Knock-down of Arp2 and Arp3 lead to larval migration defects phenocopying <i>abp1</i> knock-out.

<p>(<b>A,B</b>) Summarized migration tracks with centered common start points (bars, 5 mm) of Ubi-<i>Gal4</i>+Arp2 RNAi #2 (<b>A</b>) and Ubi-<i>Gal4</i>+Arp3 RNAi (<b>B</b>). (<b>C</b>) Mean velocities of larval migration (n = 19–20). Data represent mean±SEM. * = p<0.05 and ** = p<0.01. One way ANOVA post Tukey.</p

Abp1 is present on both the presynaptic and the postsynaptic side of NMJs.

<p>(<b>A–D</b>) Localization studies of Abp1 in HRP and anti-Dlg-stained <i>wt</i> NMJs (<b>A,B</b>) compared to anti-Abp1 immunolabeling in NMJs of flies expressing C57-induced postsynaptic Abp1 RNAi demonstrating the less obvious presynaptic pool of Abp1 (<b>C,D</b>). Lower panels represent enlargements of boxed areas. Note that presynaptic endogenous Abp1 immunolabeling was not detectable in type Is boutons but is enriched in type Ib boutons (<b>C</b>). (<b>B,D</b>) Quantitative analyses of fluorescence intensity plots across type Ib boutons along the lines shown in <b>A</b> and <b>C</b> allow for resolving spatially distinct fluorescence maxima for Dlg and HRP (marked by arrow heads in <b>B</b> and <b>D</b>). (<b>B</b>) Anti-Abp1 immunostaining spans the localization of both markers (or even extends postsynaptically) and shows segregated pre- and postsynaptic peaks, respectively. (<b>D</b>) Postsynaptic Abp1 RNAi drastically reduces the prominent postsynaptic anti-Abp1 signals and thereby reveals a remaining anti-Abp1 immunosignal overlapping with the presynaptic HRP localization. Bars in <b>A</b> and <b>C</b>, 10 µm. (<b>E</b>) Presynaptically expressed Abp1-GFP at NMJs at muscles 6/7 of abdominal segment A2 is enriched at Dlg-enriched structures, whereas Abp1 localization at interbouton axon segments and type Is boutons is low. Bars, 20 µm.</p

Abp1 is highly expressed in the central nervous system.

<p>(<b>A</b>) Immunolabeling of endogenous Abp1 at indicated stages of development. Left, confocal lateral view of stage 14 embryo; middle, confocal, ventro-lateral view of stage 15 embryo; right, confocal image of a 3^rd instar larval brain. Embryos display strong Abp1 immunoreactivity in epithelia and the CNS (left), in the commissures and longitudinal tracks of the CNS (middle) as well as in the neuropil (n) of the ventral nerve cord of 3^rd instar larvae. Bars, 100 µm. (<b>B</b>) Maximum projections of a confocal stack of a stage 15 embryo immunostained with anti-Abp1 antibodies showing that besides in the commissures and longitudinal tracks of the CNS Abp1 is present in motoneuronal growth cones marked with anti-FasII antibodies. Boxed area is presented as enlarged inset. Bar, 10 µm. (<b>C</b>) Abp1 immunoreactivity at NMJs of a 3^rd instar larvae costained with postsynaptic (Dlg) and presynaptic markers (HRP). Note that in contrast to Dlg labeling, Abp1 immunoreactivity is nearly equally strong at type Ib and type Is synaptic boutons. Bar, 10 µm. (<b>D</b>) Anti-Abp1 immunostaining of <i>wt</i> versus <i>abp1^KO</i>/Df(3 L) NMJs showing that the anti-Abp1 immunosignal at NMJs is specific. Bar, 10 µm. (<b>E</b>) Overview of anti-Abp1 immunostaining at muscles 6/7 NMJs of a 3^rd instar larvae (maximum projections of confocal stack). Note that besides a low expression in muscles, Abp1 is enriched in tracheae (example marked with T), axons of motoneurons (example marked with M) and NMJs (example marked by N). Colored panels at top and right display the intensity of the anti-Abp1 signal in Z-direction in pseudo colors encoding for the fluorescence intensity (see scale at upper right). Bar, 50 µm. (<b>F</b>) Maximum projections of stitched confocal images of anti-Dlg-stained body wall preparations show normal segmentation, muscle organization and innervations of an <i>wt</i> (left) and an <i>abp1</i>^KO/Df(3 L) fly (right). Bar, 500 µm.</p

Terminal Axonal Arborization and Synaptic Bouton Formation Critically Rely on Abp1 and the Arp2/3 Complex

