AP2 hemicomplexes contribute independently to synaptic vesicle endocytosis
- Howard Hughes Medical Institute, University of Utah, United States
- Rutgers University, United States
- University of Utah, United States
Figures
Figure 1 with 1 supplement
apa-2 cloning.
(A) Genetic map position of apa-2 on chromosome X. (B) Genomic structure of the apa-2 gene and the nature of mutant alleles. b1044 is a 925 bp deletion from the second intron to the fourth exon. ox42…
Figure 1—figure supplement 1
Western blot of α adaptin mutants apa-2(ox422) and apa-2(b1044).
Antibodies are rabbit polyclonal anti-α adaptin and mouse monoclonal anti-tubulin.
Figure 2
α-adaptin is expressed ubiquitously.
(A) Schematic of apa-2::GFP translational reporter construct. The APA-2::GFP fusion construct is expressed under the control of the apa-2 promoter (1.9 kb upstream of ATG) from an extrachromosomal …
Figure 3 with 1 supplement
Tissue-specific rescue of α-adaptin mutant.
(A) Bright field images apm-2(e840) and tissue-specific rescue of apa-2(ox422) mutants. Worms are rescued by strains carrying single-copy transgenes. The jowls are indicated by the black arrowheads; …
Figure 3—figure supplement 1
Tissue-specific expression of APA-2::GFP.
The dpy-7 promoter drives expression (skin). The rab-3 promoter drives neuronal expression in the neurons. The dpy-30 promoter drives ubiquitous expression. Worms are oriented anterior left and …
Figure 4 with 1 supplement
α- and μ2-adaptin double mutant is synthetic.
(A) Embryonic lethality (% total embryos) of AP2 mutants ± SEM: wild type 1.13 ± 0.25 n = 10, apa-2(ox422) 2.42 ± 0.96 n = 9 (p=0.1902), apm-2(e840) 5.85 ± 2.56 n = 10 (p=0.0831), apa-2(ox422) …
Figure 4—figure supplement 1
AP2 mutants exhibit slowed postembryonic development.
Mean days from L1 to L4 stage ± SEM: wild type 1.5 ± 0 n = 29, apm-2(e840) 1.97 ± 0.17 n = 28 (p=0.0068), apa-2(ox422) 2.11 ± 0.09 n = 28 (p<0.0001), apm-2(e840) apa-2(ox422) 2.75 ± 0.17 n = 29 (p<0.…
Figure 5 with 2 supplements
Endocytosis of α- and μ2-adaptin-dependent cargo.
Fluorescence images have been inverted to aid visualization of signals. (A) Yolk protein, YP170::GFP is endocytosed by maturing oocytes in the wild type and both apa-2 mutants. Black arrowheads …
Figure 5—figure supplement 1
An AP2-independent cargo is not affected by AP2 subunits mutants.
Human IL2 receptor α subunit Tac (hTAC) in the nematode intestine in the wild type, μ2 adaptin mutant (apm-2(e840) X) and an α adaptin mutant (apa-2(ox422) X). Fluorescence images are inverted to …
Figure 5—figure supplement 2
Quantification of total fluorescence intensity of hTAC in the wild type and AP2 mutants.
Total fluorescence was measured along the basolateral membrane and averaged. Fluorescence intensity mean ± SD: wild type 16,080 ± 7875 n = 18, apm-2(e840) 22,948 ± 9488 n = 18 (p=0.0240), apa-2(ox422…
Figure 6 with 2 supplements
AP2 hemicomplexes are partially stable in vivo.
All images are inverted to better visualize GFP fluorescence. (A) Synaptic localization of σ2 adaptin (APS-2::GFP) in α and μ mutants. The nerve ring is indicated by the black arrowhead. (B) …
Figure 6—figure supplement 1
μ2-adaptin is present in α-adaptin mutants.
A rescuing construct of tagged μ2-adaptin was inserted into a μ2-adaptin null strain for all genotypes. Top: μ2-adaptin (APM-2::GFP) expression in the gonad of a μ2 mutant (apm-2(e840) X, labeled as …
Figure 6—figure supplement 2
σ2-adaptin is more unstable in α-adaptin than in μ2-adaptin mutants.
