Common Membrane Trafficking Defects of Disease‐Associated Dynamin 2 Mutations

Abstract

Dynamin (Dyn) is a multidomain and multifunctional GTPase best known for its essential role in clathrin-mediated endocytosis (CME). Dyn2 mutations have been linked to two human diseases, Centronuclear Myopathy (CNM) and Charcot-Marie-Tooth (CMT) disease. Paradoxically, although Dyn2 is ubiquitously expressed and essential for embryonic development, the disease-associated Dyn2 mutants are autosomal dominant, but result in slowly progressing and tissue-specific diseases. Thus, although the cellular defects that cause disease remain unclear, they are expected to be mild. To gain new insight into potential pathogenic mechanisms we utilized mouse Dyn2 conditional knock-out cells combined with retroviral-mediated reconstitution to mimic both heterozygous and homozygous states and characterized cellular phenotypes using quantitative assays for several membrane trafficking events. Surprisingly, none of the four mutants studied exhibited a defect in CME, but all were impaired in their ability to support p75/neurotrophin receptor export from the Golgi, the raft-dependent endocytosis of cholera toxin, and clathrin-independent endocytosis of EGFR. While it will be important to study these mutants in disease-relevant muscle and neuronal cells, given the importance of neurotrophic factors and lipid rafts in muscle physiology, we speculate that these common cellular defects might contribute to the tissue-specific diseases caused by a ubiquitously expressed protein.

Expression of a dynamin 2 mutant associated with Charcot-Marie-Tooth disease leads to aberrant actin dynamics and lamellipodia formation

“…The K562E mutation leads to severe inhibition of endocytic activity [2,10,11], as shown by defects in the recruitment of the mutant protein to the plasma membrane [18]. In the present study, the recruitment of the dynamin 2 K562E mutant to endocytic pit was drastically decreased (Fig.…”

“…Dynamin 2 CMT mutations disturb clathrin-mediated endocytosis of transferrin or EGF receptors [2,10,11] and result in dynamic instability of microtubules [5].…”

“…Mutations have also been detected in the middle and PRD domains [9]. Analysis of fibroblasts derived from CMT patients or cells expressing CMT mutant dynamin 2 showed that mutations in the PH domain lead to defective endocytosis of surface receptors, including EGF and transferrin receptors [10,11]. In particular, the dynamin 2 K562E mutant shows severely inhibited endocytosis [11].…”

Dynamin 2 homozygous mutation in humans with a lethal congenital syndrome

“…Our study along with several other reports emphasizes that DNM2 has a key role in endocytosis, and that disease-causing mutations impact on this function. 9,[21][22][23] We speculate that alterations in endocytosis during development could explain the pleiotropic phenotype observed in patients with this congenital syndrome. (d) The p.Phe379Val protein exhibits B20% lower GTPase activity using a malachite green-based assay (P-value *0.05 for three independent experiments using the Student's t-test).…”

Brain Slice Biotinylation: An <em>Ex Vivo</em> Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

“…Endocytic recycling back to the plasma membrane can additionally enhance cellular signaling by increasing protein expression levels at the cell surface 3 . Moreover, membrane trafficking perturbations are implicated in numerous disease and pathological conditions 4,5 , stressing the need to investigate the molecular mechanisms that govern protein endocytic trafficking. While many proteins utilize classic clathrin-dependent internalization mechanisms, mounting evidence over the past several years demonstrates that multiple clathrin-independent endocytic mechanisms govern the endocytic potential of an increasing array of proteins 6,7 .…”