Technical Note: The Use of RNA-interference as a Tool to Find Proteins Involved in Melanosome Formation or Transport

Melanosomes are lysosome-related organelles that produce and transport the pigment melanin within melanocytes. Mutations in proteins required for melanosome transport and formation lead to a range of pigmentation defects, manifested at the cellular level as perinuclear clustering of melanosomes, or reduced sorting of melanosomal cargo such as tyrosinase-related protein 1 (TYRP1). A pilot screen was carried out to investigate whether a combination of cellular imaging and RNA interference could be used to identify new proteins involved in pigmentation pathways. In this study, eleven genes known to play a role in melanosome transport/formation or other pigmentation properties were knocked down in mouse melanocytes with shRNAmir constructs. The investigated genes were TYRP1, pallidin, cappuccino, dysbindin, HPS5, LYST, Myosin Va, melanophilin, RhoA, UBPY and mahogunin. In a blinded confocal imaging experiment, the only reproducible change observed in cells in which these targets were knocked down was a decrease in TYRP1 levels upon transfection with knockdown constructs against TYRP1 itself, or one of three constructs targeting HPS5 (Hermansky-Pudlak Syndrome 5). Upon analysis with high-content imaging software, only the knockdown construct against TYRP1 itself was detected. RT-PCR analysis showed that many of the shRNAmir constructs did not reduce mRNA and proteins levels enough to detect effects on melanosome properties. This was further examined for melanophilin, a protein necessary for melanosome transport. Altogether, the data show that this system is currently not sensitive enough for use in a screen for unknown regulators of melanosome transport and formation. The main obstacle appears to be incomplete reduction of target protein levels. Our observation that a ~50% reduction in mRNA level is not sufficient to elicit an effect is supported by the fact that heterozygous carriers of melanosomal transport disorders (Griscelli Syndrome, Hermansky-Pudlak Syndrome) do not display diseases phenotypes. A further reduction in protein levels, for example by viral infection of shRNA, may be required

Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays

Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2–4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with ∼8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases

A Blog of Her Own: Scholarly Women on the Web

The women behind the popular blogs Bitch Ph.D., Tenured Radical, Easternblot.net, and Expression Patterns discuss the interplay between their blogging and scholarship, attitudes towards blogging among their colleagues, how blogging should be valued in the academy, and blogging as a feminist act