Biology subjects

Si, M.

Publications and source records attributed to Si, M..

6 recordsLinked to original sources

bioRxiv2026

Deep-ultraviolet microscopy reveals biomolecular spatiotemporal intracellular dynamics

Intracellular dynamics span a broad range of time scales and biomolecular processes, offering insights into cell health, functional state, phenotype, and response to external perturbations. Several label-free optical imaging approaches have been used to capture intracellular dynamics but are limited by spatiotemporal resolution and biomolecular specificity required to distinguish unique subcellular and metabolic processes. In this work, we demonstrate deep-ultraviolet (UV) microscopy as a powerful, label-free, high-resolution approach for quantifying multiscale intracellular dynamics with biomolecular specificity. By leveraging power spectral analysis and phasor analysis, we capture multiscale intracellular dynamics and analyze their UV wavelength-dependent behavior predicated by the absorption of different endogenous biomolecules. We apply this technique to prostate epithelial cell lines of increasing malignancy and reveal quantitative differences in dynamic intracellular activity that correlate with increased metabolic and organelle activity between phenotypes. Furthermore, we elucidate the molecular identities of structures and activity measured via UV dynamics with broad-band coherent anti-Stokes Raman scattering spectroscopy and fluorescence microscopy. We identify lipid-specific structures, and mitochondrial-specific dynamics, among other biomolecular-specific dynamic behaviors. Together, this study demonstrates deep-UV microscopy as a powerful imaging platform for probing spatial and temporally variant intracellular dynamics with biomolecular specificity, with broad implications for cell phenotyping, tissue pathology, and studying new dynamic subcellular processes.

cell biology↗

bioRxiv2026

Label- and slide-free multispectral quantitative epi-illumination deep-UV microscopy

Label-free and slide-free imaging is highly desired in clinical pathology because it holds the potential to avoid time- and labor-intensive tissue processing and chemical staining while preserving molecular information for downstream analyses. Deep-ultraviolet (UV) microscopy offers high-resolution, label-free molecular contrast via short wavelengths and intrinsic biomolecular absorption, but prior implementations have been limited to the analysis of thin sections and cell monolayers. Here, we present a fast, low-cost, LED-based, epi-illumination deep-UV microscope (epi-DUV) for label- and slide-free imaging of fresh, thick tissues. Using 255 nm and 280 nm absorption images, and tryptophan autofluorescence, the method yields quantitative maps of nucleic acid mass, protein mass, and quantum yield. Moreover, H&E-like contrast can be generated using native 255-nm absorption images. The system achieves 0.5 {micro}m lateral resolution with an effective slice thickness of [~]6 {micro}m across a 707 {micro}m x 707 {micro}m field of view and uses [~]330-ms exposure. To the best of our knowledge, this is the first demonstration of quantitative deep-UV molecular imaging of fresh, unlabeled thick tissues. Epi-DUV has significant potential to streamline the histopathology workflow while adding objective molecular readouts, enabling point-of-care assessment of unprocessed specimens (e.g., rapid intraoperative evaluation).

bioengineering↗

bioRxiv2025

Cardiac-specific Kv1.1 deficiency alters cardiomyocyte electrophysiology without modifying overall cardiac function or arrhythmia susceptibility

The leading cause of epilepsy-related mortality is sudden unexpected death in epilepsy (SUDEP), resulting from seizure-induced cardiorespiratory arrest by mechanisms that remain unresolved. Mutations in ion channel genes expressed in both brain and heart represent SUDEP risk factors because they can disrupt neural and cardiac rhythms, providing a unified explanation for seizures and lethal arrhythmias. However, the relative contributions of brain-driven mechanisms, heart-intrinsic processes, and seizures to cardiac dysfunction in epilepsy remain unclear. Here, we investigated the heart-specific role of the Kcna1 gene, which encodes Kv1.1 voltage-gated potassium channel -subunits expressed in both neurons and cardiomyocytes, where they shape action potential firing and influence seizure and arrhythmia susceptibility. We generated cardiac-specific Kcna1 conditional knockout (cKO) mice lacking Kv1.1 selectively in cardiomyocytes and assessed their cardiac function using in vitro and in vivo electrophysiology. Cardiac Kv1.1 deficiency prolonged action potentials in atrial, but not ventricular, cardiomyocytes, demonstrating a direct role for Kv1.1 in atrial repolarization. Despite these cellular effects, cKOs exhibited normal lifespans, electrocardiographic features, heart rate variability, pacing-induced arrhythmia susceptibility, contractility, seizure susceptibility, and seizure-induced mortality. Thus, while loss of cardiac Kv1.1 was sufficient to impair atrial repolarization, it did not reproduce the broader cardiac abnormalities seen in global Kcna1 knockouts. Given the higher mortality rates of global compared with neural-specific knockouts in our previous studies, cardiac Kv1.1 deficiency, while not lethal alone, may increase vulnerability to seizure-related death when combined with neural deficiency, consistent with a brain-heart dyssynergy that lowers the threshold for fatal events.

physiology↗

bioRxiv2025

Influenza virus recruits PKCa-MEK1-ERK2 complex to regulate nuclear export of viral ribonucleoproteins and promote virus replication.

