RESUMO
Single-molecule cut-and-paste facilitates bottom-up directed assembly of nanoscale biomolecular networks in defined geometries and enables analysis with spatio-temporal resolution. However, arrangement of diverse molecules of interest requires versatile handling systems. The novel DNA-free, genetically encodable scheme described here utilises an orthogonal handling strategy to promote arrangement of enzymes and enzyme networks.
Assuntos
Enzimas Imobilizadas/química , Nanoestruturas/química , Nanotecnologia/métodos , Enzimas Imobilizadas/metabolismo , Corantes Fluorescentes , Proteínas Imobilizadas/química , Proteínas Imobilizadas/metabolismo , Microscopia de Força Atômica , Microscopia de Fluorescência , Modelos Moleculares , Nanoestruturas/ultraestruturaRESUMO
The human disease Hermansky-Pudlak syndrome results from defective biogenesis of lysosome-related organelles (LROs) and can be caused by mutations in subunits of the BLOC-1 complex. Here we show that C. elegans glo-2 and snpn-1, despite relatively low levels of amino acid identity, encode Pallidin and Snapin BLOC-1 subunit homologues, respectively. BLOC-1 subunit interactions involving Pallidin and Snapin were conserved for GLO-2 and SNPN-1. Mutations in glo-2 and snpn-1,or RNAi targeting 5 other BLOC-1 subunit homologues in a genetic background sensitized for glo-2 function, led to defects in the biogenesis of lysosome-related gut granules. These results indicate that the BLOC-1 complex is conserved in C. elegans. To address the function of C. elegans BLOC-1, we assessed the intracellular sorting of CDF-2::GFP, LMP-1, and PGP-2 to gut granules. We validated their utility by analyzing their mislocalization in intestinal cells lacking the function of AP-3, which participates in an evolutionarily conserved sorting pathway to LROs. BLOC-1(-) intestinal cells missorted gut granule cargo to the plasma membrane and conventional lysosomes and did not have obviously altered function or morphology of organelles composing the conventional lysosome protein sorting pathway. Double mutant analysis and comparison of AP-3(-) and BLOC-1(-) phenotypes revealed that BLOC-1 has some functions independent of the AP-3 adaptor complex in trafficking to gut granules. We discuss similarities and differences of BLOC-1 activity in the biogenesis of gut granules as compared to mammalian melanosomes, where BLOC-1 has been most extensively studied for its role in sorting to LROs. Our work opens up the opportunity to address the function of this poorly understood complex in cell and organismal physiology using the genetic approaches available in C. elegans.
