Defining specific protein interactions and spatially or temporally restricted local proteomes improves our understanding of all cellular processes, but obtaining such data is challenging, especially for rare proteins, cell types, or events. Proximity labeling enables discovery of protein neighborhoods defining functional complexes and/or organellar protein compositions. Recent technological improvements, namely two highly active biotin ligase variants (TurboID and miniTurbo), allowed us to address two challenging questions in plants: (1) what are in vivo partners of a low abundant key developmental transcription factor and (2) what is the nuclear proteome of a rare cell type? Proteins identified with FAMA-TurboID include known interactors of this stomatal transcription factor and novel proteins that could facilitate its activator and repressor functions. Directing TurboID to stomatal nuclei enabled purification of cell type- and subcellular compartment-specific proteins. Broad tests of TurboID and miniTurbo in Arabidopsis and Nicotiana benthamiana and versatile vectors enable customization by plant researchers.

Short linear motifs (SLiMs) drive dynamic protein-protein interactions essential for signaling, but sequence degeneracy and low binding affinities make them difficult to identify. We harnessed unbiased systematic approaches for SLIM discovery to elucidate the regulatory network of calcineurin (CN)/PP2B, the Ca2+-activated phosphatase that recognizes LxVP and PxlxIT motifs. In vitro proteome-wide detection of CN-binding peptides, in vivo SLiM-dependent proximity labeling, and in silico modeling of motif determinants uncovered unanticipated CN interactors, including NOTCH1, which we establish as a CN substrate. Unexpectedly, CN shows SLiM-dependent proximity to centrosomal and nuclear pore complex (NPC) proteins-structures where Ca2+ signaling is largely uncharacterized. CN dephosphorylates human and yeast NPC proteins and promotes accumulation of a nuclear transport reporter, suggesting conserved NPC regulation by CN. The CN network assembled here provides a resource to investigate Ca2+ and CN signaling and demonstrates synergy between experimental and computational methods, establishing a blueprint for examining SLiM-based networks.

Accurate relative quantification is critical in proteomic studies. The incorporation of stable isotope N-15 to plant-expressed proteins in vivo is a powerful tool for accurate quantification with a major advantage of reducing preparative and analytical variabilities. However, N-15 labeling quantification has several challenges. Less identifications are often observed in the heavy-labeled samples because of incomplete labeling, resulting in missing values in reciprocal labeling experiments. Inaccurate quantification can happen when there is contamination from co-eluting peptides or chemical noise in the MS1 survey scan. These drawbacks in quantification can be more pronounced in less abundant but biologically interesting proteins, which often have very few identified peptides. Here, we demonstrate the application of parallel reaction monitoring (PRM) to N-15 labeled samples on a high resolution, high mass accuracy Orbitrap mass spectrometer to achieve reliable quantification even of low abundance proteins in samples.

Interactions between physical forces and membrane proteins underpin many forms of environmental sensation and acclimation. Microbes survive osmotic stresses with the help of mechanically gated ion channels and osmolyte transporters. Plant mechanosensitive ion channels have been shown to function in defense signaling. Here, we engineered genetically encoded osmolality sensors (OzTracs) by fusing fluorescent protein spectral variants to the mechanosensitive ion channels MscL from E. coli or MSL10 from A. thaliana. When expressed in yeast cells, the OzTrac sensors reported osmolality changes as a proportional change in the emission ratio of the two fluorescent protein domains. Live-cell imaging revealed an accumulation of fluorescent sensors in internal aggregates, presumably derived from the endomembrane system. Thus, OzTrac sensors serve as osmolality-dependent reporters through an indirect mechanism, such as effects on molecular crowding or fluorophore solvation.

