A light-inducible organelle-targeting system for dynamically activating and inactivating signaling in budding yeast

Protein localization plays a central role in cell biology. Although powerful tools exist to assay the spatial and temporal dynamics of proteins in living cells, our ability to control these dynamics has been much more limited. We previously used the phytochrome B– phytochrome-interacting factor light-gated dimerization system to recruit proteins to the plasma membrane, enabling us to control the activation of intracellular signals in mammalian cells. Here we extend this approach to achieve rapid, reversible, and titratable control of protein localization for eight different organelles/positions in budding yeast. By tagging genes at the endogenous locus, we can recruit proteins to or away from their normal sites of action. This system provides a general strategy for dynamically activating or inactivating proteins of interest by controlling their localization and therefore their availability to binding partners and substrates, as we demonstrate for galactose signaling. More importantly, the temporal and spatial precision of the system make it possible to identify when and where a given protein''s activity is necessary for function, as we demonstrate for the mitotic cyclin Clb2 in nuclear fission and spindle stabilization. Our light-inducible organelle-targeting system represents a powerful approach for achieving a better understanding of complex biological systems.     

Direct Sensing and Discrimination among Ubiquitin and Ubiquitin Chains Using Solid-State Nanopores

Nanopore sensing involves an electrophoretic transport of analytes through a nanoscale pore, permitting label-free sensing at the single-molecule level. However, to date, the detection of individual small proteins has been challenging, primarily due to the poor signal/noise ratio that these molecules produce during passage through the pore. Here, we show that fine adjustment of the buffer pH, close to the isoelectric point, can be used to slow down the translocation speed of the analytes, hence permitting sensing and characterization of small globular proteins. Ubiquitin (Ub) is a small protein of 8.5 kDa, which is well conserved in all eukaryotes. Ub conjugates to proteins as a posttranslational modification called ubiquitination. The immense diversity of Ub substrates, as well as the complexity of Ub modification types and the numerous physiological consequences of these modifications, make Ub and Ub chains an interesting and challenging subject of study. The ability to detect Ub and to identify Ub linkage type at the single-molecule level may provide a novel tool for investigation in the Ub field. This is especially adequate because, for most ubiquitinated substrates, Ub modifies only a few molecules in the cell at a given time. Applying our method to the detection of mono- and poly-Ub molecules, we show that we can analyze their characteristics using nanopores. Of particular importance is that two Ub dimers that are equal in molecular weight but differ in 3D structure due to their different linkage types can be readily discriminated. Thus, to our knowledge, our method offers a novel approach for analyzing proteins in unprecedented detail using solid-state nanopores. Specifically, it provides the basis for development of single-molecule sensing of differently ubiquitinated substrates with different biological significance. Finally, our study serves as a proof of concept for approaching nanopore detection of sub-10-kDa proteins and demonstrates the ability of this method to differentiate among native and untethered proteins of the same mass.     

The giant cells produced by Telenomus remus (Hymenoptera: Scelionidae)

The parasitic larva of Telenomus remus is surrounded by giant cells throughout its first instar. These cells arise in the embryonic serosa of the parasite and grow in size, starting with a radius of 5nm and ending with 27nm. Young cells are round and mononuclear, whereas older ones are often polynuclear and have varied, irregular contours. Most cells are profusely vacuolated, the vacuoles being especially large in some of the older cells. Only a few of the giant cells are devoured by the first instar parasite larva, but all disappear at the end of this stage. No giant cells seem to be produced by supernumerary larvae. Once the parasite egg hatches, the host tissue disintegrates almost instantaneously.