Correlative SICM‐FCM reveals changes in morphology and kinetics of endocytic pits induced by disease‐associated mutations in dynamin

Abstract

Dynamin 2 (DNM2) is a GTP‐binding protein that controls endocytic vesicle scission and defines a whole class of dynamin‐dependent endocytosis, including clathrin‐mediated endocytosis by caveoli. It has been suggested that mutations in the DNM2 gene, associated with 3 inherited diseases, disrupt endocytosis. However, how exactly mutations affect the nanoscale morphology of endocytic machinery has never been studied. In this paper, we used live correlative scanning ion conductance microscopy (S1CM) and fluorescence confocal microscopy (FCM) to study how disease‐associated mutations affect the morphology and kinetics of clathrin‐coated pits (CCPs) by directly following their dynamics of formation, maturation, and internalization in skin fibroblasts from patients with centronuclear myopathy (CNM) and in Cos‐7 ce1ls expressing corresponding dynamin mutants. Using S1CM‐FCM, which we have deve1oped, we show how p.R465W mutation disrupts pit structure, preventing its maturation and internalization, and significantly increases the lifetime of CCPs. Differently, p.R522H slows down the formation of CCPs without affecting their internalization. We also found that CNM mutations in DNM2 affect the distribution of caveoli and reduce dorsal ruffling in human skin fibroblasts. Collective1y, our S1CM‐FCM findings at single CCP leve1, backed up by electron microscopy data, argue for the impairment of several forms of endocytosis in DNM2‐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. Correlative SICM‐FCM reveals changes in morphology and kinetics of endocytic pits induced by disease‐associated mutations in dynamin. FASEB J. 33, 8504–8518 (2019). http://www.fasebj.org

Dynamin-2 R465W mutation induces long range perturbation in highly ordered oligomeric structures

“…Furthermore, mutations hindering BSE bending impair endocytosis 10 , suggesting that perturbations of these regions, such as we observed with R465W mutation have physiological consequences. Our findings are in agreement with the fact that skin fibroblasts from patients bearing R465W mutation and Cos-7 cells expressing this mutation exhibit disrupted dynamin fission activity 22 , a defect that is restored by allele-specific RNA interference against R465W 33 .…”

“…Therefore, perturbations in these interactions are likely to disrupt endocytosis. Indeed, several dyn-2 mutations causing centronuclear myopathy (CNM) 1 , 18 , 19 , disrupt endocytosis 20 – 22 . However, some of them, including the mutation R465W, can also retain and even increase fission activity in vitro, and promote membrane fragmentation 13 .…”

Non-intrinsic ATP-binding cassette proteins ABCI19, ABCI20 and ABCI21 modulate cytokinin response at the endoplasmic reticulum in Arabidopsis thaliana

“…Fluorescent intensity was measured in the root tip and calculated using Image J (https://imagej.nih.gov/ij/index.html; Schneider et al 2012). The background fluorescence intensity was subtracted from the total intensity, following the method described previously (Ali et al 2019).…”

GTP and lipids control self-assembly and functional promiscuity of Dynamin2 molecular machinery

“…Thus, while the "traditional" mechano-chemical activity of dynamins is tightly coupled to a well-defined operational specialization (e.g., fusion for Atl and fission for the classical dynamins), the functional promiscuity, seen as lipid-assisted membrane tethering and lipid mixing, emerges with a drop in GTP levels. The observed functional promiscuity at low GTP is further modulated by protein partners' expression and lipidome alterations, possibly contributing to abnormal membrane morphologies during pathologies related to the dynamin superfamily members (Ali et al, 2019;Zhao et al, 2018).…”