Dynamin 2 (DNM2) is a GTP-binding protein that controls endocytic vesicle scission and defines a wholeclass of dynamin-dependent endocytosis, including clathrin-mediated endocytosis bycaveoli. It has been suggestedthat mutations in theDNM2gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactlymutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used livecorrelative scanning ion conductance microscopy (SICM) and fluorescence confocal microscopy (FCM) to study howdisease-associated mutations affect the morphology and kinetics of clathrin-coated pits (CCPs) by directly followingtheir dynamics of formation, maturation, and internalizationinskinfibroblastsfrompatients with centronuclearmyopathy (CNM) and in Cos-7 cells expressing corresponding dynamin mutants. Using SICM-FCM, which we havedeveloped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, andsignificantly increases the lifetime of CCPs. Differently,p.R522H slows down the formation of CCPs without affectingtheir internalization. We also found that CNM mutations inDNM2affect the distribution of caveoli and reduce dorsalruffling in human skin fibroblasts. Collectively, our SICM-FCM findings at single CCP level, backed up by electronmicroscopy data,argue for the impairment of several forms of endocytosis inDNM2-linked CNM.—Ali,T.,Bednarska,J.,Vassilopoulos,S.,Tran,M.,Diakonov,I.A.,Ziyadeh-Isleem,A.,Guicheney,P.,Gorelik,J.,Korchev,Y.E.,Reilly,M.M.,Bitoun,M.,Shevchuk,A.CorrelativeSICM-FCMreveals changes in morphology and kinetics of endocytic pitsinduced by disease-associated mutations in dynamin.