Biology subjects

Sitarska, E.

Publications and source records attributed to Sitarska, E..

3 recordsLinked to original sources

bioRxiv2026

Naked antisense oligonucleotides remain endolysosomally sequestered despite induced membrane damage

Antisense oligonucleotides (ASOs) enter cells efficiently, but the compartment from which productive escape occurs remains uncertain. We used live-cell microscopy, ratiometric pH measurements and 3D focused ion beam scanning electron microscopy (FIB-SEM) in U2OS cells to track a Malat1-targeting ASO from uptake to delivery. The ASO entered by endocytosis and accumulated in late endosomes, endolysosomes and lysosomes, where it induced luminal neutralization without galectin-3 recruitment or limiting-membrane rupture. Under conditions that reduced Malat1-RNA by >90%, quantitative imaging showed that less than 4% of internalized ASOs reached the nucleus. L-leucyl-L-leucine methyl ester (LLOMe)-induced membrane damage released co-internalized dextran but not ASOs, showing that ASOs remain sequestered even in damaged late endocytic compartments. In apilimod-expanded organelles, ASOs concentrated at limiting membranes and intraluminal foci with constrained motion, consistent with association with membrane and luminal structures. Although G3BP1/2 has been proposed to plug damaged endocytic membranes, we detected no recruitment of G3BP1 to endosomes or lysosomes; loss of G3BP1 and G3BP2 increased functional delivery modestly. We therefore propose that productive escape occurs earlier in endocytosis, most likely in early or recycling endosomes, where ASOs would still be unbound within the lumen and where membrane fusion and fission could generate perforations permitting release.

cell biology↗

bioRxiv2021

Curvature sensing steers actin-driven cell migration

Cell migration is a hallmark out-of-equilibrium process in biology. In addition to persistent self-propelled motion, many cells display remarkable adaptive behaviors when they navigate complex environments within the body. Combining theory and experiments, we identify a curvature-sensing mechanism underlying obstacle avoidance in immune-like cells. The genetic perturbation of this machinery leads to a reduced capacity to evade obstructions combined with faster and more persistent cell migration in obstacle-free environments. We propose that the active polymerization of the actin cytoskeleton at the advancing edge of migrating cells is locally inhibited by the curvature-sensitive BAR protein Snx33 in regions with inward plasma membrane curvature. This coupling between actin and membrane dynamics leads to a mechanochemical instability that generates complex protrusive patterns at the cellular front. Adaptive motility thus arises from two simultaneous curvature-dependent effects, i) the specific reduction of propulsion in regions where external objects deform the plasma membrane and ii) the intrinsic patterning capacity due to the membrane-actin coupling that promotes spontaneous changes in the cells protrusions. Our results show how cells utilize actin- and plasma membrane biophysics to sense their environment, allowing them to adaptively decide if they should move ahead or turn away. On the basis of our findings, we propose that the natural diversity of BAR proteins may allow cells to tune their curvature sensing machinery to match the shape characteristics in their environment.

cell biology↗

bioRxiv2020

Adaptive actin organization counteracts elevated membrane tension to ensure robust endocytosis

Clathrin-mediated endocytosis (CME) robustness under elevated membrane tension is maintained by actin assembly-mediated force generation. However, whether more actin assembles at endocytic sites in response to increased load, as has been observed in lamellipodia, has not previously been investigated. Here actin network ultrastructure at CME sites was examined under low and high membrane tension. Actin and N-WASP spatial organization indicate that actin polymerization initiates at the base of clathrin-coated pits and that the network then grows away from the plasma membrane. Actin network height at individual CME sites was not coupled to coat shape, raising the possibility that local differences in mechanical load feedback on assembly. By manipulating membrane tension and Arp2/3 complex activity we tested the hypothesis that actin assembly at CME sites increases in response to elevated load. Indeed, in response to elevated membrane tension, actin grew higher, resulting in greater coverage of the clathrin coat, and CME slowed. When membrane tension was elevated and the Arp2/3 complex was inhibited, shallow clathrin-coated pits accumulated, indicating that this adaptive mechanism is especially crucial for coat curvature generation. We propose that actin assembly increases in response to increased load to ensure CME robustness over a range of plasma membrane tensions.

cell biology↗