Dynamin photoinactivation blocks Clathrin and α-adaptin recruitment and induces bulk membrane retrieval

Abstract

Dynamin is a well-known regulator of synaptic endocytosis. Temperature-sensitive dynamin (shi(ts1)) mutations in Drosophila melanogaster or deletion of some of the mammalian Dynamins causes the accumulation of invaginated endocytic pits at synapses, sometimes also on bulk endosomes, indicating impaired membrane scission. However, complete loss of dynamin function has not been studied in neurons in vivo, and whether Dynamin acts in different aspects of synaptic vesicle formation remains enigmatic. We used acute photoinactivation and found that loss of Dynamin function blocked membrane recycling and caused the buildup of huge membrane-connected cisternae, in contrast to the invaginated pits that accumulate in shi(ts1) mutants. Moreover, photoinactivation of Dynamin in shi(ts1) animals converted these pits into bulk cisternae. Bulk membrane retrieval has also been seen upon Clathrin photoinactivation, and superresolution imaging indicated that acute Dynamin photoinactivation blocked Clathrin and α-adaptin relocalization to synaptic membranes upon nerve stimulation. Hence, our data indicate that Dynamin is critically involved in the stabilization of Clathrin- and AP2-dependent endocytic pits.

Figure 1.

Photoinactivation of Dynamin blocks synaptic vesicle recycling. (A) Genomic dynamin construct tagged in the middle domain with a Flag-tetracysteine tag (shi-4C). PH, Pleckstrin homology; PRD, Proline-rich domain; GED, GTPase effector domain. (B–D) FlAsH fluorescence after incubation of yw controls (B) and shi^12-12B; shi-4C (C) third instar fillets in FlAsH reagent shows labeling only in boutons of animals expressing Shi-4C (C). (D) Anti-Dynamin (Dyn) labeling in yw animals. Bars, 20 µm. (E) Sample EJC traces recorded from muscle 6 in 0.5 mM of extracellular CaCl₂ in yw controls and shi^12-12B; shi-4C animals that were not subjected to FALI (−) and shi^12-12B; shi-4C after FALI (+). (F) Quantification of the EJC amplitude recorded in 0.5 and 2 mM CaCl₂ in controls yw and shi^12-12B; shi-4C without (−) and with FALI (+). Error bars show SEMs; ANOVA (post hoc Tukey’s test). n for 0.5 mM CaCl₂ = 7, 7, and 10 and for 2 mM CaCl₂ = 8, 7, and 5 recordings from four to nine larvae. (G) Cumulative probability histogram of miniature EJC amplitudes measured from yw controls and shi^12-12B; shi-4C incubated with FlAsH before illumination, during light inactivation and after FALI. n = 8, 5, 5, and 5 recordings from as many larvae. (H) Relative EJP amplitude measured during 10 min of 10-Hz stimulation in controls yw (n = 8 recordings from eight larvae) and in shi^12-12B; shi-4C loaded with FlAsH (n = 5 recordings from five larvae). Recordings were made by measuring EJPs for 2 min without illuminating the samples followed by 2 min of illumination to photoinactivate Dynamin. EJP amplitudes are plotted as the means of 30 s of recording and normalized to the means of the first 15 s per genotype. (inset) Example EJP data traces of yw and shi^12-12B; shi-4C (in black). Error bars show SEMs.

Figure 2.

Photoinactivation of Dynamin results in the formation of large membrane inclusions. (A–H) FM 1-43 labeling in yw and shi^12-12B; shi-4C animals treated (+) or not treated (−) with FlAsH for 10 min and/or illumination for 2 min (±). All preparations were stimulated with KCl in the presence of FM 1-43 for 5 min. (I) Quantification of the number of FM 1-43–labeled membrane accumulations (accum.) per boutonic area in yw controls (n = 72 boutons from eight larvae) and in shi^12-12B; shi-4C after FALI (n = 72 boutons from 16 larvae). Error bars show SEMs; t test: ***, P < 0.0001. stim., stimulation. (J) Quantification of FM 1-43 labeling intensity (int.) after loading and unloading of yw controls (40 boutons from five larvae) and yw; shi-4C (n = 24 boutons from six larvae) after FALI normalized to the yw control (images shown in M–P). Error bars show SEMs. t test: ***, P < 0.0001. (K and L) FM 1-43 labeling (K) and quantification of the number of FM 1-43–labeled membrane accumulations per boutonic area (L) in yw (n = 32 boutons from five larvae) and shi^12-12B; shi-4C animals (n = 24 boutons from six larvae) after FALI when preparations were stimulated at 10 Hz for 5 min in the presence of FM 1-43. Error bars show SEMs. t test: ***, P < 0.0001. (M–P) Loading and unloading of FM 1-43 in yw and shi^12-12B; shi-4C after FALI. Preparations were loaded with FM 1-43 for 5 min in the presence of KCl (M and O, load) and unloaded using KCl stimulation for 10 min (N and P, unload; quantification in J). Bars, 5 µm.

Figure 3.

