntary action timing and readiness potential (RP) amplitude, but whether this relationship is causal or merely correlational remains unclear. Here, we experimentally manipulated breathing patterns to test if the respiratory phase causally influences the neural preparation for voluntary movement. Participants performed self-initiated button presses under four conditions: breathing in (BI), breathing out (BO), normal breathing (NB), and breath-holding (BH). Electroencephalographic recordings revealed that RP amplitude was significantly more negative during exhalation compared to inhalation and during breath-holding compared to normal breathing. These neural differences occurred without corresponding changes in behavioral measures (e.g., waiting times or retrospective timing judgments), indicating that respiratory effects on RP were not associated with altered action timing. These findings demonstrate that the respiratory phase causally modulates cortical motor preparation. We further propose that the brain optimizes voluntary action execution during respiratory phases when breathing-related motor activity is minimal, thereby revealing respiration as a fundamental organizing rhythm for voluntary behavior.

neuroscience↗

bioRxiv2024

The CYK-4 GAP domain regulates cortical targeting of centralspindlin to promote contractile ring assembly and facilitate ring dissolution

During cytokinesis, an equatorial contractile ring partitions the cell contents. Contractile ring assembly requires an equatorial zone of active GTP-bound RhoA generated by the guanine nucleotide exchange factor ECT21,2. ECT2 is activated by centralspindlin, a complex composed of two molecules each of kinesin-6 and CYK4. During anaphase, Centralspindlin is activated at the central spindle between the separating chromosomes and diffuses to the plasma membrane, where it engages with ECT2 via its N-terminal half3,4. The C-terminal half of CYK4 contains a lipid-binding C1 domain that contributes to plasma membrane targeting5 and a GTPase-activating protein (GAP) domain that has an interaction surface for a Rho family GTPase, whose functions have remained unclear 1,3,4,6,7. Here, using the one-cell stage C. elegans embryo as a model, we show that RhoA and the Rho-binding interface of the CYK4 GAP domain drive the recruitment of centralspindlin to the equatorial cortex. By contrast, a point mutant that selectively disrupts GAP activity does not prevent cortical centralspindlin recruitment but instead substantially delays dissipation of centralspindlin from the cortex. These findings suggest that positive feedback, in which centralspindlin recruitment promotes the generation of active RhoA and active RhoA drives centralspindlin recruitment, is central to the rapid assembly of the contractile ring within a narrow time window. They also indicate that the CYK4 GAP catalytic activity contributes to release of centralspindlin from the cortex, potentially to ensure timely dissolution of the contractile ring.

cell biology↗

bioRxiv2024

The Taiwan Precision Medicine Initiative: A Cohort for Large-Scale Studies

The Taiwan Precision Medicine Initiative (TPMI), a project initiated by the Academia Sinica in collaboration with 16 major medical centers around Taiwan, has recruited 565,390 participants who consented to provide DNA samples for genetic profiling and grant access to their electronic medical records (EMR) for studies to develop precision medicine. Access to the EMR is both retrospective and prospective, allowing researchers to conduct prospective studies over time. Genetic profiling is done with population-optimized SNP arrays for the Han Chinese populations that enable genetic analyses such as genome-wide association, phenome-wide association, and polygenic risk score studies to evaluate common disease risk and pharmacogenetic response. Furthermore, the TPMI participants agree to be contacted for future research opportunities related to their genetic risks and receive personalized genetic risk profiles with health management recommendations. TPMI has established the TPMI Data Access Platform (TDAP), a central database and analysis platform that both safeguards the security of the data and facilitates academic research. The TPMI is the largest non-European cohort that merges genetic profiles with EMR in the world. With a cohort that can be followed over time, it can be utilized to validate genetic risk prediction models, conduct clinical trials to show the efficacy of risk-based health management, and optimize health policies based on genetic risks. In this report, we describe the TPMI study design, the population and genetic characteristics of the TPMI cohort, and the power it provides to conduct crucial studies in developing precision medicine on a population and personal level. As Han Chinese represent almost 20% of the worlds population, the results of TPMI studies will benefit >1.4 billion people around the world and serve as a model for developing population-based precision medicine.

genetics↗

bioRxiv2023

Self-dyeing textiles grown from cellulose-producing bacteria with engineered tyrosinase expression