In symbioses established through horizontal transmission, evolution has selected for mechanisms that promote the recruitment of symbionts from the environment. Using the binary association between the Hawaiian bobtail squid, Euprymna scolopes, and its symbiont, Vibrio fischeri, we explored the first step of symbiont enrichment around sites where V. fischeri cells will enter host tissues. Earlier studies of the system had shown that, within minutes of hatching in natural seawater, ciliated epithelia of the nascent symbiotic tissue secrete a layer of mucus in response to exposure to the cell-wall biomolecule peptidoglycan (PGN) from non-specific bacterioplankton. We hypothesized that a peptidoglycan recognition protein, EsPGRP4, is the receptor that mediates host mucus secretion by sensing the environmental PGN; earlier studies of this protein family had shown that this is the only member predicted to behave as a membrane receptor. Immunocytochemistry localized EsPGRP4 to the superficial ciliated fields of the juvenile organ. We found that production of EsPGRP4 increased over the first 48 h after hatching if the light organ remained uncolonized. When colonized by V. fischeri, the levels of the protein in light-organ tissue remained similar to that of hatchling organs. Pharmacologically curing the initially colonized light organ with antibiotics resulted in return of EsPGRP4 production to levels similar to light organs that had remained uncolonized since hatching. Furthermore, we found that preincubation of the tissues with an EsPGRP4 antibody decreased light organ mucus production and colonization. These findings provide evidence of an innate mechanism that underlies a crucial first step in the horizontal recruitment of bacterial symbionts.

Sewage released from lakeside development can reshape ecological communities. Nearshore periphyton can rapidly assimilate sewage-associated nutrients, leading to increases of filamentous algal abundance, thus altering both food abundance and quality for grazers. In Lake Baikal, a large, ultra-oligotrophic, remote lake in Siberia, filamentous algal abundance has increased near lakeside developments, and localized sewage input is the suspected cause. These shifts are of particular interest in Lake Baikal, where endemic littoral biodiversity is high, lakeside settlements are mostly small, tourism is relatively high (similar to 1.2 million visitors annually), and settlements are separated by large tracts of undisturbed shoreline, enabling investigation of heterogeneity and gradients of disturbance. We surveyed sites along 40 km of Baikal's southwestern shore for sewage indicators-pharmaceuticals and personal care products (PPCPs) and microplastics-as well as periphyton and macroinvertebrate abundance and indicators of food web structure (stable isotopes and fatty acids). Summed PPCP concentrations were spatially related to lakeside development. As predicted, lakeside development was associated with more filamentous algae and lower abundance of sewage-sensitive mollusks. Periphyton and macroinvertebrate stable isotopes and essential fatty acids suggested that food web structure otherwise remained similar across sites; yet, the invariance of amphipod fatty acid composition, relative to periphyton, suggested that grazers adjust behavior or metabolism to compensate for different periphyton assemblages. Our results demonstrate that even low levels of human disturbance can result in spatial heterogeneity of nearshore ecological responses, with potential for changing trophic interactions that propagate through the food web.

We discuss the field retermination of high-fiber count MTP fiber connectors used with the APOGEE spectrograph at Apache Point Observatory (APO) in 2021. We address lessons-learned, wear-analysis of removed MTPs, and throughput of the fiber train with the newly terminated fibers in SDSS-V. For the past decade the spectrograph at APO, as part of multiple incarnations of the Sloan Digital Sky Survey (SDSS), has relied upon rapid changes of ten MTP connectors, each containing 30 terminated fibers, and all contained within a custom gang connector system. These rapid changes enable the iterative plugging of the gang connector into multiple cartridges with different plug plates to observe various survey fields throughout the night. While robotic Focal Plane Systems have been developed for SDSS-V to replace plug plates, which will minimize the fiber connector cycles, we nonetheless reterminated the most heavily used MTP connectors. The connector cycles had far exceeded manufacturer lifetimes and the overall system throughput was degrading.

The GMT-Consortium Large Earth Finder (G-CLEF) is a fiber-fed, optical echelle spectrograph that will be a first light instrument for the Giant Magellan Telescope (GMT). G-CLEF is a general-purpose echelle spectrograph with precision radial velocity (PRV) capability. The radial velocity (RV) precision goal of G-CLEF is 10 cm/sec; necessary for detection of Earth-sized exoplanets orbiting Solar-type stars in their habitable zone. This imposes challenging stability requirements on the optical mounts and spectrograph support structures especially when considering the instrument's operational environment. G-CLEF's accuracy will be influenced by thermal effects, ambient air pressure, vibration, and micro gravity-vector variations caused by normal telescope slewing.