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Results for “subcellular organization”

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NASA NTRS1997

Intracellular distributions and putative functions of calcium-binding proteins in the bullfrog vestibular otolith organs

Hair cells in the bullfrog vestibular otolith organs were immunolabeled by monoclonal and polyclonal antisera against calbindin (CaB), calmodulin (CaM), calretinin (CaR), and parvalbumin (PA). S-100, previously shown to immunolabel striolar hair cells in fish vestibular organs, only weakly immunolabeled hair cells in the bullfrog vestibular otolith organs. Immunolabeling was not detected in supporting cells. With the exception of CaR, myelinated axons and unmyelinated nerve terminals were immunolabeled by all of the above antisera. Immunolabeling was seen in all saccular hair cells, although hair cells at the macular margins were immunolabeled more intensely for CaB, CaM, and PA than more centrally located hair cells. As the macula margins are known to be a growth zone, this labeling pattern suggests that marginal hair cells up-regulate their calcium-binding proteins during hair cell development. In the utriculus, immunolabeling for CaM and PA was generally restricted to striolar hair cells. CaR immunolabeling was restricted to the stereociliary array. Immunolabeling for other calcium-binding proteins was generally seen in both the cell body and hair bundles of hair cells, although this labeling was often localized to the stereociliary array and the apical portion of the cell body. CaM and PA immunolabeling in the stereociliary array in saccular and utricular striolar cells suggests a functional role for these proteins in mechanoelectric transduction and adaptation.

NASA Discipline Number 40-10↗

DOE OSTI2021

Incorporation and Assembly of a Light-Emitting Enzymatic Reaction into Model Protein Condensates

Eukaryotic cells partition enzymes and other cellular components into distinct subcellular compartments to generate specialized biochemical niches. A subclass of these compartments form in the absence of lipid membranes, via liquid-liquid phase separation of proteins to form biomolecular condensates or “membraneless organelles” such as nucleoli, stress granules, and P-bodies. Because of their ability to form compartments from simple starting materials, membraneless organelles are an attractive target for engineering new functionalities in both living cells and protocells. In this work, we demonstrate incorporation of novel enzymatic activity in protein coacervates with a light-generating enzyme, NanoLuc, to produce bioluminescence. Using condensates comprised of the disordered RGG domain of C. elegans LAF-1, we show functionalization of condensates with enzymatic activity in vitro and that localization to protein coacervates enhances the assembly and activity of split enzymes. To build condensates that function as light emitting reactors, we designed a NanoLuc enzyme flanked by RGG domains. Furthermore, the resulting condensates concentrated NanoLuc by 10-fold over bulk solution and display significantly increased net reaction rates. We further show that condensate viscosity impacts light emission due to diffusion-limited behavior. By splitting NanoLuc enzyme into its constituent components, we demonstrate that NanoLuc activity can be reconstituted via co-condensation. Further, we demonstrate control of the spatial localization of enzyme within condensates by targettng NanoLuc to the surface of in vitro condensates. Collectively, this work demonstrates that membraneless organelles can be endowed with localized enzymatic activity, and that this activity can be spatially and temporally controlled via enzyme reconstitution and design of protein surfactants.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

NASA NTRS1999

Regiochemical control of monolignol radical coupling: a new paradigm for lignin and lignan biosynthesis

BACKGROUND: Although the lignins and lignans, both monolignol-derived coupling products, account for nearly 30% of the organic carbon circulating in the biosphere, the biosynthetic mechanism of their formation has been poorly understood. The prevailing view has been that lignins and lignans are produced by random free-radical polymerization and coupling, respectively. This view is challenged, mechanistically, by the recent discovery of dirigent proteins that precisely determine both the regiochemical and stereoselective outcome of monolignol radical coupling. RESULTS: To understand further the regulation and control of monolignol coupling, leading to both lignan and lignin formation, we sought to clone the first genes encoding dirigent proteins from several species. The encoding genes, described here, have no sequence homology with any other protein of known function. When expressed in a heterologous system, the recombinant protein was able to confer strict regiochemical and stereochemical control on monolignol free-radical coupling. The expression in plants of dirigent proteins and proposed dirigent protein arrays in developing xylem and in other lignified tissues indicates roles for these proteins in both lignan formation and lignification. CONCLUSIONS: The first understanding of regiochemical and stereochemical control of monolignol coupling in lignan biosynthesis has been established via the participation of a new class of dirigent proteins. Immunological studies have also implicated the involvement of potential corresponding arrays of dirigent protein sites in controlling lignin biopolymer assembly.

NASA Discipline Plant Biology↗