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Expanding the palette of fluorescent dyes is vital to push the frontier of biological imaging. Although rhodamine dyes remain the premier type of small-molecule fluorophore owing to their bioavailability and brightness, variants excited with far-red or near-infrared light suffer from poor performance due to their propensity to adopt a lipophilic, nonfluorescent form. We report a framework for rationalizing rhodamine behavior in biological environments and a general chemical modification for rhodamines that optimizes long-wavelength variants and enables facile functionalization with different chemical groups. This strategy yields red-shifted ‘Janelia Fluor’ (JF) dyes useful for biological imaging experiments in cells and in vivo. A general tuning strategy is introduced for improving the utility of rhodamines for biological imaging applications. The strategy yielded bright, versatile and bioavailable far-red and near-infrared ‘Janelia Fluor’ dyes.

During starvation the transcriptional activation of catabolic processes is induced by the nuclear translocation and consequent activation of transcription factor EB (TFEB), a master modulator of autophagy and lysosomal biogenesis. However, how TFEB is inactivated upon nutrient refeeding is currently unknown. Here we show that TFEB subcellular localization is dynamically controlled by its continuous shuttling between the cytosol and the nucleus, with the nuclear export representing a limiting step. TFEB nuclear export is mediated by CRM1 and is modulated by nutrient availability via mTOR-dependent hierarchical multisite phosphorylation of serines S142 and S138, which are localized in proximity of a nuclear export signal (NES). Our data on TFEB nucleo-cytoplasmic shuttling suggest an unpredicted role of mTOR in nuclear export. On amino acid deprivation TFEB translocates from the cytoplasm to the nucleus. Here the authors identify a nuclear export signal in TFEB that is recognized by the exportin CRM1, and show that dual phosphorylation at S142 and S138 by mTOR accelerates export of TFEB.

The ability of naturally occurring proteins to change conformation in response to environmental changes is critical to biological function. Although there have been advances in the de novo design of stable proteins with a single, deep free-energy minimum, the design of conformational switches remains challenging. We present a general strategy to design pH-responsive protein conformational changes by precisely preorganizing histidine residues in buried hydrogen-bond networks. We design homotrimers and heterodimers that are stable above pH 6.5 but undergo cooperative, large-scale conformational changes when the pH is lowered and electrostatic and steric repulsion builds up as the network histidine residues become protonated. The transition pH and cooperativity can be controlled through the number of histidine-containing networks and the strength of the surrounding hydrophobic interactions. Upon disassembly, the designed proteins disrupt lipid membranes both in vitro and after being endocytosed in mammalian cells. Our results demonstrate that environmentally triggered conformational changes can now be programmed by de novo protein design.

Antibiotics target specific biochemical mechanisms in bacteria. In response to new drugs, pathogenic bacteria rapidly develop resistance. In contrast, antimicrobial peptides (AMPs) have retained broad spectrum antibacterial potency over millions of years. We present single-cell fluorescence assays that detect reactive oxygen species (ROS) in the Escherichia coli cytoplasm in real time. Within 30 s of permeabilization of the cytoplasmic membrane by the cationic AMP CM15 [combining residues 1–7 of cecropin A (from moth) with residues 2–9 of melittin (bee venom)], three fluorescence signals report oxidative stress in the cytoplasm, apparently involving O ₂⁻, H ₂O ₂, and •OH. Mechanistic studies indicate that active respiration is a prerequisite to the CM15-induced oxidative damage. In anaerobic conditions, signals from ROS are greatly diminished and the minimum inhibitory concentration increases 20-fold. Evidently the natural human AMP LL-37 also induces a burst of ROS. Oxidative stress may prove a significant bacteriostatic mechanism for a variety of cationic AMPs. If so, host organisms may use the local oxygen level to modulate AMP potency. Significance Antimicrobial peptides (AMPs) help to kill invading bacteria on skin and lung surfaces. We are developing fluorescence microscopy assays that reveal the mechanisms of action of AMPs in real time. It is increasingly clear AMP damage to bacterial cells goes far beyond permeabilization of membranes. Here we demonstrate that for Escherichia coli in aerobic conditions, the peptide CM15 [combining residues 1–7 of cecropin A (from moth) with residues 2–9 of melittin (bee venom)], induces a burst of biochemically harmful reactive oxygen species within 30 s of membrane permeabilization. In anaerobic conditions, CM15 is 20-fold less potent. AMP efficacy in vivo may be tuned to the local level of oxygenation.

Photoactivatable derivatives of the bright and photostable Janelia Fluor dyes enable improved multicolor single-particle tracking and facile localization microscopy in cells. Small-molecule fluorophores are important tools for advanced imaging experiments. We previously reported a general method to improve small, cell-permeable fluorophores which resulted in the azetidine-containing 'Janelia Fluor' (JF) dyes. Here, we refine and extend the utility of these dyes by synthesizing photoactivatable derivatives that are compatible with live-cell labeling strategies. Once activated, these derived compounds retain the superior brightness and photostability of the JF dyes, enabling improved single-particle tracking and facile localization microscopy experiments.

