Biology subjects

Kasberg, W.

Publications and source records attributed to Kasberg, W..

4 recordsLinked to original sources

bioRxiv2026

mRNAbow: A versatile gene expression system for multiplexed fluorescent imaging using optimized in vitro transcribed mRNA

Messenger RNA (mRNA) transfection enables rapid, transient protein expression without nuclear entry, providing a powerful alternative to DNA or viral delivery in post-mitotic and otherwise difficult-to-transfect cells. Although in vitro transcribed (IVT) mRNAs have revolutionized therapeutic applications, their adoption in experimental biology remains limited by challenges in synthesis, variability across cell types, and concerns about cytotoxicity. Here, we define design principles that maximize IVT mRNA performance across diverse cellular and organismal systems. Through systematic comparison of capping strategies and base modifications, including N1-methyl-pseudouridine, 5-methylcytidine, and 5-methoxyuridine, we identify modifications that enhance translation while minimizing activation of cellular stress responses. Optimized transcripts drive robust protein expression within four hours, persist for up to one week, and support multiplexed expression of structurally and functionally distinct proteins in mammalian cells, including cancer cell lines, iPSC-derived systems, primary cells, and organoids, as well as in vivo in zebrafish embryos and in less genetically tractable models such as Danionella cerebrum and sea urchin embryos. To further expand accessibility for community use, we developed mRNAbow, a platform for generating low-toxicity mRNAs encoding organelle-targeted fluorescent proteins and biosensors for multiplex imaging, with corresponding plasmids made publicly available. Together, these advances establish a generalizable framework for IVT mRNA design and expand experimental access to synthetic mRNA technologies for dissecting cellular architecture and dynamics.

cell biology↗

bioRxiv2025

ER-to-Golgi Trafficking is a Nutrient-Sensitive Checkpoint Linking Glucose Starvation to Cell Surface Remodeling

Cancer cells adapt to nutrient stress by remodeling the repertoire of proteins on their surface, enabling survival and progression under starvation conditions. However, the molecular mechanisms by which nutrient cues reshape the cell surface proteome to influence cell behavior remain largely unresolved. Here, we show that acute glucose starvation, but not amino acid deprivation or mTOR inhibition, selectively impairs ER-to-Golgi export of specific cargoes, such as E-cadherin, in a SEC24C-dependent manner. Quantitative cell surface proteomics reveal that glucose deprivation remodels the cell surface proteome, notably reducing surface expression of key adhesion molecules. This nutrient-sensitive reprogramming enhances cell migration in vitro and promotes metastasis in vivo. Mechanistically, we show that AMPK and ULK1 signaling orchestrate this process independent of autophagy, with ULK1-mediated phosphorylation of SEC31A driving SEC24C-dependent COPII reorganization. These findings establish ER-to-Golgi trafficking as a nutrient-sensitive regulatory node that links metabolic stress to cell surface remodeling and metastatic potential.

cell biology↗

bioRxiv2025

A privileged ER compartment for post-translational heteromeric assembly of an ion channel

Mechanisms underlying heterotypic subunit assembly of ion channels and other oligomeric complexes are poorly understood. In the human heart, heteromeric assembly of two isoforms encoded by the human ether-a-go-go related gene (hERG) is essential for the normal function of cardiac IKr in ventricular repolarization, with loss of hERG1b contributing to arrhythmias associated with long QT-syndrome (LQTS). While hERG1a homomers traffic efficiently to the plasma membrane, hERG1b homomers are retained in the endoplasmic reticulum (ER). When expressed together, the two subunits avidly associate during biogenesis. Seeking rules specifying heteromeric association, we characterized the fate of hERG1b proteins using confocal and superresolution imaging in fixed and live HeLa cells. We found hERG1b sequestered in punctate intracellular structures when expressed alone in HeLa cells. These puncta, which depend on the presence of an N-terminal "RXR" ER retention signal, represent a privileged ER sub-compartment distinct from that containing ER-retained, type 2 (hERG-based) LQTS mutant proteins, which were rapidly degraded by the proteasome. Introducing hERG1a to cells with preformed hERG1b puncta dissolved these puncta by rescuing extant hERG1b. Rescue occurred by association of fully translated hERG1b with 1a, a surprising finding given previous studies demonstrating cotranslational heteromeric association. We propose that sequestration limits potentially deleterious surface expression of hERG1b homomeric channels while preserving hERG1b for an alternative mode of heteromeric hERG1a/1b channel assembly post-translationally. These findings reveal a surprising versatility of biosynthetic pathways promoting heteromeric assembly. Significance StatementhERG potassium channels are essential for repolarizing the ventricular action potential. Heteromeric hERG channels, which conduct cardiac IKr, are formed by association of hERG1a and 1b subunits. However, mechanisms governing the assembly of hERG and other heteromeric protein complexes are poorly understood. We identify a noncanonical ER compartment that sequesters hERG1b, distinct from previously described ER quality control pathways that degrade misfolded proteins, including LQTS-associated hERG variants. Instead, this compartment preserves hERG1b and facilitates its post-translational assembly with hERG1a, challenging the current view of simultaneous, cotranslational heteromeric assembly. Our findings redefine ER retention as an active regulatory step in protein complex formation that may broadly govern hetero-oligomeric assembly in many systems and facilitate therapeutic approaches to arrhythmia and other diseases.

cell biology↗

bioRxiv2023

Defining Cardiac Nerve Architecture During Development, Disease, and Regeneration

Cardiac nerves regulate neonatal mouse heart regeneration and are susceptible to pathological remodeling following adult injury. Understanding cardiac nerve remodeling can lead to new strategies to promote cardiac repair. Our current understanding of cardiac nerve architecture has been limited to two-dimensional analysis. Here, we use genetic models, whole-mount imaging, and three-dimensional modeling tools to define cardiac nerve architecture and neurovascular association during development, disease, and regeneration. Our results demonstrate that cardiac nerves sequentially associate with coronary veins and arteries during development. Remarkably, our results reveal that parasympathetic nerves densely innervate the ventricles. Furthermore, parasympathetic and sympathetic nerves develop synchronously and are intertwined throughout the ventricles. Importantly, the regenerating myocardium reestablishes physiological innervation, in stark contrast to the non-regenerating heart. Mechanistically, reinnervation during regeneration is dependent on collateral artery formation. Our results reveal how defining cardiac nerve remodeling during homeostasis, disease, and regeneration can identify new therapies for cardiac disease.

developmental biology↗