Environmental concerns are driving interests in post-petroleum synthetic textiles produced from microbial and fungal sources. Bacterial cellulose is a promising sustainable leather alternative, on account of its material properties, low infrastructure needs and biodegradability. However, for alternative textiles like bacterial cellulose to be fully sustainable, alternative ways to dye textiles need to be developed alongside alternative production methods. To address this, we here use genetic engineering of Komagataeibacter rhaeticus to create a bacterial strain that grows self-dyeing bacterial cellulose. Dark black pigmentation robust to material use is achieved through melanin biosynthesis in the bacteria from recombinant tyrosinase expression. Melanated bacterial cellulose production can be scaled up for the construction of prototype fashion products, and we illustrate the potential of combining engineered self-dyeing with tools from synthetic biology, via the optogenetic patterning of gene expression in cellulose-producing bacteria. With this work, we demonstrate that combining genetic engineering with current and future methods of textile biofabrication has the potential to create a new class of textiles.

synthetic biology↗

bioRxiv2018

Ancestral Roles of the Fam20C Family of Secreted Protein Kinases Revealed by Functional Analysis in C. elegans

Fam20C is a secreted protein kinase mutated in Raine syndrome, a human skeletal disorder. In vertebrates, bone and enamel proteins are major Fam20C substrates. However, Fam20 kinases are conserved in invertebrates lacking bone and enamel, suggesting other ancestral functions. We show that FAMK-1, the C. elegans Fam20C ortholog, contributes to fertility, embryogenesis, and development. These functions are not fulfilled when FAMK-1 is retained in the early secretory pathway. During embryogenesis, FAMK-1 maintains inter-cellular partitions and prevents multinucleation; notably, temperature elevation or lowering cortical stiffness reduces requirement for FAMK-1 in this context. FAMK-1 is expressed in multiple adult tissues that undergo repeated mechanical strain, and selective expression in the spermatheca restores fertility. Informatic, biochemical and functional analysis implicate lectins as FAMK-1 substrates. These findings suggest that FAMK-1 phosphorylation of substrates, including lectins, in the late secretory pathway is important in embryonic and tissue contexts where cells are subjected to mechanical strain.

cell biology↗

bioRxiv2017

CYK-4 functions independently of its centralspindlin partner ZEN-4 to cellularize oocytes in germline syncytia

Throughout metazoans, germ cells undergo incomplete cytokinesis to form syncytia connected by intercellular bridges. Formation of gametes ultimately requires bridge closure. Here, we investigate the contribution of the conserved bridge component centralspindlin to oocyte production in C. elegans. Centralspindlin is composed of the Rho family GTPase-activating protein (GAP) CYK-4/MgcRacGAP and the microtubule motor ZEN-4/kinesin-6, which are both essential for cytokinesis. In contrast, we show that oocyte production by the syncytial germline requires CYK-4 but not ZEN-4. Longitudinal imaging after conditional CYK-4 inactivation revealed a role in oocyte cellularization, rather than in generation of syncytial compartments. CYK-4s lipid-binding C1 domain and the GTPase-binding interface of its GAP domain were individually important for oocyte cellularization and for targeting CYK-4 to bridges, where it contributes to enrichment of active RhoA. These results identify a C1-GAP module in CYK-4 that recruits it to bridges in the germline and directs their closure to produce oocytes.\n\nIMPACT STATEMENTThe CYK-4 subunit of centralspindlin, a broadly conserved component of intercellular bridges across metazoa, is required for the cytokinesis-like closure of intercellular bridges that cellularizes oocytes to separate them from germline syncytia.\n\nMAJOR SUBJECT AREASCell Biology, Developmental Biology & Stem Cells

cell biology↗

bioRxiv2017

Systematic selection of reference genes for normalization of circulating RNA transcripts in pregnant women based on RNA-seq data

RNA transcripts circulating in peripheral blood represent an important source of non-invasive biomarkers. To accurately quantify the levels of a circulating transcript, one needs to normalize the data with internal control reference genes, which are detected at relatively constant levels across different blood samples. A few stably-expressed reference gene candidates have to be selected from transcriptome data before validation of their stable expression by reverse-transcription quantitative polymerase chain reaction. However, there is a lack of transcriptome, let alone whole-transcriptome, data from maternal blood. To overcome this shortfall, we performed RNA-seq on blood samples from women presented with preterm labor. Of 11215 exons detected in the maternal blood whole-transcriptome, we systematically identified a panel of 395 genes comprising exons that were detected at a coefficient of variation (CV) ranging from 7.75%-17.7%. Their levels were considerably less variable than any GAPDH exon (minimum CV, 27.3%). Upon validation, selected genes from this panel remained as more stably expressed than GAPDH in maternal blood. This panel is over-represented with genes involved with actin cytoskeleton, macromolecular complex and the integrin signaling pathway. This groundwork provides a starting point for systematically selecting reference gene candidates for normalizing the levels of circulating RNA transcripts in maternal blood.

genomics↗