Dynamin FlAsH-FALI is specific. (A–C) Labeling of controls (dicer-2/+;; nSybGal4/+; A) and larvae expressing shi RNAi (dicer-2/+; shi RNAi/+; nSybGal4/+; B) third instar larval boutons with anti-Dynamin (Dyn) and anti-HRP and quantification of boutonic anti-Dynamin labeling intensity. Error bars show SEMs; t test: **, P < 0.001 (n = 10 NMJs from five larvae per genotype). (D–F) FM 1-43 dye uptake measured after 5 min of stimulation with KCl in controls (dicer-2/+;; nSybGal4/+; D), larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+; E) and in heterozygous mutant shi larvae that express RNAi to shi (shi^12-12B/dicer-2; shi RNAi/+; nsybGAL4/+; F). (G and H) Quantification of the number of FM 1-43–labeled accumulations (Accum.) per boutonic area (G) and relative FM 1-43 labeling intensity (int.; H) in controls (dicer-2/+;; nSybGal4/+), in larvae expressing RNAi to shi (dicer-2/+; shi RNAi/+; nSybGal4/+), and in heterozygous mutant shi larvae that express RNAi to shi (shi^12-12B/dicer-2; shi RNAi/+; nsybGAL4/+). Error bars show SEMs; ANOVA (post hoc Tukey’s test): **, P < 0.001. In G, n = 24, 36, and 60 boutons from three, seven, and five animals. In H, n = 24, 36, and 20 boutons from three, five, and four animals. (I) Strategy used to generate a genomic Endo-4C construct. The 4C is inserted between the BAR and SH3 domain. ATG is the start codon. (J–L) FM 1-43 labeling in w and w; endo-4C; endo¹ animals treated (+) or not treated (−) with FlAsH for 10 min and/or illumination for 5 min (±). All preparations were stimulated with KCl in the presence of FM 1-43 for 1 min, washed, and imaged. (M) Quantification of FM 1-43 labeling intensity after loading of w and w; endo-4C; endo¹ before and after FALI normalized to the w control. Error bars show SEMs; ANOVA (post hoc Tukey’s test): ***, P < 0.0001. n = 24, 60, and 64 boutons from six, four, and seven animals. Bars, 5 µm.

Figure 4.

Photoinactivation of Dynamin results in massive bulk membrane uptake, whereas shi^ts1 mutant boutons at restrictive temperature show an accumulation of invaginated pits. (A and B) Electron micrographs of yw (A) and shi^12-12B; shi-4C (B) control boutons stimulated for 5 min with KCl without FALI. Arrows, T bar; m, mitochondria. (C–H) Electron micrographs of shi^12-12B; shi-4C boutons in which Dynamin was photoinactivated using FALI and subsequently stimulated for 5 min with KCl. High magnifications of the membrane with an active zone decorated with a T bar (E) and of inner membrane inclusions (F–H). Arrowheads, submembrane; arrow, T bar; m, mitochondria. (I) Cumulative probability distributions of vesicular profile diameter size in yw (n = 1,291 vesicles from three larvae), shi^12-12B; shi-4C controls not treated with FALI (n = 1,291 vesicles from three larvae), and shi^12-12B; shi-4C treated with FALI (n = 2,824 vesicles from three larvae). (J–L) Quantification of different boutonic features: the number of synaptic vesicles with a diameter <80 nm per area (J), the number of synaptic vesicles with a diameter >80 nm per area (K), and the number of invaginated pits per area (L) in yw controls (n = 33 bouton profiles from three larvae), shi^12-12B; shi-4C after FALI (n = 27 bouton profiles from three larvae), and shi^ts1 at a restrictive temperature (33°C; n = 16 profiles from three larvae). Error bars show SEMs; ANOVA (post hoc Tukey’s test): ***, P < 0.0001. (M and N) Electron micrographs of shi^ts1 boutons stimulated for 5 min with KCl at permissive (25°C; M) and restrictive (33°C) temperature (N). Asterisks, invaginated pits; arrow, T bar; m, mitochondria. (O–Q) Higher magnification of the active zones in shi^ts1 boutons stimulated for 5 min with KCl at permissive (O) and restrictive temperature (P) and of invaginated pits in shi^ts1 boutons at restrictive temperature (Q). Note the lack of synaptic vesicles around the active zone in shi^ts1 at restrictive temperature. Bars: (A–H, M, and N) 0.5 µm; (O–Q) 0.1 µm.

Figure 5.

