Biology subjects

Ashby, G.

Publications and source records attributed to Ashby, G..

4 recordsLinked to original sources

bioRxiv2024

Poly-ubiquitylated transmembrane proteins outcompete other cargo for limited space inside clathrin-coated vesicles

Endocytic recycling of transmembrane proteins is essential to cell signaling, ligand uptake, protein traffic and degradation. The intracellular domains of many transmembrane proteins are ubiquitylated, which promotes their internalization by clathrin-mediated endocytosis. How might this enhanced internalization impact endocytic uptake of transmembrane proteins that lack ubiquitylation? Recent work demonstrates that diverse transmembrane proteins compete for space within highly crowded endocytic structures, suggesting that enhanced internalization of one group of transmembrane proteins may come at the expense of other groups. Here we show that preferential internalization of poly-ubiquitylated transmembrane proteins results in reduced endocytosis of mono-ubiquitylated and non-ubiquitylated proteins. Using a combination of live-cell imaging and ligand uptake assays, we confirmed that increased ubiquitylation correlates with increased internalization by clathrin-coated vesicles. Further, poly-ubiquitylated receptors significantly outcompeted their mono-ubiquitylated and non-ubiquitylated counterparts for localization to endocytic sites and uptake of extracellular ligands. These findings demonstrate the inherent interdependence of transmembrane protein recycling, suggesting that clathrin-coated vesicles act as selective filters, prioritizing highly ubiquitylated transmembrane proteins for uptake while leaving proteins with little or no ubiquitylation behind. Given that poly-ubiquitylation is thought to signal protein aging and damage, our findings suggest a mechanism for selective internalization of high priority cargo proteins, with simultaneously exclusion and protection of functional proteins that lack poly-ubiquitylation.

cell biology↗

bioRxiv2024

A live cell imaging-based assay for tracking particle uptake by clathrin-mediated endocytosis

A popular strategy for therapeutic delivery to cells and tissues is to encapsulate therapeutics inside particles that cells internalize via endocytosis. The efficacy of particle uptake by endocytosis is often studied in bulk using flow cytometry and Western blot analysis and confirmed using confocal microscopy. However, these techniques do not reveal the detailed dynamics of particle internalization and how the inherent heterogeneity of many types of particles may impact their endocytic uptake. Toward addressing these gaps, here we present a live-cell imaging-based method that utilizes total internal reflection fluorescence (TIRF) microscopy to track the uptake of a large ensemble of individual particles in parallel, as they interact with the cellular endocytic machinery. To analyze the resulting data, we employ an open-source tracking algorithm in combination with custom data filters. This analysis reveals the dynamic interactions between particles and endocytic structures, which determine the probability of particle uptake. In particular, our approach can be used to examine how variations in the physical properties of particles (size, targeting, rigidity), as well as heterogeneity within the particle population, impact endocytic uptake. These data impact the design of particles toward more selective and efficient delivery of therapeutics to cells.

biophysics↗

bioRxiv2023

Ubiquitin-driven protein condensation promotes clathrin-mediated endocytosis

Clathrin-mediated endocytosis is an essential cellular pathway that enables signaling and recycling of transmembrane proteins and lipids. During endocytosis, dozens of cytosolic proteins come together at the plasma membrane, assembling into a highly interconnected network that drives endocytic vesicle biogenesis. Recently, multiple groups have reported that early endocytic proteins form flexible condensates, which provide a platform for efficient assembly of endocytic vesicles. Given the importance of this network in the dynamics of endocytosis, how might cells regulate its stability? Many receptors and endocytic proteins are ubiquitylated, while early endocytic proteins such as Eps15 contain ubiquitin-interacting motifs. Therefore, we examined the influence of ubiquitin on the stability of the early endocytic protein network. In vitro, we found that recruitment of small amounts of polyubiquitin dramatically increased the stability of Eps15 condensates, suggesting that ubiquitylation could nucleate endocytic assemblies. In live cell imaging experiments, a version of Eps15 that lacked the ubiquitin-interacting motif failed to rescue defects in endocytic initiation created by Eps15 knockout. Furthermore, fusion of Eps15 to a deubiquitylase enzyme destabilized nascent endocytic sites within minutes. In both in vitro and live cell settings, dynamic exchange of Eps15 proteins, a hallmark of liquid-like systems, was modulated by Eps15-Ub interactions. These results collectively suggest that ubiquitylation drives assembly of the flexible protein network responsible for catalyzing endocytic events. More broadly, this work illustrates a biophysical mechanism by which ubiquitylated transmembrane proteins at the plasma membrane could regulate the efficiency of endocytic recycling. Significance StatementThe assembly of proteins into dynamic, liquid-like condensates is an emerging principle of cellular organization. During clathrin-mediated endocytosis, a liquid-like protein network catalyzes vesicle assembly. How do cells regulate these assemblies? Here we show that ubiquitin and endocytic proteins form a dynamic, mutually-reinforcing protein network in vitro and in live cells. To probe the impact of ubiquitylation on the dynamics of endocytosis, we engineered opto-genetic control over recruitment of proteins to nascent endocytic sites. While recruitment of wildtype proteins promoted endocytosis, recruitment of deubiquitylases, enzymes capable of removing ubiquitin, resulted in disassembly of endocytic sites within minutes. These results illustrate that ubiquitylation can regulate the fate of endocytic structures, elucidating a functional connection between protein condensates, endocytosis, and ubiquitin signaling.

cell biology↗

bioRxiv2023

Selective endocytic uptake of targeted liposomes occurs within a narrow range of liposome diameter

Cell surface receptors facilitate signaling and nutrient uptake. These processes are dynamic, requiring receptors to be actively recycled by endocytosis. Due to their differential expression in disease states, receptors are often the target of drug-carrier particles, which are adorned with ligands that bind specifically to receptors. These targeted particles are taken into the cell by multiple routes of internalization, where the best-characterized pathway is clathrin-mediated endocytosis. Most studies of particle uptake have utilized bulk assays, rather than observing individual endocytic events. As a result, the detailed mechanisms of particle uptake remain obscure. To address this gap, we have employed a live-cell imaging approach to study the uptake of individual liposomes as they interact with clathrin-coated structures. By tracking individual internalization events, we find that the size of liposomes, rather than the density of the ligands on their surfaces, primarily determines their probability of uptake. Interestingly, targeting has the greatest impact on endocytosis of liposomes of intermediate diameters, with the smallest and largest liposomes being internalized or excluded, respectively, regardless of whether they are targeted. These findings, which highlight a previously unexplored limitation of targeted delivery, can be used to design more effective drug carriers.

bioengineering↗