Vesicle Size Regulates Nanotube Formation in the Cell

Biological membrane nanotubes are tubular structures that are commonly observed within the cell and between cells. The formation and dynamics of nanotubes are increasingly recognized to play important roles in a multitude of biological progresses, such as endosomal antigen delivery in polarized T-cells1, transportation between ER and Golgi2 and between Golgi and the plasma membrane3. We recently also demonstrated the role of nanotube dynamics in autophagic lysosome reformation during autophagy4,5 and mitochondrial network remodeling6. Nanotube formation is usually an active process that requires works. McMahon and Gallop have reviewed the factors that drive biological membrane deformation, including the lipid composition, membrane proteins and scaffolding proteins as well as cytoskeleton and its associated motor proteins7. Up to now, mechanistic understanding of membrane tubulation is mainly based on in vitro reconstitution assays, which comprise two approaches8. In the first approach, components that are thought to be involved in membrane tubulation are either synthesized, purified or even kept in cell extracts and used to reconstitute the tubulation process. This approach has helped to make reduction of required components for nanotube formation9. As an example, such assays have established that kinesin motors and microtubules are sufficient to induce membrane tubes from vesicles in the absence of any other machinery or proteins10. The other approach focuses more on the mechanical response of membrane tubulation and is thus often conducted with model liposomes, namely Giant Unilamellar Vesicles (GUVs)11,12. Usually, direct single vesicle manipulation techniques are used in these assays, including hydrodynamic flow13, micropipettes14 and optical tweezers15,16. As these techniques allow to control the load precisely, the mechanical response of membrane tubulation may be studied in details. Despite all the findings, it is not clear at what extent the size of a vesicle affects its tubulation. This question is of great interest as the size of vesicles and organelles is heterogeneous in the cell. Although a previous in vitro study indicates that the force required to initiate tubulation is scaled with the vesicle size, the conclusion was limited to GUVs which are about 20 μm in diameter15, much larger than most intracellular vesicles and organelles. In contrast, little has been accomplished for understanding tubulation of small vesicles that are more physiologically relevant. In this work, our goal is to determine how vesicle size affects membrane tubulation within living cells using lysosomes and autolysosomes as a set of model systems. Autolysosomes are degradative compartments formed by fusion of an autophagosome and multiple lysosomes during autophagy. We recently discovered that at the late stage of autophagy, tubular structures are extruded and pinched off from autolysosomes to form small proto-lysosomes, which become functional lysosomes after a maturation process. This process, namely autophagic lysosomal reformation (ALR), is crucial for cells to retain their lysosome level when they exit the autophagic stage4. While the membrane composition of autolysosomes is similar with lysosomes, their sizes are quite different. It is known that the range of lysosome diameter is between 50 nm and 500 nm17. For autolysosomes, their size ranges from a few hundred nanometers to several micrometers, larger and more varied than that of lysosomes17. We speculate that cells may use vesicle size to distinguish lysosomes and autolysosomes in promoting tubule formation specifically on autolysosomes. To prove this idea, we first quantify the tubulation percentage of lysosomes and autolysosomes in the cell. The in vivo observations reveal that autolysosomes show much higher probability of tubulation than lysosomes. The size-dependence hypothesis is further enforced by a sucrose-induced enlargement method in the cell. In addition, with the knowledge that tubulation of both autolysosomes and lysosomes is driven by associated kinesin motors, we reconstitute the tubulation process in vitro using purified lysosomes, autolysosomes and artificial liposomes. Precise tuning of the kinesin motor concentration allows to separate the size effect on tubulation from other factors. Lastly, we apply Atomic Force Microscopy (AFM) to quantitatively measure the force barrier during the tubulation process and carry a simple computation based on the measurements. Overall, these results suggest that vesicle size indeed affects the tubulation probability for lysosomes and autolysosomes, whose size ranges between 50 nm and several micrometers. Our assays built for lysosomes and autolysosomes may be easily extended to other vesicle deformation systems. Importantly, the size-dependence effect may be one of the mechanisms for the cell to regulate cellular processes involving membrane-deformation.