Ajmail, K. | bioRxiv Science

bioRxiv2026

Optogenetic cross-linking of the actin cytoskeleton using DARPins

Precise tools to control actin filament organization and dynamics are essential for studying how the actin cytoskeleton regulates fundamental cell processes, such as morphological changes, migration and intracellular transport. Here, we present an optogenetic system for the reversible, spatiotemporal manipulation of actin cross-linking in living cells. Our approach employs actin-binding Designed Ankyrin Repeat Proteins (DARPins) fused to the improved Light-Induced Dimer (iLID) system, enabling actin cross-linking upon light-triggered dimerization. Following validation of cross-linking of bifunctional DARPins in reconstituted networks, the iLID system was integrated to create light-controlled associations inside cells. Optogenetic DARPin dimerization is rapid, reversible, and locally inducible. Activation of the DARPin-based actin cross-linkers revealed significant inhibition of cell traction forces when triggered throughout the cell. Our findings establish a versatile tool for investigating cytoskeletal dynamics with high spatial and temporal precision, paving the way for controlled manipulation of cellular architecture and mechanics. Graphical Abstract/Table of Contents Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/726659v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@c27701org.highwire.dtl.DTLVardef@112e82borg.highwire.dtl.DTLVardef@4892e4org.highwire.dtl.DTLVardef@a3d5a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2025

Arrhythpy: An Automated Tool to Quantify and Classify Arrhythmias in Ca2+ Transients of iPSC-Cardiomyocytes

Arrhythmias constitute an intricate and clinically significant phenomenon of great importance in various research areas. Ca2+ homeostasis plays a pivotal role in forming rhythmic contractions in the heart, and its dysregulation has emerged as a critical component in developing arrhythmias. Until now, however, the quantification of arrhythmias has been limited to indirect measurements via Ca2+ sparks, electrophysiological parameters, or manual classification, which can lead to human bias. We aimed to develop an analysis platform that directly and automatically analyzes arrhythmias in human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Here, we present Arrhythpy, a robust and automated open-source program to quantify and classify confocal microscopy-based Fluo-4 Ca2+ transients to generate a measure of arrhythmia. In contrast to other automated and semi-automated analysis tools, that measure established parameters such as time-to-peak, Arrhythpy directly analyzes the degree of arrhythmia in a Ca2+ transient. We demonstrate its utility in monitoring Ca2+ transient-based arrhythmias in atrial and ventricular iPSC-CMs of healthy individual and cardiac disease patients, including dilated cardiomyopathy (DCM) and Takotsubo syndrome (TTS). Arrhythpy was validated by analyzing drug-treated iPSC-CMs, confirming the beating frequency effects of compounds that directly activate (Isoprenaline) or inhibit (Metoprolol) beta-adrenergic signaling. Arrhythpy analysis of iPSC-CMs of TTS patients recapitulated TTS phenotypes, including atrial arrhythmia that could be normalized with beta-blocker treatment. The programs adaptable framework enables arrhythmic pattern analysis in various cell types using periodic dye-based line scan measurement techniques, applicable to single cells or layered cultures.

pharmacology and toxicology↗