Biology subjects

Lim, K. S.

Publications and source records attributed to Lim, K. S..

9 recordsLinked to original sources

bioRxiv2026

Intrafilament nucleotide exchange in a prokaryotic actin homolog

Polymerization and disassembly govern the cellular functions of cytoskeletal proteins. Canonical nucleotide-dependent polymers, including actin and microtubules, renew their nucleotide state through subunit subunit dissociation, nucleotide exchange in solution and repolymerization. Yet the assembly dynamics of the membrane-bound bacterial actin MreB remain unresolved. Here, using total internal reflection fluorescence microscopy and high-speed atomic force microscopy, we visualise Bacillus subtilis MreB assembly on supported lipid bilayers in real time. ATP binding drives polymerization into symmetrically elongating filaments, whereas ATP hydrolysis within filaments promotes disassembly. Unexpectedly, nucleotides continuously exchange within membrane-bound filaments without detectable subunit turnover, coupling filament stability to the surrounding nucleotide pool: ATP exchange stabilizes filaments, whereas ADP exchange triggers rapid fragmentation and disassembly. Monte-Carlo modelling and single-cell in vivo imaging support this mechanism. Thus, intrafilament nucleotide exchange enables MreB filaments to renew their nucleotide state without polymer turnover, revealing nucleotide-state rejuvenation as a new mode of biological polymer regulation.

microbiology↗

bioRxiv2026

ATP-driven membrane binding and polymerization of bacterial actin MreB promotes local membrane fluidization

The bacterial actin homologue MreB plays a key role in rod cell shape determination. We recently showed that MreB from the Gram-positive bacterium Geobacillus stearothermophilus (MreBGs) polymerizes into straight pairs of protofilaments in the presence of both ATP and a lipid surface. Membrane interaction is thought to be mediated by electrostatic interactions with anionic lipids, with final anchoring relying on two spatially close hydrophobic motifs that protrude from the MreBGs monomers, forming a putative membrane-insertion domain. Here, we determined the binding properties of ATP and ADP to MreBGs using fluorescence anisotropy, and monitored ATP-mediated binding and polymer formation on lipid bilayers using liposome binding assays and AFM, respectively. Finally, we used solid-state NMR to visualize the interaction between the membrane and MreBGs at the atomic level. Our findings reveal that MreBGs has similar affinity for both ATP and ADP, unlike eukaryotic actin. We also show that monomeric MreBGs establishes peripheral contacts with the membrane likely through electrostatic interactions, while ATP-induced MreBGs filaments insert into the lipid bilayer without interfering with the membrane lamellar phase and have a significant local fluidifying effect. Statement of significanceBacteria rely on the actin-like protein MreB to determine and maintain their cell shape, like actin does in eukaryotic cells. To perform its tightly regulated morphogenetic function, MreB forms membrane-associated nanofilaments in vivo, which control the cell wall biosynthetic machinery. We recently demonstrated that, in vitro, MreB from the Gram-positive bacterium Geobacillus stearothermophilus requires both ATP and lipids to polymerize into pairs of filaments. Here, we show that in the presence of ATP, Geobacillus MreB forms membrane-bound filaments that directly impact local membrane fluidity, which could translate into a regulatory effect on cell wall synthetic enzymes. We further reveal that MreB binds both ATP and ADP with similar affinity, unlike eukaryotic actin, which preferentially binds ATP over ADP, and speculate that this could be a mechanism modulating the pool of polymerization-competent MreB in bacteria.

biophysics↗

bioRxiv2026

Dityrosine photocrosslinking of native collagen bioinks for controlled shape-fidelity of bioprinted cardiac tissue constructs: probing the interplay between fibrillogenesis and covalent bond formation

Collagen bioinks are widely used in biofabrication, but their relatively soft mechanical properties can lead to structural instabilities under cell-generated contraction forces. While synthetic functional groups can be conjugated for covalent crosslinking, these methods often disrupt natural protein fibrillogenesis, thereby compromising collagen fibre architecture. This work presents a strategy for the direct covalent stabilisation of native collagen bioinks with dityrosine bonds via visible-light photocrosslinking with ruthenium (Ru) and sodium persulfate (SPS), avoiding the need for polymer pre-functionalisation. Multimodal characterisation, including high-resolution microscopy, spectroscopy, mass spectrometry, and nanoindentation, identified photocrosslinking conditions that enhance collagen fibrillogenesis and reduce off-target polymer oxidation. Interestingly, the biofabrication process itself affected ultimate collagen fibre architecture, with shear-induced alignment during extrusion enhancing fibril proximity and self-assembly, overcoming inhibitory effects the crosslinkers had on fibrillogenesis via ionic and electrostatic interactions. Leveraging these insights, embedded bioprinting was used to fabricate cardiac constructs with high cell viability (>80%), where dityrosine crosslinking could be tuned to modulate geometric shape changes under cell-generated forces (1-15% shrinkage). Finally, the platform was used to bioprint anatomically accurate double-ventricle human heart models with robust shape fidelity. This research establishes a versatile photocrosslinking framework for bioprinting cardiac constructs with tunable shape stability using native collagen bioinks.

