摘要The transmembrane protein CD47,an innate immune checkpoint protein,plays a pivotal role in preventing healthy erythrocytes from immune clearance.Our study utilized stochastic optical reconstruction microscopy(STORM)and single-molecule analysis to investigate the distribution of CD47 on the human erythrocyte membrane.Contrary to previous findings in mouse erythrocytes,we discovered that CD47 exists in randomly distributed monomers rather than in clusters across the human erythrocyte membrane.Using second antibody-induced crosslinking,we found that CD47 aggregates into stable clusters within minutes.By comparing these STORM results with those of the fully mobile protein CD59 and the cytoskeleton-bound membrane protein glycophorin C under similar conditions,as well as devising two-color STORM co-labeling and co-clustering experiments,we further quantitatively re-vealed an intermediate,self-limiting clustering behavior of CD47,elucidating its fractional(～14%)attachment to the cytoskeleton.Moreover,we report reductions in both the amount of CD47 and its clustering capability in aged erythrocytes,providing new insight into erythrocyte senescence.Together,the combination of STORM and second antibody-based crosslinking unveils the unique self-limiting clustering behavior of CD47 due to its fractional cytoskeleton attachment.