Mutations in DCTN1 a component of the dynactin complex are linked

Mutations in DCTN1 a component of the dynactin complex are linked to neurodegenerative diseases characterized by a broad collection of neuropathologies. membrane binding by Arfaptin mediates membrane association of the dynactin complex in motor neurons and is required for normal synapse growth. Arfaptin represents a novel dynactin complex-binding protein that specifies dynactin complex function during synapse growth. INTRODUCTION Dynamic cellular processes such as membrane trafficking are predicted to use the precise spatial and temporal regulation of a vast array of proteins including motor proteins such as kinesin and dynein. The mechanisms underlying the precise regulation of motor protein function in neurons are incompletely described. One strategy used to specify motor function is usually illustrated by the kinesins which constitute a large and diverse family of proteins that have unique motor properties (Hirokawa homologue of Arfaptin2 ((and during synapse growth we find that Arfaptin (Arfip) also biochemically interacts with dynactin complexes in flies and mice demonstrating a conserved biochemical conversation. Mechanistically Arfip is usually a membrane-binding protein localized at the Golgi that is required for binding of the dynactin complex to membranes in the motor neuron. This membrane-binding property of Arfip is required for normal synapse growth supporting a role for Arfip and the dynactin complex at the Golgi for normal motor neuron development and function. RESULTS Enhancer/suppressor screen for modifiers of dynactin complex function in motor neurons A genetic screen of 3663 P-element insertion lines was performed to identify P-element insertions that specify dynactin complex function in motor neurons. To enrich for the recovery of insertions required for normal motor neuron function we performed initial viability screens in backgrounds in which the function of the dynactin complex is usually impaired in motor neurons. Specifically F1 flies were generated harboring a single-copy P-element insertion in-to a recombinant third chromosome harboring both the transgene and the Gal4 driver (in fly motor neurons using the driver results in animals with delayed larval development reduced eclosion rates and decreased survival providing useful metrics for screening. Because a portion of the P-elements screened have the ability to drive the expression of nearby genes in response to the presence of the Gal4 the recovery of both loss-of-function and gain-of-function alleles was possible (R?rth encodes Arfaptin a modifier of dynactin complex function during synapse growth. (A) An enhancer-suppressor screen of 3663 individual P-element insertion lines identified 40 candidate insertions that LG 100268 enhance or suppress the toxicity of dominant … Insertion lines were categorized as either enhancers or suppressors based on the effects around the lethality compared with heterozygote controls. We LG 100268 performed two rounds of primary screening under low- and high-stringency conditions resulting in the identification of 65 insertion lines that enhanced or TNFRSF9 suppressed viability in mutants. Control screens found that 25 lines interacted only with the Gal4 driver (to the heterozygote allele for effects on known dynactin-dependent processes within the motor neuron: LG 100268 synaptic growth (determined by counting the number of 1b boutons at the neuromuscular junction [NMJ]) synaptic stability (determined by quantifying the number of presynaptic retraction events) and axonal transport (determined by quantifying the extent axonal blockage). These analyses found that 14 insertions significantly enhanced the synapse growth phenotype resulting in significantly LG 100268 fewer boutons compared with the heterozygote control (Physique 1 B and ?andC C and Table 1). Five of these also enhanced the number of synaptic retractions (footprints) a measure of synapse stability and an established synaptic phenotype of dynactin complex mutations in flies and mice (Physique 1C indicated by E above bar; Table 1; Eaton gene encodes a modifier of dynactin-dependent synapse growth Our screen recovered two P-element insertions ((Figures 1C and ?and2A2A and Table 1). BLAST LG 100268 analysis of the peptide sequence obtained from the.