Top: σ2::GFP is localized to the plasma membrane in the wild type and μ2-adaptin mutant but not in α-adaptin mutants or α-μ2 adaptin double mutants. The oocyte cell surface is indicated by black …
Figure 7 with 1 supplement
α-adaptin mutants exhibit weak defects in synaptic vesicle protein localization.
(A) All images are inverted to better visualize GFP fluorescence. Synaptic localization of synaptic vesicle proteins in apa-2 and apm-2(e840) apa-2(ox422) double mutants (an escaper with no skin …
Figure 7—figure supplement 1
α-adaptin colocalizes with synaptobrevin at synapses.
Young adult hermaphrodites were used for imaging. Left: α-adaptin (APA-2::GFP) and synaptobrevin (SNB-1::tagRFP) colocalize at synapses in the dorsal nerve cord of GABA motor neurons. The …
Figure 8 with 4 supplements
Large vesicles accumulate at synapses in AP2 mutants.
(A) Representative images of acetylcholine neuromuscular junctions in the ventral nerve cord from the wild type, apa-2(ox422), ubiquitously-rescued apa-2(ox422), neuronally-rescued apa-2(ox422), …
Figure 8—figure supplement 1
Synaptic vesicles are reduced at GABA synapses in α-adaptin mutants and α-adaptin μ2-adaptin double mutants.
The number of synaptic vesicles in GABA neurons n = synapses: wild type 36.9 ± 1.5 n = 36, apa-2(ox422) 10.7 ± 0.7 n = 46 (p<0.0001), ubiquitously-rescued apa-2(ox422) 33.9 ± 1.4 n = 33 (p=0.1504), n…
Figure 8—figure supplement 2
Synaptic vesicle diameters are larger in α-adaptin mutants.
Median size of synaptic vesicles per GABA synapse profile containing a dense projection n = synapses: wild type 28.4 nm n = 8, apa-2(ox422) 30.9 nm n = 9 (p<0.0001), ubiquitously-rescued apa-2(ox422)…
Figure 8—figure supplement 3
Large vesicles accumulate in α-adaptin mutants and α-adaptin μ2-adaptin double mutants.
The number of large vesicles in GABA neurons n = synapses: Wild type GABA 0.9 ± 0.1 n = 41, apa-2(ox422) GABA 4.9 ± 0.5 n = 43 (p<0.0001), ubiquitously-rescued apa-2(ox422) GABA 0.9 ± 0.2 n = 32 …
Figure 8—figure supplement 4
In some cases, the large vacuole remains associated with the plasma membrane in α-adaptin μ2-adaptin double mutants.
(A–C) additional images of acetylcholine neuromuscular junctions in the ventral nerve cord from the skin-rescued apa-2(ox422) apm-2(e840) in adult hermaphrodites. (D–F) Zoomed-in images of large …
Figure 9
Synaptic vesicle fusion is reduced in α adaptin mutants.
(A) Sample traces of miniature postsynaptic current (minis) recorded from the wild type, apa- 2(ox422), apa-2(ox422) neuronal-rescued, apa-2(ox422) skin-rescued and apa-2(ox422) apm-2(e840) …
Tables
Table 1
GFP fluorescence of tagged AP2 subunits in the nerve ring (quantification for Figure 6). Average GFP intensity in the nerve ring (percentage of the wild type)
| wild type | apa-2 | apm-2 | |
| σ2::GFP | 3543 ± 169 (100%) | 389 ± 28 (11%) | 1391 ± 51 (39%) |
| β1::GFP | 3881 ± 31 (100%) | 2329 ± 123 (60%) | 1360 ± 62 (35%) |
| μ2::GFP | 2783 ± 142 (100%) | 532 ± 73 (19%) | – |
| α::GFP | 3230 ± 132 (100%) | – | 1296 ± 126 (40%) |
-
The data are mean ± SEM of averaged fluorescence, n = 5 worms each. The p value for all pair-wise comparisons (wild type vs mutants or apa-2 vs apm-2) is p<0.0001. Student's t test. Note the beta subunit in C. elegans is shared by both the AP1 and AP2 complexes. Beta levels are reduced in apm-2 mutants compared to apa-2 mutants; however, beta is still present and stable in AP1 complexes in apm-2 mutants. In particular, beta is highly expressed in pharyngeal muscle, which is included in the region of interest.
Additional files
-
Supplementary file 1
Strains.
- https://doi.org/10.7554/eLife.00190.025
