Cross-link assisted spatial proteomics to map sub-organelle proteomes and membrane protein topologies.

The functions of cellular organelles and sub-compartments depend on their protein content, which can be characterized by spatial proteomics approaches. However, many spatial proteomics methods are limited in their ability to resolve organellar sub-compartments, profile multiple sub-compartments in parallel, and/or characterize membrane-associated proteomes. Here, we develop a cross-link assisted spatial proteomics (CLASP) strategy that addresses these shortcomings. Using human mitochondria as a model system, we show that CLASP can elucidate spatial proteomes of all mitochondrial sub-compartments and provide topological insight into the mitochondrial membrane proteome. Biochemical and imaging-based follow-up studies confirm that CLASP allows discovering mitochondria-associated proteins and revising previous protein sub-compartment localization and membrane topology data. We also validate the CLASP concept in synaptic vesicles, demonstrating its applicability to different sub-cellular compartments. This study extends the scope of cross-linking mass spectrometry beyond protein structure and interaction analysis towards spatial proteomics, and establishes a method for concomitant profiling of sub-organelle and membrane proteomes.

Reproductive isolation between Stigmaeopsis celarius and its sibling species sympatrically inhabiting bamboo (Pleioblastus spp.) plants.

Stigmaeopsis celarius Banks (hereafter Sc) is a spider mite living and feeding on the leaves of various bamboo species such as Moso bamboo [Phyllostachys edulis (=P. pubescens)] and Pleioblastus spp. (Poaceae). A previous phylogenetic study revealed a cryptic, phylogenetic sister species to Sc (hereafter Ss). Although its life type appears to be similar to that of Sc, individuals of Ss make much smaller nests compared with Sc, and the nests have been found mostly on Nezasa bamboo (Pleioblastus argenteostriatus). To investigate whether Sc and Ss are reproductively isolated, we explored their populations in southwestern Japan, and crossed them to examine mating behaviors and fertilization success. Field surveys revealed that the nests of these two species occur on the same leaves and, thus, the individuals of these species may make frequent contact. Reciprocal crosses suggested that the two species are reproductively isolated. Though Sc males have tried to mate with Ss females, copulation seldom occurred because of their long opisthosoma (hind body), which prevented the insertion of the aedeagus into the genitalia of Ss females. In contrast, most Ss males ignored Sc females, and eggs were not fertilized even in the few cases where copulation appeared to occur. These results suggest that strong selection pressure is imposed on body length to prevent interspecific hybridization in the contact area of these species.

Tailoring the supramolecular structure of aminated polyrotaxanes toward enhanced cellular internalization.

The effects of the supramolecular polyrotaxane (PRX) structure on cellular internalization are investigated by flow cytometry and confocal laser scanning microscopy. AF-545-labeled aminated PRXs (APRXs) containing different numbers of threaded α-cyclodextrins (CDs) and amino groups are synthesized; their cellular uptakes are analyzed using HeLa cells in serum. The APRX threaded CD number is discovered to be a more critical factor for enhancing cellular internalization than the APRX amine content. Additionally, APRXs are demonstrated to be more easily internalized than conventional linear cationic macromolecules. Because increased numbers of threaded CDs are related to increased PRX rigidity, the PRX rigid frame resulting from CD molecules threaded on a poly(ethylene glycol) (PEG) chain is suitable for intracellular tools in therapy and diagnosis.