bioengineering↗

bioRxiv2025

Tissue resident memory T cells populate the human uveal tract

The current concept is that the eye is an immune privileged site endowed with innate immune regulatory networks to maintain organ function. We now have evidence that resident T cells occupy intraocular tissues. In immune-mediated inflammatory diseases, such as psoriasis and rheumatoid arthritis, tissue resident T cells trigger disease flares in the skin and joints. This suggests resident T cells in the uvea may have similar functions in non-infectious immune-mediated uveitis, a collective term for autoinflammatory and autoimmune diseases of the uveal tract causing intraocular inflammation. Here, we demonstrate by spectral cytometry and immunofluorescence imaging that non-inflamed uveal tissue contains multiple T cell subtypes including CD8+ CD103+ tissue resident memory T (TRM) cells. Using single cell RNA & T cell receptor (TCR) sequencing to profile aqueous humour cells from donors with acute, active uveitis, we identify clonally expanded T cells which are enriched for TRM -associated genes. We further show that in donors with active uveitis, CD8+ CD103+ T cells persist within tissue in the uveal tract. Using bulk RNA sequencing and weighted gene co-expression network analysis (WGCNA) we show that quiescent iris tissue from donors with a history of uveitis are enriched for genes associated with T cell activation and antigen presentation. Finally, we demonstrate that TRM cells persist in the anterior uvea in mice following resolution of experimental autoimmune uveoretinitis (EAU). Our results show that the human eye contains T cells both in health and during active inflammation. Our findings challenge the dogma that the eye is devoid of lymphocytes and supports the concept of resident T cell involvement in the pathogenesis of non-infectious immune-mediated uveitis and as promising targets for therapy. One Sentence SummaryT cells infiltrate aqueous humour during intraocular inflammation and have capacity to migrate into uveal tissue where they remain long-lived.

immunology↗

bioRxiv2023

Tuneable hydrogel stiffness in a 3D in vitro model induces epithelial to mesenchymal transition in MCF7 but not MDA-MB-231 breast cancer cells

The study of in vitro models of breast cancer is crucial for understanding and treating the malignancy in patients, with 3D in vitro models providing researchers with more biomimetic systems to overcome limitations of current to 2D cultures and in vivo animal models. Ex vivo patient tissues have shown that malignant breast tissues are stiffer than healthy or benign tissues, and that the stiffness corresponds with increasing tumour grade. Stiffening of the breast tumour environment alters tumour cell phenotype and facilitates tumour progression, invasion and metastasis. Better understanding of the relationship between extracellular matrix stiffness and breast cancer cell phenotype, and how that is important in the initiation of metastasis, should lead to designing 3D models that mimic the breast tumour microenvironment at different stages of breast cancer progression. This study investigated phenotypic response of two breast cancer cell lines that are representative of clinical breast cancer subtypes (MCF7, Luminal A; MDA-MB-231, Triple Negative Breast Cancer) in gelatin-methacryloyl (GelMA) hydrogels of varying stiffness. A visible light photoinitiation system was adopted to provide a tuneable photocrosslinking platform to systematically control hydrogel stiffness and tumour microenvironment. This allowed rapid fabrication of biocompatible hydrogels supporting high cell viability over long-term culture. The impact of a clinically relevant range of microenvironmental stiffness on breast cancer cell behaviour and phenotype was examined over a 21-day culture period using GelMA hydrogels. Results showed that MCF7 cells cultured for 21 days in high stiffness hydrogels (10 wt%; 28 kPa) responded by downregulating the epithelial marker E-cadherin and upregulating mesenchymal markers N-cadherin and Vimentin, whereas MDA-MB-231 cells showed no changes in EMT-markers when cultured in hydrogels of corresponding stiffness (10 wt%; 33 kPa). Culturing both cell lines in soft hydrogels (5 wt%; 11 kPa) maintained their phenotype over 21 days, highlighting the importance of controlling hydrogel mechanical properties when studying breast cancer cell phenotype.