Host kinases had long been implicated in regulating various steps of influenza virus replication cycle. Previous studies showed that the protein kinase C (PKC) activated mitogen activated protein kinase (MEK)/ extracellular signal-regulated kinase (ERK) cascade promotes nuclear export of viral ribonucleoprotein complexes (RNPs), by triggering phosphorylation of viral nucleoprotein (NP), the major constituent of RNPs. But, the molecular mechanism by which PKC coordinates with specific members of the MAPK pathway to regulate NP phosphorylation remained obscure. Additionally, the molecular interactions of these kinases with NP and the spatiotemporal dynamics of such interactions have never been investigated. Here we unravel the existence of a tripartite PKC-MEK1-ERK2 complex that associates with influenza virus NP and mediates its phosphorylation during the course of infection. Using an analogue sensitive kinase, we show that ERK2 can directly phosphorylate NP at specific serine-threonine residues, which promote vRNP nuclear export and are indispensable for virus propagation. PKC not only activates the MAP kinase, but acts as a scaffold for mediating stable interactions between MEK1, ERK2 and NP, thereby forming an NP associated multi-kinase complex that facilitate its phosphorylation. This multiprotein complex localizes in the nucleus early during infection but into cytoplasm at later stages and overexpression of a dominant negative PKC blocks this complex formation, NP phosphorylation, vRNP export and progeny virus production. These data not only unravel a complex network of virus-host interactions supporting different influenza A and B virus replication, but also shade light upon an unexplored avenue of PKC mediated regulation MAPK pathway. SignificanceThe substrate specificity of kinases is the key for precise regulation of various signaling cascades and can be governed by direct kinase-substrate interaction or indirect interaction mediated through scaffolding or anchoring proteins. This study shows how influenza virus exploit a well-known cellular kinase, PKC, as a scaffold protein to activate and recruit other cellular kinases, MEK1 and ERK2, upon viral replication machinery, RNPs. This RNP associated multi-kinase complex facilitates ERK2 mediated phosphorylation of viral NP protein, which regulates RNP nuclear export and ultimately the production of new virion particles. This work elucidates an unconventional mechanism of kinase-substrate interaction which is critical for influenza virus replication and hence paves the way towards the development of novel host directed anti-influenza therapy.

molecular biology↗

bioRxiv2023

ROCK1 activates mitochondrial fission leading to oxidative stress and muscle atrophy

Chronic kidney disease (CKD) is often associated with protein-energy wasting (PEW), which is characterized by a reduction in muscle mass and strength. Although mitochondrial dysfunction and oxidative stress have been implicated to play a role in the pathogenesis of muscle wasting, the underlying mechanisms remain unclear. In this study, we used transcriptomics, metabolomics analyses and mouse gene manipulating approaches to investigate the effects of mitochondrial plasticity and oxidative stress on muscle wasting in mouse CKD models. Our results showed that the expression of oxidative stress response genes was increased, and that of oxidative phosphorylation genes was decreased in the muscles of mice with CKD. This was accompanied by reduced oxygen consumption rates, decreased levels of mitochondrial electron transport chain proteins, and increased cellular oxidative damage. Excessive mitochondrial fission was also observed, and we found that the activation of ROCK1 was responsible for this process. Inducible expression of muscle-specific constitutively active ROCK1 (mROCK1ca) exacerbated mitochondrial fragmentation and muscle wasting in CKD mice. Conversely, ROCK1 depletion (ROCK1-/-) alleviated these phenomena. Mechanistically, ROCK1 activation promoted the recruitment of Drp1 to mitochondria, thereby facilitating fragmentation. Notably, the pharmacological inhibition of ROCK1 mitigated muscle wasting by suppressing mitochondrial fission and oxidative stress. Our findings demonstrate that ROCK1 participates in CKD-induced muscle wasting by promoting mitochondrial fission and oxidative stress, and pharmacological suppression of ROCK1 could be a therapeutic strategy for combating muscle wasting in CKD conditions. Translational StatementProtein-energy wasting (PEW) is a prevalent issue among patients with chronic kidney disease (CKD) and is characterized by the loss of muscle mass. Our research uncovers a critical role that ROCK1 activation plays in muscle wasting induced by CKD. We found that ROCK1 is instrumental in causing mitochondrial fission, which leads to increased oxidative stress in muscle cells. By employing a pharmacological inhibitor, hydroxyfasudil, we were able to effectively curb ROCK1 activity, which in turn mitigated muscle wasting by reducing both mitochondrial fission and oxidative stress. These findings suggest that pharmacological inhibition of ROCK1 presents a promising therapeutic strategy for combating the muscle wasting associated with CKD.

cell biology↗

bioRxiv2021

A transferrable and integrative type I-F Cascade for heterologous genome editing and transcription modulation

The Class 1 type I CRISPR-Cas systems represent the most abundant and diverse CRISPR systems in nature. However, their applications for generic genome editing have been hindered by difficulties of introducing the class-specific, multi-component effectors in heterologous hosts for functioning. Here we established a transferrable Cascade system that enables stable integration and expression of a complete and highly active I-F Cascade in the notoriously recalcitrant and diverse P. aeruginosa genomes by conjugation. The transferred Cascade displayed substantially higher DNA interference activity and greater editing capacity than the Cas9 system in diverse genetic backgrounds, including removal of the large (21-kb) integrated cassette with efficiency and simplicity. An advanced {lambda}red-I-F system enabled editing in genotypes with poor homologous recombination capacity, clinical isolates lacking sequence information, and cells containing anti-CRISPR elements Acrs. Lastly, an all-in-one I-F Cascade-mediated CRISPRi platform was developed for transcription modulation by simultaneous introduction of the Cascade and the mini-CRISPR array expressing desired crRNA in one-step. This study provides a framework for expanding the diverse type I Cascades for widespread, heterologous genome editing and establishment of editing techniques in non-model isolates of pathogens.

microbiology↗