Coordinated Regulation of Axonal Microtubule Organization and Transport by Drosophila Neurexin and BMP Pathway

Abstract

Neurexins are well known trans-synaptic cell adhesion molecules that are required for proper synaptic development and function across species. Beyond synapse organization and function, little is known about other roles Neurexins might have in the nervous system. Here we report novel phenotypic consequences of mutations in Drosophila neurexin (dnrx), which alters axonal microtubule organization and transport. We show that dnrx mutants display phenotypic similarities with the BMP receptor wishful thinking (wit) and one of the downstream effectors, futsch, which is a known regulator of microtubule organization and stability. dnrx has genetic interactions with wit and futsch. Loss of Dnrx also results in reduced levels of other downstream effectors of BMP signaling, phosphorylated-Mad and Trio. Interestingly, postsynaptic overexpression of the BMP ligand, Glass bottom boat, in dnrx mutants partially rescues the axonal transport defects but not the synapse undergrowth at the neuromuscular junctions. These data suggest that Dnrx and BMP signaling are involved in many diverse functions and that regulation of axonal MT organization and transport might be distinct from regulation of synaptic growth in dnrx mutants. Together, our work uncovers a novel function of Drosophila Neurexin and may provide insights into functions of Neurexins in vertebrates.

Figure 1

dnrx mutants show defects in axonal microtubule cytoskeleton and transport. (A–J’) Confocal images of 3^rd instar larval nerve fibers labeled with antibodies against Syt (green) and Futsch (red) in indicated genotypes. (K,L) Quantification of Syt puncta per 80 μm nerve length (K) and per μm² nerve area of 3^rd instar larval segmental nerve 5–8 passing through abdominal segments A4–7 of specified genotypes. n = 15 animals. Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant). Scale bar: (A–J’) = 10 μm.

Figure 2

Genetic interactions between dnrx and wit and their influence on axonal transport. (A–F’) Confocal images of portions of nerve fibers showing axonal MT stained with anti-Futsch (red) and anti-Syt (green) from 3^rd instar larvae of indicated genotypes. (G) Quantification Syt puncta per 80 μm of segmental nerve 5–8 passing through A4-7 in all indicated genotypes. n = 15 animals. Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant). Scale bar: (A–F’) = 10 μm.

Figure 3

Ultrastructural changes in axonal microtubule organization in dnrx and wit mutants. (A–C’) Longitudinal sections at low (A–C) and high magnifications (A’–C’) of segmental nerves of +/+ (A,A’), dnrx (B,B’) and wit (C,C’) mutants. Black arrows (A’–C’) represent axonal MT filaments in indicated genotypes and blue arrows (wit mutants, C) point to intersecting MT filaments. (A”–C’”) Higher magnification images showing mitochondria (A”–C”) and septate junctions (A’”–C’”) in wild type (A”,A’”), dnrx (B”,B’”) and wit (C”,C’”) mutant nerves. (D) Quantification of percent MT breaks in axons of wild type, dnrx and wit mutants. Scale bars: (A–C) = 1 μM, (A’–C’) = 200 nm, (A”–C”) = 600 nm and (A’”–C’”) = 200 nm. n = 5 animals/genotype. Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant).

Figure 4

dnrx and wit mutants show defects in larval locomotion. (A–D) Wild type, dnrx and wit mutant larval locomotor behavior assayed by measuring the number of 0.5 cm² grids crossed in 30 seconds (A), time taken in seconds for larvae to exit a circle of 1.5 cm in diameter (B), number of full body peristaltic contractions in 1 minute (C) and time taken in seconds for larvae to right themselves when turned on their dorsal surface (D). n = 50 animals/genotype. Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant).

Figure 5

Axon Transport Defects in futsch Mutants and Genetic Interactions between dnrx and futsch. (A–D’) Portions of segmental nerves labeled with anti-Syt (green) and anti-Futsch (red) in indicated genotypes. (E) Quantification of Syt puncta along 80 μM of segmental nerves of indicated genotypes. Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant). Scale bar: (A–D’) = 10 μm.

Figure 6

Postsynaptic Overexpression of Gbb in dnrx mutants partially rescues the axonal transport defects. (A–D) Immunoblots of brain lobes/VNC lysates from wild type, dnrx and wit mutants showing levels of phosphorylated Mad (pMad, 55 kDa, A) and the cytoskeletal RhoGEF, Trio (250 kDa, B), together with their corresponding Actin levels as loading controls. (Note the breaks in panels separated by white space are due to removal of irrelevant lanes, see Fig. S4). Ratio of band intensity was quantified by measuring levels of pMad to Actin (C) and Trio to Actin (D) in indicated genotypes. (E–H’) Confocal images of portions of segmental nerves from 3^rd instar larvae stained against Futsch (red) and Syt (green) in various genotypes. (I–N) Confocal images of larval NMJ synaptic boutons stained with antibodies against Dlg (red) and Hrp (green) in indicated genotypes. (O,P) Quantification of Syt puncta (O) and total bouton numbers in NMJ 6/7 of A3 (P) in indicated genotypes. n = 15 animals/genotype in (E–N). Error bars represent mean ± SEM (***p < 0.001, **p ≤ 0.01, *p ≤ 0.5, ns – not significant). Scale bars: (A–H’ and I–N) = 10 μm.