Source was adult heads, or purified synaptic membrane fraction from adult heads, of wild-type fly line; protein produced from endogenous gene.

orb2 forms SDS-resistant oligomers, in addition to the 82kDa monomer, migrating between 160 and 420kDa and resistant to treatment with various denaturing agents: detergents, urea or guanidine. This high molecular weight band is definitively orb2 because is not visible upon orb2 RNAi-mediated knockdown or in orb2 mutants, and reappears in orb2 mutants upon rescue with a genomic fragment. This oligomer is not due to phosphorylation, glycosylation, ubiquitination, sumoylation or acetylation, as it persists after various treatments that remove these modifications. This oligomer is not due to association with other proteins as it persists after treatment with various denaturing agents, RNaseA or high salt. This oligomeric form is only observed in head extracts, not in body extracts. The oligomeric form of orb2 is upregulated by neuronal stimulation of the mushroom body or feeding starved flies neurotransmitters (tyramine, dopamine, octopamine or serotonin) and is enriched in the synaptic membrane fraction. While oligomers contain both the short (Orb2A, orb2-PA) and long (Orb2B, orb2-PB) isoforms, oligomer formation in vivo requires the presence of the low abundance short isoform (Orb2A, orb2-PA) - since this short isoform has a greater propensity for oligomer formation, it may seed formation of oligomers by the more abundant long isoform.

Protein was immunoprecipitated and resolved by SDS-PAGE.

Source was adult heads of wild-type fly line; protein produced from endogenous gene.

orb2 oligomeric forms cosediment with an apparent molecular weight of 670kDa.

Protein lysate was separated on 5-30% sucrose gradient, then orb2 was immunoprecipitated and subjected to SDS-PAGE.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

Both short and long isoforms of orb2 form large amyloid-like fibers in vitro, visible by electron microscopy.

Purified recombinant orb2 was incubated for 4hr in urea.

The short orb2 isoform (Orb2A, orb2-PA) forms oligomers in vivo much more efficiently than the longer orb2 isoform (Orb2B, orb2-PB) which has a 162 residue N-terminal extension. While 30% of the short isoform gets incorporated into oligomers, only 5% of the long isoform gets incorporated into oligomers. Tagged transgenic short isoform, but not the long isoform, is visible in a punctate form under immunofluorescence.

Source was adult heads of transgenic fly line; protein produced from tagged transgenic construct.

Tagged, isoform-specific transgenes were expressed in neurons to observe the isoform-dependence of oligomer formation.

Source was adult heads, of transgenic fly line; protein produced from transgenic construct.

The short 8 residue N-terminal region of the orb2-PA isoform is required for aggregation in yeast, and mutation of the conserved Phe residue at position 5 in this short isoform-specific N-terminal region reduces oligomerization of transgenic orb2 in flies, as characterized by immunoprecipitation and western blot of adult head protein lysates.

Protein was immunoprecipitated and resolved by SDS-PAGE.

orb2 protein was immunoprecipitated by the GFP tag, and samples were separated by SDD-AGE to separate oligomeric complexes.

Source was cell extract of S2 cell line; protein produced from transfected construct.

The glutamine-rich Q-domain is necessary for orb2 protein oligomerization.

orb2 protein was immunoprecipitated by it\'s GFP tag, and samples were separated by SDD-AGE to separate oligomeric complexes.

The glutamine-rich Q-domain of the Orb2A isoform is necessary for orb2 protein oligomerization.

Neuronal stimulation of flies by dopamine or tyramine feeding induces orb2 oligomerization.

Source was adult heads of transgenic fly line; protein produced from tagged transgenic construct.

The glutamine-rich Q-domain is necessary for orb2 protein oligomerization.

Source was adult heads of transgenic fly line; bait produced from tagged transgenic construct; prey produced from untagged transgenic construct.

orb2 protein (Orb2A isoform) was immunoprecipitated by it\'s GFP tag, and it\'s ability to pull down the untagged Orb2B isoform (distinguished by its unique N-terminus) was detected by mass spectrometry.

orb2 protein (Orb2A isoform) was immunoprecipitated by it\'s GFP tag, and it\'s ability to pull down the untagged Orb2B isoform (distinguished by its unique N-terminus) was detected by mass spectrometry.

Source was adult heads of transgenic fly line; bait produced from tagged transgenic construct; prey produced from untagged transgenic construct.

The glutamine-rich Q-domain is necessary for orb2 protein oligomerization.

The short orb2 "A" isoform coimmunoprecipitates both "A" and "B" isoforms of orb2, as well as orb2 oligomers.

Source was adult heads of transgenic fly line; bait produced from tagged transgenic construct; prey produced from endogenous gene.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

orb2 forms large amyloid-like unbranched fibers in vitro, visible by electron microscopy. These aggregates have many characteristics of canonical amyloids, such as binding to thioflavin and Congo Red, and a secondary structure that goes from being helix-rich in soluble form to beta-sheet rich as aggregation occurs (as monitored by turbidity and spectroscopy). These oligomers are also recognized by the conformational A11 antibody and the amyloid fiber-specific OC antibody.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

The N-terminal domain of orb2 can bind Ni2+. In the absence of Ni2+, orb2 aggregates into fibrils while in the presence of Ni2+, it aggregates into non-fibril structures.

Source was live S2 cells; proteins produced from transfected constructs.

Positive control.

Interaction in vitro; protein produced as a recombinant fusion protein in bacterial system.

The orb2-A full length isoform and M9I domain spontaneously forms amyloid-like fibrils in vitro. The F5Y abd F8Y mutations result in shorter, wider fibrils.

The F5Y mutation results in poor alignment of fibrils.

Interaction in vitro; protein produced as a recombinant fusion proteins in bacterial system.

Interaction in vitro; protein produced as a recombinant fusion proteins in bacterial system.

The F5Y mutation results in poor alignment of fibrils.