Biology subjects

Lesiak, L.

Publications and source records attributed to Lesiak, L..

4 recordsLinked to original sources

bioRxiv2025

Single-dye, transfection-free FLIM multiplexing via bioorthogonal chemistry

Fluorescence lifetime imaging microscopy (FLIM) can visualize multiple targets in a single spectral window, making it a powerful tool to overcome multiplexing limitations during live cell fluorescence microscopy. Here we show that small molecule probes-which are well-suited for imaging applications due to high specificity, low toxicity, and the elimination of transfection requirements-can be fine-tuned via bioorthogonal chemistry to exhibit predictably different fluorescent lifetimes suitable for FLIM multiplexing.

biochemistry↗

bioRxiv2023

Design rules for efficient endosomal escape

The inefficient translocation of proteins across biological membranes limits their application as therapeutic compounds and research tools. In most cases, translocation involves two steps: uptake into the endocytic pathway and endosomal escape. Certain charged or amphiphilic molecules promote protein uptake but few enable efficient endosomal escape. One exception is ZF5.3, a mini-protein that exploits natural endosomal maturation machinery to translocate across endosomal membranes. Although certain ZF5.3-protein conjugates are delivered efficiently into the cytosol or nucleus, overall delivery efficiency varies widely with no obvious design rules. Here we evaluate the role of protein size and thermal stability in the ability to efficiently escape endosomes when attached to ZF5.3. Using fluorescence correlation spectroscopy, a singlemolecule technique that provides a precise measure of intra-cytosolic protein concentration, we demonstrate that delivery efficiency depends on both size and the ease with which a protein unfolds. Regardless of size and pI, low-Tm cargos of ZF5.3 (including intrinsically disordered domains) bias its endosomal escape route toward a high-efficiency pathway that requires the homotypic fusion and protein sorting (HOPS) complex. Small protein domains are delivered with moderate efficiency through the same HOPS portal even if the Tm is high. These findings imply a novel protein- and/or lipid-dependent pathway out of endosomes that is exploited by ZF5.3 and provide clear guidance for the selection or design of optimally deliverable therapeutic cargo. Significance StatementThe results described in this paper provide new insights into how protein delivery works and how it can be best utilized in the future. Although intracellular protein delivery has been studied for decades, this paper describes the first interrogation of why certain protein cargos are privileged for efficient endosomal escape. These results represent a fundamental advance in the long-awaited goal of efficient protein delivery and provide design rules to overcome one of the most significant challenges for the future of biotechnology.

cell biology↗

bioRxiv2023

A Bright, Photostable Dye that Enables Multicolor, Time Lapse, and Super-Resolution Imaging of Acidic Organelles

Lysosomes have long been known for their acidic lumen and efficient degradation of cellular byproducts. In recent years it has become clear that their function is far more sophisticated, involving multiple cell signaling pathways and interactions with other organelles. Unfortunately, their acidic interior, fast dynamics, and small size makes lysosomes difficult to image with fluorescence microscopy. Here we report a far-red small molecule, HMSiR680-Me, that fluoresces only under acidic conditions, causing selective labeling of acidic organelles in live cells. HMSiR680-Me can be used alongside other far-red dyes in multicolor imaging experiments and is superior to existing lysosome probes in terms of photostability and maintaining cell health and lyso-some motility. We demonstrate that HMSiR680-Me is compatible with overnight time lapse experiments, as well as time lapse super-resolution microscopy with a fast frame rate for at least 1000 frames. HMSiR680-Me can also be used alongside silicon rhodamine dyes in a multiplexed super-resolution microscopy experiment to visualize interactions between the inner mitochondrial membrane and lysosomes with only a single excitation laser and simultaneous depletion. We envision this dye permitting more detailed study of the role of lysosomes in dynamic cellular processes and disease.

cell biology↗

bioRxiv2022

Long-term, super-resolution HIDE imaging of the inner mitochondrial membrane in live cells with a cell-permeant lipid probe

The densely packed inner mitochondrial membrane (IMM) is vital for bioenergy generation and its dynamics control mitochondrial health and cellular homeostasis. IMM structure is complex, however, and imaging its dynamics with high temporal and spatial resolution is complicated by the photosensitivity of IMM-resident enzymes. Here we describe the cell-permeant, lipid-like acridine orange derivative MAO-N3 and use it to assemble high-density, environmentally sensitive (HIDE) probes that selectively label and image the IMM in live cells. MAO-N3 pairs with multiple SPAAC-reactive fluorophores to support HIDE imaging via confocal, Structured Illumination, Single Molecule Localization, and Stimulated Emission Depletion microscopy, all with significantly improved resistance against photobleaching. The HIDE probes generated using MAO-N3 require no genetic manipulations, are non-toxic in model cell lines and primary cardiomyocytes, even under conditions that amplify the effects of mitochondrial toxins, and visualize the IMM for up to 12.5 hours with unprecedented spatial and temporal resolution.

cell biology↗