Stimulus-dependent Chc and α-Ada recruitments are blocked upon Dynamin photoinactivation. (A–H) Superresolution imaging of HA-Chc fusion proteins with anti-HA antibodies, using structured illumination microscopy in not stimulated (−KCl) and stimulated (+KCl; 90 mM for 5 min) preparations. (A and B) Labeling of yw; HA-chc controls (yw) and shi^12-12B/Y; HA-chc/shi-4C without FALI at rest (−KCl). Note the presence of Chc in the bouton center and at the bouton periphery as quantified in F (n = 10 boutons from three larvae); see Materials and methods and also Fig. S3. rel., relative. (C–E) Labeling of yw; HA-chc (yw) and shi^12-12B/Y; HA-chc/shi-4C, stimulated with KCl without Dynamin inactivation (blue; C and D) and with Dynamin inactivation using FALI (E). Note that in the stimulated controls (C and D), Chc becomes more concentrated in the bouton periphery than in animals in which Dynamin was inactivated (E) as quantified in G and H (n = 11–12 boutons from four to five larvae); see Materials and methods and also Fig. S3. (I–P) Superresolution imaging of α-Ada using structured illumination microscopy in not stimulated (−KCl) and stimulated (+KCl; 90 mM for 5 min) preparations. (I and J) Labeling of yw controls (yw) and shi^12-12B/Y; shi-4C/+ without FALI at rest (−KCl). Note the presence of α-Ada in the bouton center and at the bouton periphery as quantified in N (n = 9–10 boutons from three to four larvae); see Materials and methods and also Fig. S3. (K–M) Labeling of yw (yw) and shi^12-12B/Y; shi-4C/+, stimulated with KCl without Dynamin inactivation (blue; K and L) and with Dynamin inactivation using FALI (M). Note that in the stimulated controls (K and L), α-Ada becomes more concentrated in the bouton periphery than in animals in which Dynamin was inactivated (M) as quantified in O and P (n = 9–10 boutons from four to five larvae); see Materials and methods and also Fig. S3. SEM is shown in the lighter shade. Arrows, plasma membrane. Bars, 2 µm.

Figure 6.

Photoinactivation of Dynamin converts invaginated pits in shi^ts1 mutants into bulk cisternae. (A and B) Quantification of the HA-Chc (A, n = 11 boutons from seven larvae each) and α-Ada (B, n = 7–10 boutons from six to seven larvae) labeling intensity in a line over the (largest) bouton diameter (and normalized to the length of the bouton diameter; see Materials and methods) in KCl-stimulated shi^ts1/Y; HA-chc/+ at permissive (RT) temperature and shi^ts1/Y; HA-chc/+ at restrictive temperature (33°C); see Materials and methods. Averages are in black lines, and SEMs are in gray shades. rel., relative. (C) Model of the distribution of different Dynamin molecules (Shi-4C and Shi^ts1) in endocytic pits (based on anti-Dynamin labeling intensity—see Stimulus-dependent Clathrin and α-Ada recruitment in shi^ts1 mutants in the Results section) and calculation of the probability that Dynamin rings consist only of Shi^ts1 or only of Shi-4C in shi^ts1/Y; shi-4C/+ larvae. (D–I) FM 1-43 labeling in shi^ts1/Y; shi-4C/+ larvae treated (+; F–I) or not treated (−; D and E) with FlAsH for 10 min and illuminated for 2 min (+; G and I) at permissive (D, F, and G) or restrictive (E, H, and I) temperature. All preparations were stimulated for 5 min with KCl in the presence of FM 1-43. Note that shi^ts1/Y; shi-4C/+ animals at restrictive temperature without FALI phenocopy shi^ts1 animals and do not internalize FM 1-43, whereas shi^ts1/Y; shi-4C/+ animals at restrictive temperature after FALI also internalize FM 1-43 in cisternal inclusions, similar to shi^12-12B; shi-4C animals in which Dynamin was photoinactivated. Bar, 5 µm.

Figure 7.

Invaginated pits in shi^ts1 mutants are converted into bulk cisternae upon Dynamin photoinactivation at the ultrastructural level. (A–D) Electron micrographs of shi^ts1/Y; shi-4C larvae treated with FlAsH for 10 min (+; B–D) or not treated (−; A) and illuminated for 2 min (+; C and D) or not illuminated (−; A and B) at permissive (RT; A) or restrictive (33°C) temperature (B–D) stimulated for 5 min with KCl. Bars: (A–C) 0.5 µm; (D) 0.25 µm. Arrowheads, submembrane inclusions; arrows, T bar; m, mitochondria; asterisks, invaginated pits. (E and F) Model of a bouton after surface rendering of a tomogram of shi^ts1/Y; shi-4C larvae after FALI (+/+) at restrictive temperature 33°C stimulated for 5 min with KCl (see also Video 1). (E) Note that some of the membrane inclusions are so massive that they are intertwined and folded inside each other. (F) Individual tomography models of different membrane inclusions. Gray, plasma membrane; blue, red, yellow, purple, and green, membrane inclusions. (G–I) Quantification of the number of synaptic vesicles <80 nm per area (G), the number of synaptic cisternae >80 nm per area (H), and the number of invaginated pits per area (I) in shi^ts1/Y; shi-4C larvae at 22°C (RT) not treated with FALI (−/−), at 33°C not treated with FALI (−/−), and at 33°C after FALI (+/+). Error bars show SEMs; ANOVA (post hoc Tukey’s test): *, P < 0.01; ***, P < 0.0001. n = 13, 10, and 14 bouton profiles from three larvae each.