cancer biology↗

bioRxiv2023

Hypothalamic neuronal activation in primates drives naturalistic goal-directed eating behavior

Eating addiction is the primary cause of modern obesity. Although the causal role of the lateral hypothalamic area (LHA) for eating is demonstrated in rodents, there is no evidence in primates regarding naturalistic eating behaviors. We investigated the role of LHA GABAergic (LHAGABA) neurons in eating by chemogenetics in three macaques. LHAGABA neuron activation significantly increased naturalistic goal-directed behaviors and food motivation, which was specific for palatable food. PET and MRS validated the chemogenetic activation. Rs-fMRI result revealed that functional connectivity (FC) between the LHA and frontal areas was increased, while the FC between the frontal cortices was decreased after the LHAGABA neuron activation. Thus, our study elucidates the role of LHAGABA neurons in eating and obesity therapeutics for primates and humans.

neuroscience↗

bioRxiv2022

Diversity-oriented synthesis encoded by deoxyoligonucleotides

Diversity-oriented synthesis (DOS)is a powerful strategy to prepare molecules with underrepresented features in commercial screening collections, resulting in the elucidation of novel biological mechanisms. In parallel to the development of DOS, DNA-encoded libraries (DELs) have emerged as an effective, efficient screening strategy to identify protein binders. Despite recent advancements in this field, most DEL syntheses are limited by the presence of sensitive DNA-based constructs. Here, we describe the design, synthesis, and validation experiments performed for a 3.7 million-member DEL, generated using diverse skeleton architectures with varying exit vectors, derived from DOS, to achieve structural diversity beyond what is possible by varying appendages alone. We will make this DEL available to the academic scientific community to increase access to novel structural features and accelerate early-phase drug discovery.

biochemistry↗

bioRxiv2020

Impaired hand dexterity function in a non-human primate model with chronic Parkinson's disease

Symptoms of Parkinsons disease (PD) caused by loss of dopaminergic neurons are accompanied by movement disorders, including tremors, rigidity, bradykinesia, and akinesia. Non-human primate (NHP) models with PD play an essential role in the analysis of PD pathophysiology and behavior symptoms. As impairments of hand dexterity function can affect activities of daily living in patients with PD, research on hand dexterity function in NHP models with chronic PD is essential. Traditional rating scales previously used in the evaluation of animal spontaneous behavior were insufficient due to factors related to subjectivity and passivity. Thus, experimentally designed applications for an appropriate apparatus are necessary. In this study, we aimed to longitudinally assess hand dexterity function using hand dexterity task (HDT) in NHP-PD models. To validate this assessment, we analyzed an alteration in Parkinsonian tremor symptoms, loss of dopaminergic neuron, and positron emission tomography (PET) imaging of dopamine transporters (DAT) in these models. HDT latency significantly increased in NHP-PD models. In addition, a significant inverse correlation between HDT and DAT was identified, but no local bias was found. The correlation with intention tremor symptoms was lower than the resting tremor. In conclusion, the evaluation of HDT may reflect behavioral symptoms of NHP-PD models. Furthermore, HDT was effectively used to experimentally distinguish intention tremors from other tremors.

neuroscience↗

bioRxiv2020

Gait analysis of healthy rhesus monkeys using a pressure-sensing walkway system

Gait analysis in non-human primate models has been performed to elucidate the neural systems involved in controlling quadrupedal locomotor behavior. This study aimed to use a pressure-sensing walkway to identify characteristics of species-specific quadrupedal locomotion in rhesus monkeys. A total of nine healthy adult female rhesus monkeys (Macaca mulatta) were used for gait analysis. We measured the temporo-spatial and kinetic parameters of quadrupedal gait using a custom-built pressure-sensing walkway and compared the left- and right-side parameters to assess the symmetry of the gait pattern. All temporo-spatial and kinetic parameter values showed no significant differences among the nine rhesus monkeys for both the forelimbs and hindlimbs. However, significant differences were observed between forelimb and hindlimb kinetic parameters such as peak vertical force, vertical impulse, and the percentage of body weight distribution. All kinetic parameter values were higher for the forelimbs than for the hindlimbs. These data indicated that the center of gravity in healthy rhesus monkeys is located at the forelimbs rather than at the hindlimbs while walking. Furthermore, the symmetry indices considered for symmetric gait pattern showed a low variability. Most median symmetry index values were nearly zero, indicating no difference between the right and left sides. This study described valid methods for assessing gait parameters and demonstrated rhesus-specific characteristics of quadrupedal locomotion, providing a basis for the assessment of gait normality in rhesus monkeys.

animal behavior and cognition↗