<div><p>Neuronal network formation depends on properly timed and localized generation of presynaptic as well as postsynaptic structures. Although of utmost importance for understanding development and plasticity of the nervous system and neurodegenerative diseases, the molecular mechanisms that ensure the fine-control needed for coordinated establishment of pre- and postsynapses are still largely unknown. We show that the F-actin-binding protein Abp1 is prominently expressed in the <i>Drosophila</i> nervous system and reveal that Abp1 is an important regulator in shaping glutamatergic neuromuscular junctions (NMJs) of flies. STED microscopy shows that Abp1 accumulations can be found in close proximity of synaptic vesicles and at the cell cortex in nerve terminals. <i>Abp1</i> knock-out larvae have locomotion defects and underdeveloped NMJs that are characterized by a reduced number of both type Ib synaptic boutons and branches of motornerve terminals. Abp1 is able to indirectly trigger Arp2/3 complex-mediated actin nucleation and interacts with both WASP and Scar. Consistently, Arp2 and Arp3 loss-of-function also resulted in impairments of bouton formation and arborization at NMJs, i.e. fully phenocopied <i>abp1</i> knock-out. Interestingly, neuron- and muscle-specific rescue experiments revealed that synaptic bouton formation critically depends on presynaptic Abp1, whereas the NMJ branching defects can be compensated for by restoring Abp1 functions at either side. In line with this presynaptic importance of Abp1, also presynaptic Arp2 and Arp3 are crucial for the formation of type Ib synaptic boutons. Interestingly, presynaptic Abp1 functions in NMJ formation were fully dependent on the Arp2/3 complex, as revealed by suppression of Abp1-induced synaptic bouton formation and branching of axon terminals upon presynaptic Arp2 RNAi. These data reveal that Abp1 and Arp2/3 complex-mediated actin cytoskeletal dynamics drive both synaptic bouton formation and NMJ branching. Our data furthermore shed light on an intense bidirectional functional crosstalk between pre- and postsynapses during the development of synaptic contacts.</p></div

Genotypes generated for knock-down, overexpression and rescue experiments.

<p>Genotypes generated for knock-down, overexpression and rescue experiments.</p

Abp1 deficiency causes defects in NMJ development.

<p>(<b>A</b>) Examples of NMJs at muscles 6/7 of abdominal segment A2 from <i>wt</i>, <i>abp1</i>^KO/Df(3 L) and hemizygous <i>abp1^WH-rev</i> larvae, respectively, stained for pre- and postsynaptic markers HRP (green) and Dlg (red). Note that the NMJs of <i>abp1</i>^KO/Df(3 L) flies (middle) appear condensed and less arborized and contain fewer synaptic boutons type Ib when compared to <i>wt</i> and to revertants generated to restore <i>abp1</i> expression by precise excision to demonstrate the specificity of <i>abp1</i> knock-out phenotypes. Bars, 20 µm. (<b>B,C</b>) Quantification of type Ib bouton numbers (<b>B</b>) and terminal axonal branch points (<b>C</b>). The significant reduction in the number of boutons and branch points observed in all <i>abp1</i>-mutant allelic combinations are restored in the revertant <i>abp1^WH-rev</i>. (<b>D</b>) HRP and anti-Dlg-stained NMJs of <i>abp1</i>-deficient larvae with postsynaptic Abp1 re-expression (C57<i>-Gal4</i>+Abp1+<i>abp1</i>^KO/Df(3 L)) and of larvae with a presynaptic rescue of Abp1 functions (OK371-<i>Gal4</i>+Abp1+<i>abp1</i>^KO/Df(3 L)). Bar, 20 µm. (<b>E,F</b>) Quantitative analyses of type Ib boutons and of terminal axonal branch points show that only presynaptic expression of Abp1 rescues bouton number whereas the number of terminal axonal branch points can be rescued by both pre- and postsynaptic expression of Abp1. Data represent mean±SEM. n = 19–42 NMJs/genotype. * = p<0.05, ** = p<0.01 and *** = p<0.001. One way ANOVA post Tukey.</p

<i>Abp1</i> knock-out leads to impaired larval migration.

<p>(<b>A</b>) Genomic scheme of <i>abp1</i> (<i>CG10083</i>) locus 3 L 70B2 with PBac insertion WH f05024, of <i>abp1</i> knock-out (<i>abp1</i>^KO) <i>flies</i> generated by FRT-mediated recombination of PBac insertions, of the deficiency Df(3 L) and of the revertant <i>abp1</i>^WH-rev. Shown are exons with translated (red) and untranslated regions (grey). (<b>B–D</b>) Summarized presentation of migration tracks with centered common start points (bars, 5 mm) of <i>wt</i> (<b>B</b>), <i>abp1</i>^KO/Df(3 L) (<b>C</b>) and <i>abp1</i>^WH-rev/Df(3 L) (<b>D</b>). (<b>E</b>) Mean velocities of larval migration (n = 17–38). Data represent mean±SEM. ** = p<0.01. One way ANOVA post Tukey.</p

Arp2 and Arp3 loss-of-function mirror the defects in NMJ development observed upon <i>abp1</i> deficiency.

