Role of Drosophila α-Adaptin in Presynaptic Vesicle Recycling Marcos González-Gaitán, Herbert Jäckle  Cell  Volume 88, Issue 6, Pages 767-776 (March 1997) DOI: 10.1016/S0092-8674(00)81923-6

Figure 1 Molecular Characterization of the Drosophila α-Adaptin Gene (A) In situ hybridization using the 4.8 kb λzap cDNA as a probe to salivary gland chromosomes indicating that D-αAdaptin is located in the cytogenetic interval 21C1-2 at the left arm of the second chromosome. (B) Northern blot analysis with polyA+ RNA from 0- to 18-hr-old embryos (Em) and adults (Ad) using the cDNA as a probe, indicating that D-αAdaptin codes for two transcripts (4.8 and 6.3 kb), which are present both in embryos and adults. Both transcripts were also detected with a probe encompassing exon 1 and 2 (data not shown). Thus, they share the exons deleted in the D-αAda3 mutation. The two transcripts can also be found in third instar larvae (data not shown). (C) Genomic organization and molecular characterization of the D-αAdaptin mutants. Locations of intronic sequences corresponding to the smaller 4.8 kb transcript are shown. Solid bar indicates the open reading frame of the transcript. P element insertion in the first exon (at position 81 of the 4.8 kb transcript, as determined by sequencing) and the extent of DNA deletions caused by imprecise excisions of the P element (determined by genomic Southern blot analysis of the mutants) are indicated. The resulting mutant alleles D-αAda1, D-αAda2, and D-αAda3 are described in the text. B refers to BglII; K, KpnI; A, ApaI; N, NheI. Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 2 Sequence of Drosophila α-Adaptin and Comparison with the Vertebrate Homologs D-αAda refers to Drosophila α-Adaptin; M-αAda-C, mouse α-Adaptin-C; R-αAda-C, rat α-adaptin-C; M-αAda-A, mouse α-adaptin-A. Numbers above the sequence denote the position of the seven introns within the open reading frame. The regions corresponding to the head, hinge, and ear domains are indicated by empty, solid, and dotted lines above the sequence, respectively. Sequence identity between Drosophila α-Adaptin and its vertebrate homologs is highest in the head (αAda-C, 82% similarity; αAda-A, 80%), intermediate in the ear (55%), and lowest in the hinge domain (αAda-C, 36%; αAda-A, 11%). Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 3 D-αAdaptin Embryonic Expression Pattern and Localization of the Protein in Larval Motoneurons (A) Whole-mount RNA in situ hybridization of a stage 17 embryo (stage according toCampos-Ortega and Hartenstein 1985) using the cDNA as a probe. The transcript is restricted to the embryonic brain (br) and the ventral cord (vc) of the CNS. Staining can also be detected in the garland cells (gc) around the proventriculus. No other location of D-αAdaptin transcript was observed. (B–D) Confocal images show wild-type larval neuromuscular junctions (type I) (Johansen et al. 1989) stained with antibodies against Drosophila α-adaptin ([B]; anti-αAda) and anti-horseraddish peroxidase ([C]; anti-hrp), and the superimposed image of both (D). Anti-hrp antibodies recognize a membrane and cytoskeleton-associated β subunit of the Na+/K+-ATPase (Sun and Salvaterra 1995; van de Goor et al. 1995), which is localized at the membrane of both the axons (arrowheads) and the presynaptic terminals (arrows). α-adaptin is restricted to the presynaptic terminal (arrow in [B] and [D]). (E) Confocal images of wild-type larval neuromuscular junction (Type II) (Johansen et al. 1989) stained with anti-αAda (green) and anti-hrp (red) antibodies. Anti-αAda and anti-hrp colocalize at the small presynaptic boutons (arrow; yellow), while anti-αAda is absent from the interconnecting axons (arrowhead; red). (F) High magnification confocal image of a larval neuromuscular junction (type I) stained with anti-αAda. Fluorescence is associated with the presynaptic membrane. Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 4 Drosophila α-Adaptin-Deficient Embryos Develop a Normal CNS (A) Ventral view of a stage 17 D-αAda3 mutant embryo stained with anti-Fasciclin II antibodies. The six longitudinal tracks of the ventral cord in the CNS and the segmental and intersegmental nerves are indistinguishable from wild type. (B) Lateral view indicating that the transversal nerve (TN), intersegmental nerve (ISN), the different branches of the segmental nerve (SNa, SNc, and SNd), as well as the axons of the RP1, RP3, RP4, and RP5 motoneurons (SNb branch innervating the ventral longitudinal muscles 6, 7, 12, and 13) are intact (for details on the muscle and innervation patterns in wild type, seeGoodman and Doe 1993). (C) Ventral view of a stage 17 D-αAda3 mutant embryo stained with anti-repo antibodies showing that the pattern of glial cells in the ventral cord is normal (for details of glial patterns, seeHalter et al. 1995). Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 5 D-αAdaptin Mutations Impair the Formation of Endocytotic Synaptic Vesicles (A–D) FM1-43 assay applied to wild-type and D-αAda1 mutant presynaptic terminals. (A and B) Wild-type presynaptic terminals at the larval neuromuscular junction after 5 s (A) and 60 s (B) FM1-43 loading pulses. (C and D) D-αAda1 presynaptic terminals at the larval neuromuscular junction after 5 s (C) and 60 s (D) loading pulses. The 5 s FM1-43 pulse is sufficient to load the wild-type but not the mutant presynaptic terminals, while the presynaptic boutons of both are labeled after the 60 s loading pulse. Thus, vesicle formation is not blocked in the mutant but is impaired. This is consistent with the finding that D-αAda1 is not a lack-of-function allele (see text). (E and F) Ultrastructure of a presynaptic terminal in the CNS neuropile of wild type (E) and homozygous D-αAda3mutant embryos (F). Note vesicles in the synaptic boutons of wild-type embryos that are absent in the mutant and also the deep folds of the presynaptic membrane of the mutant (arrowheads in [F]). The example shown represents 1 out of more than 40 boutons of 6 different D-αAda3 mutant neuropiles, all showing the characteristics outlined in (F). db stands for dense body; mt, mitochondria; m, microtubules; sv, synaptic vesicles. Scale bars = 0.1 μm. Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 6 Subcellular Localization of α-Adaptin and Dynamin (A) Confocal image of a neuromuscular junction (type I) of wild-type larvae stained with anti-α-adaptin antibodies. α-adaptin is not homogeneously distributed throughout the presynaptic membrane but forms a network-like structure that leaves α-adaptin-free islands (about 0.5 μm in diameter) at the membrane. (B–D) Confocal images of a neuromuscular junction (type I) of wild-type larvae stained with anti-Drosophila α-adaptin ([B]; green) and anti-dynamin ([C]; red) antibodies, and the superimposed images (D). Note in the highly magnified inset a pattern of higher intensities of dynamin staining corresponding to the pattern of α-adaptin staining and lower intensities of dynamin staining covering the α-adaptin-free islands. In order to visualize the α-adaptin pattern at the plasma membrane, tangential sections of the presynaptic terminal at the side facing the muscle were selected. For the pattern in a nontangential section, see Figure 3F. Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)

Figure 7 Dynamin Is Recruited to Prelocalized α-Adaptin (A–C) Tangential confocal images of shits2 presynaptic terminals (type I) of larvae, showing the patterns of α-adaptin ([A]; αAda, green), dynamin ([B]; Dyn, red), or both (C). In the shits2 mutant presynaptic terminals, dynamin colocalizes with α-adaptin and disappears from the α-adaptin-free islands. (D–I) Corresponding images showing the distribution of α-adaptin ([D]; αAda, green), synaptotagmin ([E]; Syt, red), or both (F), and the patterns of α-adaptin ([G], αAda, green), cystein string protein ([H]; CSP, red), or both (I). The networklike structure of α-adaptin is not affected in the shits2 mutant presynaptic terminal, while synaptotagmin and CSP become restricted to the membrane (arrowhead in [E]), where they are homogeneously distributed as previously described (Littleton et al. 1993; Ramaswami et al. 1994; Zinsmaier et al. 1994; Estes et al. 1996). Note that the patterns of dynamin in wild-type and shits2 mutants are identical at the permissive temperature of 18°C, and that the redistribution at the nonpermissive temperature involves a mutant dynamin protein. Data were obtained from stimulated synapses maintained at nonpermissive temperatures. Cell 1997 88, 767-776DOI: (10.1016/S0092-8674(00)81923-6)