Antimicrobial peptides (AMPs) are thought to kill bacterial cells by permeabilizing their membranes. However, some antimicrobial peptides inhibit E. coli growth more efficiently in aerobic than in anaerobic conditions. In the attack of the human cathelicidin LL-37 on E. coli, real-time, single-cell fluorescence imaging reveals the timing of membrane permeabilization and the onset of oxidative stress. For cells growing aerobically, a CellROX Green assay indicates that LL-37 induces rapid formation of oxidative species after entry into the periplasm, but before permeabilization of the cytoplasmic membrane (CM). A cytoplasmic Amplex Red assay signals a subsequent burst of oxidative species, most likely hydrogen peroxide, shortly after permeabilization of the CM. These signals are much stronger in the presence of oxygen, a functional electron transport chain, and a large proton motive force (PMF). They are much weaker in cells growing anaerobically, by either fermentation or anaerobic respiration. In aerobic growth, the oxidative signals are attenuated in a cytochrome oxidase-bd deletion mutant, but not in a -bo3 deletion mutant, suggesting a specific effect of LL-37 on the electron transport chain. The AMPs melittin and LL-37 induce strong oxidative signals and exhibit O2-sensitive MICs, while the AMPs indolicidin and cecropin A do not. These results suggest that AMP activity in different tissues may be tuned according to the local oxygen level. This may be significant for control of opportunistic pathogens while enabling growth of commensal bacteria.

In vitro assays find that ribosomes form peptide bonds to proline (Pro) residues more slowly than to other residues. Ribosome profiling shows that stalling at Pro-Pro-X triplets is especially severe but is largely alleviated in Escherichia coli by the action of elongation factor EF-P. EF-P and its eukaryotic/archaeal homolog IF5A enhance the peptidyl transfer step of elongation. Here, a superresolution fluorescence localization and tracking study of EF-P–mEos2 in live E. coli provides the first in vivo information about the spatial distribution and on-off binding kinetics of EF-P. Fast imaging at 2 ms/frame helps to distinguish ribosome-bound (slowly diffusing) EF-P from free (rapidly diffusing) EF-P. Wild-type EF-P exhibits a three-peaked axial spatial distribution similar to that of ribosomes, indicating substantial binding. The mutant EF-P K34A exhibits a homogeneous distribution, indicating little or no binding. Some 30% of EF-P copies are bound to ribosomes at a given time. Two-state modeling and copy number estimates indicate that EF-P binds to 70S ribosomes during 25 to 100% of translation cycles. The timescale of the typical diffusive search by free EF-P for a ribosome-binding site is τ free ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τ bound ≈ 7 ms. Evidently, EF-P binds to ribosomes during many or most elongation cycles, much more often than the frequency of Pro-Pro motifs. Emptying of the E site during part of the cycle is consistent with recent in vitro experiments indicating dissociation of the deacylated tRNA upon translocation. IMPORTANCE Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle. Ribosomes translate the codon sequence within mRNA into the corresponding sequence of amino acids within the nascent polypeptide chain, which in turn ultimately folds into functional protein. At each codon, bacterial ribosomes are assisted by two well-known elongation factors: EF-Tu, which aids binding of the correct aminoacyl-tRNA to the ribosome, and EF-G, which promotes tRNA translocation after formation of the new peptide bond. A third factor, EF-P, has been shown to alleviate ribosomal pausing at rare Pro-Pro motifs, which are translated very slowly without EF-P. Here, we use superresolution fluorescence imaging to study the spatial distribution and ribosome-binding dynamics of EF-P in live E. coli cells. We were surprised to learn that EF-P binds to and unbinds from translating ribosomes during at least 25% of all elongation events; it may bind during every elongation cycle.

Minimum inhibitory concentrations (MICs) against E. coli were measured for three nylon-3 polymers using Luria-Bertani broth (LB), brain-heart infusion broth (BHI), and a chemically defined complete medium (EZRDM). The polymers differ in the ratio of hydrophobic to cationic subunits. The cationic homopolymer is inert against E. coli in BHI and LB, but becomes highly potent in EZRDM. A mixed hydrophobic/cationic polymer with a hydrophobic t-butylbenzoyl group at its N-terminus is effective in BHI, but becomes more effective in EZRDM. Supplementation of EZRDM with the tryptic digest of casein (often found in LB) recapitulates the LB and BHI behavior. Additional evidence suggests that polyanionic peptides present in LB and BHI may form electrostatic complexes with cationic polymers, decreasing activity by diminishing binding to the anionic lipopolysaccharide layer of E. coli. In contrast, two natural antimicrobial peptides show no medium effects. Thus, the use of a chemically defined medium helps to reveal factors that influence antimicrobial potency of cationic polymers and functional differences between these polymers and evolved antimicrobial peptides.

We present in this paper several efficient numerical schemes for the magneto-hydrodynamic (MHD) equations. These semi-discretized (in time) schemes are based on the standard and rotational pressure-correction schemes for the Navier-Stokes equations and do not involve a projection step for the magnetic field. We show that these schemes are unconditionally energy stable, present an effective algorithm for their fully discrete versions and carry out demonstrative numerical experiments.

Nanobubbles have been vigorously investigated in the last decade. However, the dynamics of hydrogen gas nanobubbles are yet to be fully understood due to the experimental limitations of high spatial and temporal resolutions. The novel imaging technology, in-situ TEM, facilitated the direct observation of dynamic nanobubble evolution at nanometer spatial resolution and millisecond temporal resolution, which enabled our investigation of nanobubble growths and interactions. In this thesis, the growth of individual hydrogen nanobubbles under electron beam radiolysis of water has been investigated using an in-situ TEM. The oversaturation of hydrogen gas in liquid water has been analyzed to understand the conditions that nanobubbles nucleated and grew, and a contact line pinning mechanism has been identified as one major mechanism in the diffusively-controlled growth of the single nanobubbles.Furthermore, both the Ostwald ripening and reverse-Ostwald ripening phenomenon were observed and analyzed. The results demonstrated the pressure difference between two neighboring nanobubbles played a critical role in creating a concentration gradient between the two differently sized nanobubbles, influencing the dynamics of neighboring nanobubbles.