<p>(<b>A</b>) The immobilized SH3 domain of Abp1 (GST-SH3) specifically precipitates endogenous WASP and Scar from fly head extracts whereas GST and a point-mutated SH3 domain (GST-SH3*; P523A mutation), respectively, do not. (<b>B</b>) Model depicting that Abp1 interfaces with both WASP and Scar and may regulate Arp2/3 complex dependent actin dynamics at the NMJ using both Arp2/3 complex activation pathways. (<b>C</b>) NMJs at muscles 6/7 of abdominal segment A2 of <i>wt</i>, Arp2 RNAi #1, Arp2 RNAi #2 and Arp3 RNAi expressing larvae stained with HRP (green) and anti-Dlg (red). (<b>D,E</b>) Quantitative analyses of type Ib boutons and of terminal axonal branch points visualize that Arp2/3 complex deficiency phenocopies <i>abp1</i> knock-out in reducing type Ib bouton number and terminal branch points of the axon within NMJs. (<b>F,G</b>) Quantitative analyses revealing that larvae with presynaptic Arp2 RNAi #2 or Arp3 RNAi phenotypically mirror presynaptic Abp1 RNAi, as specifically type Ib bouton formation but not the formation of terminal axonal branch points is affected. Data represent mean±SEM. n = 18–36/genotype. * = p<0.05, ** = p<0.01 and *** = p<0.001. One way ANOVA post Tukey.</p

Super-resolution 2-channel STED microscopy shows distinct localization of Abp1-GFP in presynaptic nerve terminals at NMJs.

<p>Maximum intensity projections of deconvolved confocal (<b>B,D</b>) as well as STED images (<b>A,C,F</b>) recorded from NMJs at muscle 6/7 (A2). (<b>A</b>) STED images of Abp1-GFP and Futsch (outlining presynaptic microtubules) show no colocalization between the two proteins. Abp1 often displays enrichments at the cell cortex. Bar, 2 µm. (<b>B</b>) Confocal imaging of Bruchpilot (BRP) and HRP shows the localization of Abp1-GFP near the plasma membrane but not in the middle of boutons. There is no overlap with BRP marking the active zone core structure, as seen in the merge and in the magnified inset representing as single image plane. Bar, 5 µm; bar in inset, 1 µm. (<b>C</b>) STED microscopy confirms the lack of spatial overlap of Abp1 and BRP. Bars, 2 µm; bar in insert (single slice image), 500 nm. (<b>D</b>) Confocal microscopy of immunostainings for CSP suggests a strong colocalization of Abp1-GFP with CSP. Bar, 5 µm; in inset (single image plane), 1 µm. (<b>E</b>) However, 2-channel STED microscopy shows no colocalization of Abp1 with CSP but Abp1 rather localizes adjacent to individual anti-CSP-stained synaptic vesicles. Bars, 2 µm; bar in inset (single image plane), 500 nm.</p

Presynaptic Abp1-dependent functions in NMJ development critically rely on presynaptic Arp2/3 complex.

<p>(<b>A</b>) Anti-GFP, HRP and anti-Dlg-stained NMJs at muscles 6/7 of abdominal segment A2 from larvae expressing OK371-<i>Gal4</i>+Arp2 RNAi #2, OK371-<i>Gal4</i>+Abp1-GFP and OK371-<i>Gal4</i>+Abp1-GFP+Arp2 RNAi #2, respectively. Bar, 20 µm. (<b>B–E</b>) Quantitative analyses of type Ib boutons and of nerve terminal branch points show that the presynaptic expression of Abp1-GFP leads to both an increase in bouton number (<b>B</b>) and to an increase in terminal axonal branch points (<b>C</b>), i.e. to phenotypes opposite to Abp1 knock-out. (<b>D,E</b>) Presynaptic knock-down of Arp2 suppresses both of the Abp1-induced effects. Abp1's functions in the presynapse of NMJs thus are critically dependent on presynaptic Arp2/3 complex functions. Data represent mean±SEM. n = 20–22/genotype. * = p<0.05 and *** = p<0.001. Student's t-test (<b>B,C</b>) and One way ANOVA post Tukey (<b>D,E</b>).</p