Sakuta, H.; Kanakubo, Y.; Takada, S.; Yanagisawa, N.; Oda, T.; Mayumi, K.; Sadakane, K.; Fujiwara, K.; Yanagisawa, M.

Intracellular molecular organization is often explained by attractive interactions driving clustering and phase separation. Although consideration of repulsive forces is essential in physics, their roles remain unclear in cellular contexts. Here, we demonstrated the fundamental role of repulsion in regulating protein diffusion within cell-size space. By analyzing negatively charged protein diffusion in bulk solutions and in cell-size spaces with membranes, we revealed that membrane-enhanced repulsion inhibited protein diffusion in cell-size spaces. This was due to the amplified electrostatic interactions among proteins because of the large membrane area-to-volume ratio. Notably, ATP, a cellular central energy source, further inhibited protein diffusion in cell-size spaces, whereas protein wave propagation on the membrane counteracted this inhibition. These findings suggest an active regulatory mechanism restoring molecular mobility by dynamically adjusting membrane-enhanced repulsive forces. Our study challenges the traditional emphasis on attractive interactions, highlighting repulsion as a critical tunable factor governing molecular transport and spatial organization in cells.\n\nTeaserProtein diffusion within crowded cell-size spaces is tuned by membrane-enhanced repulsion and its active regulation.