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 knockout 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 the clathrin-independent endocytosis of epidermal growth factor receptor (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.

Figure 1. Generation of stable cell lines expressing disease-related Dyn2 mutants

(A) Dyn2 domain architecture and mutants chosen for cell line generation. (B, C) Structures of dynamin-like MxA stalk region (PDB 3LJB) and Dyn2 PH domain (PDB 2YS1), purple coloring indicates location of the disease-related mutant residues we have studied. (D, E) Western blot with α-Dyn2 showing expression levels of both endogenous and exogenous Dyn2-GFP in Dyn2+/− (D) or Dyn2KO (E). Dyn2-GFP expression levels as a percentage of endogenous Dyn2 are shown on the bottom of (D).

Figure 2. Subcellular localization of disease-related Dyn2 mutants

(A) Colocalization of GFP-tagged WT and Dyn2 mutants in Dyn2KO cells with clathrin coated pits on the plasma membrane detected with anti-AP2 and Alexa 594-conjugated secondary antibodies. Cells were fixed, permeabilized and stained as described in Methods. (B) Golgi targeting efficiency of disease-related Dyn2 mutants. Dyn2KO cells expressing GFP-tagged WT and Dyn2 mutants were simultaneously fixed and permeabilized in 0.5% TX-100 to extract background cytosolic Dyn2 as described in Methods and then labeled with anti-AP-1 to detect TGN-localized coated pits and Alexa-594 conjugated secondary antibodies. Bar indicates 10 μm.

Figure 3. Effects of exogenous Dyn2-GFP on endogenous Dyn2 localization

Dyn2+/− cells expressing GFP-tagged WT and mutant Dyn2 were simultaneously fixed and permeabilized in 0.5% TX-100 and then labeled with anti-Dyn2 antibody to detect total Dyn2 within each cell line. Bar indicates 10 μm.

Figure 4. Disease-related Dyn2 mutants are defective in p75/neurotrophin receptor transport

(A) Diagram of the biotinylated p75-GFP construction. Signal sequence (SS) corresponds to the N-terminal 28 amino acids of p75. AP is a 15 amino acid insert that is recognized and biotinylated by E. coli-derived BirA. (B) Biotinylated p75-GFP transport validation. To monitor whether biotinylation alters p75 trafficking along the secretory pathway, samples from each step indicated in the time-line were fixed and observed directly by epifluorescence microscopy. After 5h induction by washing out tetracycline and adding biotin, most p75-GFP are in either the ER or Golgi (a). Following incubation for 3h at 20°C in the presence of 100μg/ml cycloheximide (CHX), p75-GFP accumulates in the Golgi (b), and is subsequently transported to the PM after shift to 32°C for 0.5, 1 or 1.5 h (c–e). Scale bar indicates 10 μm. (C) Quantification of biotinylated p75-GFP transport to PM in the Dyn2KO cells expressing Dyn2 mutants. p75 export was assayed as in (B) with 40 min incubation at 32°C and the plasma membrane localized biotinylated p75-GFP was stained with Streptavidin-Alexa568. Fluorescence intensity was measured after cell lysis with 1% TX-100 as described in Methods. The data are normalized to levels obtained in KO cells reconstituted with WT Dyn2. (D) Quantification of biotinylated p75-GFP transport to PM in HeLa cells expressing Dyn2 mutants. HeLa cells co-infected with adenoviruses to express p75-GFP and disease-related Dyn2 mutants were assayed for the p75-GFP export as describe in (C). Data are shown as averages ± Std. Dev. of n=3 experiments. Paired student t test was used to analyze the probabilities of these data. *p<0.05, **p<0.01 compared to the cells expressing exogenous Dyn2WT.

Figure 5. BTfn internalization in Dyn2+/−, Dyn2KO or HeLa cells expressing mutant Dyn2

(A) Dominant negative effect of mutant Dyn2 on CME. BTfn uptake is shown as a fraction of internalized bTfn divided by surface bound bTfn in each condition. Data are normalized to the amount of bTfn internalized at 6 min in control Dyn2+/− cells reconstituted with WT Dyn2-GFP. (B) HeLa cells expressed about 5 fold endogenous level of Dyn2WT or mutants (overnight expression with the presence of 5 ng/ml tetracyclin) were analyzed for the bTfn uptake efficiency as in (A). (C) The ability of mutant Dyn2 to rescue the bTfn uptake defect in Dyn2KO cells, assayed as described above. Data are normalized to the amount of bTfn internalized at 6 min in KO cells reconstituted with WT Dyn2-GFP. **p<0.01 compared to the cells expressing exogenous Dyn2WT with paired student t test. n≥3.

Figure 6. Disease-related Dyn2 mutants are defective in clathrin-independent endocytosis of EGF and cholera toxin

(A) Dominant negative effect of mutant Dyn2 on bEGF internalization. After 4 h serum starvation, mutant Dyn2 expressing Dyn2+/− cells were incubated with 10 ng/ml bEGF for 10 min. Data are normalized and compared to the amount of bEGF internalized at 10 min into Dyn2 +/− cells reconstituted with WT Dyn2-GFP. (B) Dominant negative effect of mutant Dyn2 on CTB internalization. After 10 min incubation with 1μg/ml CTB-594 at 37°C, cells were washed with acid buffer to remove surface bound CTB and fixed for microscopy. Bar indicates 10 μm. (C) CTB uptake quantification. To quantify internalized CTB, surface bound and internalized CTB fluorescence intensity was measured from the cell lysate with fluorescence plate reader, and the fraction of internalized intensity of WT Dyn2-GFP expressing cells is taken as 1. *p<0.05, **p<0.01 compared to Dyn2WT expressing cells with paired student t test. n=3.

Figure 7. EGF-stimulated EGFR degradation is delayed in Dyn2 mutant cells

(A) EGFR degradation in Dyn2+/− cells. The degradation of EGFR was quantified by Western blot analysis of lysates after 90min EGF (10 ng/ml) incubation. After normalization with tubulin, the residual EGFR percentage was shown in (B).