No abstract

SummaryLincomycin (LIN)‐mediated inhibition of protein synthesis in chloroplasts prevents the greening of seedlings, represses the activity of photosynthesis‐related genes in the nucleus, including LHCB1.2, and induces the phenylpropanoid pathway, resulting in the production of anthocyanins. In genomes uncoupled (gun) mutants, LHCB1.2 expression is maintained in the presence of LIN or other inhibitors of early chloroplast development. In a screen using concentrations of LIN lower than those employed to isolate gun mutants, we have identified happy on lincomycin (holi) mutants. Several holi mutants show an increased tolerance to LIN, exhibiting de‐repressed LHCB1.2 expression and chlorophyll synthesis in seedlings. The mutations responsible were identified by whole‐genome single‐nucleotide polymorphism (SNP) mapping, and most were found to affect the phenylpropanoid pathway; however, LHCB1.2 expression does not appear to be directly regulated by phenylpropanoids, as indicated by the metabolic profiling of mutants. The most potent holi mutant is defective in a subunit of cellulose synthase encoded by IRREGULAR XYLEM 3, and comparative analysis of this and other cell‐wall mutants establishes a link between secondary cell‐wall integrity and early chloroplast development, possibly involving altered ABA metabolism or sensing.

and intermediate filaments (IFs). While the roles of actin-based networks and MT in cell mechanics and cell function have been extensively studied (reviewed, e.g., in refs. [2, 3]), the contribution of IFs to cell mechanics is still relatively opaque, beyond their function in bearing large tensile forces. Of the cytoskeletal proteins, IF constituent proteins have the unique feature of being able to undergo molecular structural changes in response to external loads, at least in vitro, as shown by single-molecule experiments and molecular dynamics simulations. [4-7] This structural polymorphism observed outside of a cellular context has led to the hypothesis that IFs could play a role in intracellular mechanotransduction-relaying information about the cell's mechanical state into biochemical changes that influence cell shape [8-10] and phenotype. [11] IF proteins consist dimeric building blocks ≈45 nm in length that assemble into ≈60 nm antiparallel tetrameters (due to dimer overlap), which laterally associate into unit length filaments that assemble longitudinally to make apolar filaments. These filaments can further bundle into fibers and form networks. [7,12,13] Crystal structures show that IF protein are at least 67% α-helical. [14,15] Individual IFs self-assemble into quite diverse networks with location-specific architectures and composition in cells. [8] IFs proteins can form both ionic and hydrophobic molecular contacts resulting in multiple stabilizing interactions. In addition, proteins such as plectin help bundling and cross-bridging between IFs and to stress fibers and MT. [8,16] Vimentin is one IF protein that forms cytoplasmic IF networks and is particularly interesting because of its important role in cell adhesion, mechanical stability, [17-19] and cell migration, as well as intracellular signaling. [8,20] Specific demonstrations of laser ablation of super-stretched cells showed that the IF network is load bearing; however, this effect was not observed on relaxed cells grown on very soft substrates. [21] Moreover, vimentin has been established as a marker of the epithelial-to-mesenchymal transitions (EMT) in embryogenesis and in tumor metastasis, as the cytoplasmic IF network in epithelial cells mostly consists of keratin that is converted to a vimentin-rich IF network during EMT. [10,22-24] A complex multi-regime response of IF protein structure to deformation has been revealed via computational [7,25,26] and in vitro experimental [4,5] studies of IF protein mechanics.

Intermediate filament (IF) proteins are a class of proteins that constitute different filamentous structures in mammalian cells. As such, IF proteins are part of the load-bearing cytoskeleton and support the nuclear envelope. Molecular dynamics simulations have shown that IF proteins undergo secondary structural changes to compensate mechanical loads, which has been confirmed by experimental in vitro studies on IF hydrogels. However, the structural response of intracellular IF to mechanical load has yet to be elucidated in cellulo. Here, we use in situ nonlinear Raman imaging combined with multivariate data analysis to quantify the intracellular secondary structure of the IF cytoskeletal protein vimentin under different states of cellular tension. We find that cells under native cellular tension contain more unfolded vimentin than chemically or physically relaxed specimens. This indicates that unfolding of IF proteins occurs intracellularly when sufficient forces are applied, suggesting that IF structures act as local force sensors in the cell to mark locations under large mechanical tension.