Nanoscopic spontaneous poration as a precursor to protein-based transport in early protocells

Understanding the mechanisms of material transport in protocells before the emergence of proteins is crucial to uncovering the origins of cellular life. While previous research has demonstrated that direct permeation is a feasible transport mechanism for protocells with fatty acid-based membranes, this process becomes less efficient as membranes evolve to include phospholipids-before the advent of protein transport systems. To address this knowledge gap, we investigated fundamental processes that could have facilitated molecular transport in such protein-free systems. In this study, we identify and characterize nanoscopic transient pores spontaneously forming in phospholipid vesicle membranes, likely driven by osmotic imbalances. We for the first time pinpointed individual pore formation events by observing intermittent fluorescence bursts resulting from the brief influx of fluorescent tracers into the vesicular interior. Kinetic analysis of these burst profiles reveals that these membrane pores possess lifespans of about fourteen milliseconds and radii of around twenty nanometers, suggesting that they are sufficiently large and long-lived to enable the transport of essential nutrients and metabolic products. These findings are confirmed by conventional pore-sizing methods using tracers of various sizes and supported by numerical simulations. Importantly, this transient pore formation does not compromise the integrity of the membrane, nor does it require the participation of proteins or peptides. Our results indicate that spontaneous transient poration provides a viable mechanism for molecular transport through the membrane of primitive cellular entities, offering an alternative to simple diffusion or direct permeation. This study sheds light on potential evolutionary strategies employed by pre-protein protocellular entities to facilitate material transport, contributing to our understanding of the early mechanisms that may have driven the origin of life.