Biology subjects

Nasufovic, V.

Publications and source records attributed to Nasufovic, V..

5 recordsLinked to original sources

bioRxiv2026

Fast and Luminous: CLIP-tag2

CLIP-tag is a self-labeling protein tag used for the specific fluorescence labeling of proteins. However, its low labeling speed and the poor cell permeability of its substrates result in low labeling efficiencies in live-cell applications. Here, we introduce a substrate optimized for live-cell applications, as well as an engineered CLIP-tag variant, CLIP-tag2, which reacts with the new substrate almost 1000-fold faster than the original CLIP-tag-substrate pair. CLIP-tag2 fusion proteins can be specifically and efficiently fluorescently labeled in cells within minutes at nanomolar substrate concentrations, and can be multiplexed with other self-labeling tags such as SNAP-tag2 and HaloTag7. These advances establish CLIP-tag2 as a powerful tagging platform for high-performance live-cell bioimaging.

biochemistry↗

bioRxiv2025

Spindle-localized F-actin regulates polar MTOC organization and the fidelity of meiotic spindle formation

Mammalian oocytes are notoriously prone to chromosome segregation errors leading to aneuploidy. The spindle provides the machinery for accurate chromosome segregation during cell division. Mammalian oocytes lack centrioles and, therefore, the meiotic spindle relies on the organization of numerous acentriolar microtubule organizing centers into two poles (polar MTOCs, pMTOCs). The traditional view is that, in mammalian oocytes, microtubules are the sole cytoskeletal component responsible for regulating pMTOC organization and spindle assembly. We identified a novel F-actin pool that surrounds pMTOCs, forming F-actin cage-like structure. We demonstrated that F-actin localization on the spindle depends on unconventional myosins X and VIIb. Selective disruption of spindle-localized F-actin, using myosin X/VIIb knockdown oocytes or photoswitchable Optojasp-1, perturbed pMTOC organization, leading to unfocused spindle poles and chromosome missegregation. Here, we unveil an important function of F-actin in regulating pMTOC organization, a critical process for ensuring the fidelity of meiotic spindle formation and proper chromosome segregation.

cell biology↗

bioRxiv2025

SiR-XActin: A fluorescent probe for imaging actin dynamics in live cells

Imaging actin-dependent processes in live cells is important for understanding numerous biological processes. However, currently used natural-product based fluorescent probes for actin filaments affect the dynamics of actin polymerization and can induce undesired cellular phenotypes. Here, we introduce SiR-XActin, a simplified jasplakinolide-based, far-red fluorescent probe that enables bright and photostable staining in various cell types without requiring genetic modifications. Due to its relatively weak binding affinity, the probe exhibits minimal cytotoxicity and labels actin filaments without significantly altering actin dynamics. Furthermore, SiR-XActin is suitable for time-resolved, live-cell super-resolution STED microscopy. Exchanging the SiR fluorophore in SiR-XActin for other fluorophores yields probes in different colors. All these properties make SiR-XActin and its analogs powerful tools for studying actin dynamics using live-cell fluorescence microscopy.

biochemistry↗

bioRxiv2024

SNAP-tag2: faster and brighter protein labeling

SNAP-tag is a powerful tool for labeling proteins with synthetic fluorophores in bioimaging. However, its utility in live-cell applications can be constrained by its relatively slow labeling kinetics and the limited cell permeability of its substrates. Here we introduce new labeling substrates and an engineered SNAP-tag for faster labeling in vitro and in live cells. SNAP-tag2 presents a second-order rate constant with rhodamine substrates that approaches 107 s-1 M-1, a 100-fold improvement over the corresponding SNAP-tag-substrate pairs. When labeled with highly fluorogenic dyes, SNAP-tag2 also shows a 5-fold increase in fluorescence brightness relative to currently used SNAP-tag. The increased labeling kinetics and brightness of SNAP-tag2 translates into a greatly improved performance in various live-cell (super-resolution) imaging applications.

biochemistry↗

bioRxiv2022

Next Generation Opto-Jasplakinolides Enable Local Remodeling of Actin Networks

The natural product jasplakinolide is a widely used tool compound to stabilize F-actin and influence actin dynamics. We have previously introduced photoswitchable jasplakinolides (optojasps) that are activated with violet light and deactivated with blue light. Based on insights from cryo-electron microscopy and structure-activity relationship (SAR) studies, we now developed a new generation of functionally superior optojasps that are better suited for biological investigations. These compounds are procured through chemical total synthesis and feature rationally designed red-shifted azobenzene photoswitches. Our new optojasps can be activated with longer wavelengths in the visible range (e.g. 440-477 nm) and rapidly return to their inactive state through thermal relaxation. This has enabled the reversible control of F-actin dynamics, as shown through live-cell imaging and cell migration, as well as cell proliferation assays. Brief sub-cellular activation with blue-green light resulted in highly localized F-actin clusters that gradually dissolved in the dark. Our light-responsive tools can be useful in diverse fields to study actin dynamics with outstanding spatiotemporal precision. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/480923v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@1883e3corg.highwire.dtl.DTLVardef@1ae0c4borg.highwire.dtl.DTLVardef@1a92f8forg.highwire.dtl.DTLVardef@718d87_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