The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes

Abstract

Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

Dynamin-2 Mutations Linked to Neonatal-onset Centronuclear Myopathy impair exocytosis and endocytosis in adrenal chromaffin cells

“…Other dynamin-2 mutations that cause CNM, such as the mutants R369W and R465W, or mutations linked to Charcot-Marie-Tooth neuropathy like the K562E, also lead to aberrant actin dynamics (Yamada et al, 2016b; González-Jamett et al, 2017; Hamasaki et al 2022; Arriagada-Diaz et al, 2023). Interestingly, the R465W mutation further perturbs the synaptic plasticity and cognitive function in a mouse model of CNM (Arriagada-Diaz et al, 2023), supporting the pleiotropic role of these mutations.…”

Dynamin-dependent entry of Chlamydia trachomatis is sequentially regulated by the effectors TarP and TmeA

“…Determining the exact mechanism of compound-mediated rescue following TmeA deletion will require elucidating which oligomeric species of Dyn2 is induced by either Ryngo or TmeA signaling. Mutations that prevent dynamin self-assembly (i.e., Dyn1 I670K 51 ) or membrane association (i.e., Dyn2 K562E 52 ) could be informative toward this end, as these mutants are scission-incompetent and are not rescued by Ryngo 41 , 53 . Our working model predicts that these mutants are likewise unresponsive to TmeA signaling.…”

Annexin A1 may contribute to the morphological changes in podocytes by mediating endocytic vesicle fusion and transport via promotion of SNARE assembly in idiopathic membranous nephropathy

“…Vesicle formation plays an important role in maintaining the structure of foot processes in glomerular podocytes. 22 However, increased vesicle transcytosis is associated with foot process effacement in podocytes in nephrotic syndrome. 15 In a rat model of nephrotic syndrome induced by puromycin aminonucleoside, accumulation of blue-labelled albumin endocytic vesicles is observed in podocytes.…”

“…Vesicle formation plays an important role in maintaining the structure of foot processes in glomerular podocytes 22 . However, increased vesicle transcytosis is associated with foot process effacement in podocytes in nephrotic syndrome 15 .…”