Cryopreservation method for Drosophila melanogaster embryos.

The development of a widely adopted cryopreservation method remains a major challenge in Drosophila research. Here we report a robust and easily implemented cryopreservation protocol of Drosophila melanogaster embryos. We present innovations for embryo permeabilization, cryoprotectant agent loading, and rewarming. We show that the protocol is broadly applicable, successfully implemented in 25 distinct strains from different sources. We demonstrate that for most strains, >50% embryos hatch and >25% of the resulting larvae develop into adults after cryopreservation. We determine that survival can be significantly improved by outcrossing to mitigate the effect of genetic background for strains with low survival after cryopreservation. We show that flies retain normal sex ratio, fertility, and original mutation after successive cryopreservation of 5 generations and 6-month storage in liquid nitrogen. Lastly, we find that non-specialists are able to use this protocol to obtain consistent results, demonstrating potential for wide adoption.

Methods to identify and analyze gene products involved in neuronal intracellular transport using Drosophila.

Proper neuronal function critically depends on efficient intracellular transport and disruption of transport leads to neurodegeneration. Molecular pathways that support or regulate neuronal transport are not fully understood. A greater understanding of these pathways will help reveal the pathological mechanisms underlying disease. Drosophila melanogaster is the premier model system for performing large-scale genetic functional screens. Here we describe methods to carry out primary and secondary genetic screens in Drosophila aimed at identifying novel gene products and pathways that impact neuronal intracellular transport. These screens are performed using whole animal or live cell imaging of intact neural tissue to ensure integrity of neurons and their cellular environment. The primary screen is used to identify gross defects in neuronal function indicative of a disruption in microtubule-based transport. The secondary screens, conducted in both motoneurons and dendritic arborization neurons, will confirm the function of candidate gene products in intracellular transport. Together, the methodologies described here will support labs interested in identifying and characterizing gene products that alter intracellular transport in Drosophila.

Spectrin mutations that cause spinocerebellar ataxia type 5 impair axonal transport and induce neurodegeneration in Drosophila.

Spinocerebellar ataxia type 5 (SCA5) is an autosomal dominant neurodegenerative disorder caused by mutations in the SPBTN2 gene encoding beta-III-spectrin. To investigate the molecular basis of SCA5, we established a series of transgenic Drosophila models that express human beta-III-spectrin or fly beta-spectrin proteins containing SCA5 mutations. Expression of the SCA5 mutant spectrin in the eye causes a progressive neurodegenerative phenotype, and expression in larval neurons results in posterior paralysis, reduced synaptic terminal growth, and axonal transport deficits. These phenotypes are genetically enhanced by both dynein and dynactin loss-of-function mutations. In summary, we demonstrate that SCA5 mutant spectrin causes adult-onset neurodegeneration in the fly eye and disrupts fundamental intracellular transport processes that are likely to contribute to this progressive neurodegenerative disease.

Dynein and Star interact in EGFR signaling and ligand trafficking.

Intracellular transport and processing of ligands is critical to the activation of signal transduction pathways that guide development. Star is an essential gene in Drosophila that has been implicated in the trafficking of ligands for epidermal growth factor (EGF) receptor signaling. The role of cytoplasmic motors in the endocytic and secretory pathways is well known, but the specific requirement of motors in EGF receptor transport has not been investigated. We identified Star in a screen designed to recover second-site modifiers of the dominant rough eye phenotype of the Glued mutation Gl(1). The Glued (Gl) locus encodes the p150 subunit of the dynactin complex, an activator of cytoplasmic dynein-driven motility. We show that alleles of Gl and dynein genetically interact with both Star and EGFR alleles. Similarly to mutations in Star, the Gl(1) mutation is capable of modifying the phenotypes of the EGFR mutation Ellipse. These genetic interactions suggest a model in which Star, dynactin and dynein cooperate in the trafficking of EGF ligands. In support of this model, overexpression of the cleaved, active Spitz ligand can partially bypass defective trafficking and suppress the genetic interactions. Our direct observations of live S2 cells show that export of Spitz-GFP from the endoplasmic reticulum, as well as the trafficking of Spitz-GFP vesicles, depends on both Star and dynein.

The Drosophila tctex-1 light chain is dispensable for essential cytoplasmic dynein functions but is required during spermatid differentiation.

Variations in subunit composition and modification have been proposed to regulate the multiple functions of cytoplasmic dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila cytoplasmic dynein and use P element excision to generate flies that completely lack this dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear "cap" of cytoplasmic dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other dynein subunits and is not required for any essential functions in early development or in the adult organism.

The third P-loop domain in cytoplasmic dynein heavy chain is essential for dynein motor function and ATP-sensitive microtubule binding.

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.