RNA Granules Hitchhike on Lysosomes for Long-Distance Transport, Using Annexin A11 as a Molecular Tether.

Long-distance RNA transport enables local protein synthesis at metabolically-active sites distant from the nucleus. This process ensures an appropriate spatial organization of proteins, vital to polarized cells such as neurons. Here, we present a mechanism for RNA transport in which RNA granules "hitchhike" on moving lysosomes. In vitro biophysical modeling, live-cell microscopy, and unbiased proximity labeling proteomics reveal that annexin A11 (ANXA11), an RNA granule-associated phosphoinositide-binding protein, acts as a molecular tether between RNA granules and lysosomes. ANXA11 possesses an N-terminal low complexity domain, facilitating its phase separation into membraneless RNA granules, and a C-terminal membrane binding domain, enabling interactions with lysosomes. RNA granule transport requires ANXA11, and amyotrophic lateral sclerosis (ALS)-associated mutations in ANXA11 impair RNA granule transport by disrupting their interactions with lysosomes. Thus, ANXA11 mediates neuronal RNA transport by tethering RNA granules to actively-transported lysosomes, performing a critical cellular function that is disrupted in ALS.

A general method to optimize and functionalize red-shifted rhodamine dyes.

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.

Bright photoactivatable fluorophores for single-molecule imaging.

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.

Oxidative stress induced in E. coli by the human antimicrobial peptide LL-37.

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.

Melittin-Induced Permeabilization, Re-sealing, and Re-permeabilization of E. coli Membranes.

The permeabilization of model lipid bilayers by cationic peptides has been studied extensively over decades, with the bee-sting toxin melittin perhaps serving as the canonical example. However, the relevance of these studies to the permeabilization of real bacterial membranes by antimicrobial peptides remains uncertain. Here, we employ single-cell fluorescence microscopy in a detailed study of the interactions of melittin with the outer membrane (OM) and the cytoplasmic membrane (CM) of live Escherichia coli. Using periplasmic green fluorescent protein (GFP) as a probe, we find that melittin at twice the minimum inhibitory concentration first induces abrupt cell shrinkage and permeabilization of the OM to GFP. Within ∼4 s of OM permeabilization, the CM invaginates to form inward facing "periplasmic bubbles." Seconds later the bubbles begin to leak periplasmic GFP into the cytoplasm. Permeabilization is localized, consistent with possible formation of toroidal pores. Within ∼20 s, first the OM and then the CM re-seals to GFP. Some 2-20 min later, both CM and OM are re-permeabilized to GFP. We invoke a mechanism based on curvature stress concepts derived from model bilayer studies. The permeabilization and re-sealing events involve sequential, time-dependent build-up of melittin density within the outer and inner leaflets of each bilayer. We also propose a mechanical explanation for the early cell shrinkage event induced by melittin and a variety of other cationic peptides. As peptides gain access to the periplasm, they bind to the anionic peptido-crosslinks of the lipopolysaccharide layer, increasing its longitudinal elastic modulus. The cell wall shrinks because it can withstand the same turgor pressure with smaller overall extension. Shrinkage in turn induces invagination of the CM, preserving its surface area. We conclude by comparing the behavior of different peptides.

Single-cell, real-time detection of oxidative stress induced in Escherichia coli by the antimicrobial peptide CM15.

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 O2 (-), H2O2, 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.

Time-dependent effects of transcription- and translation-halting drugs on the spatial distributions of the Escherichia coli chromosome and ribosomes.

Previously observed effects of rifampicin and chloramphenicol indicate that transcription and translation activity strongly affect the coarse spatial organization of the bacterial cytoplasm. Single-cell, time-resolved, quantitative imaging of chromosome and ribosome spatial distributions and ribosome diffusion in live Escherichia coli provides insight into the underlying mechanisms. Monte Carlo simulations of model DNA-ribosome mixtures support a novel nucleoid-ribosome mixing hypothesis. In normal conditions, 70S-polysomes and the chromosomal DNA segregate, while 30S and 50S ribosomal subunits are able to penetrate the nucleoids. Growth conditions and drug treatments determine the partitioning of ribosomes into 70S-polysomes versus free 30S and 50S subunits. Entropic and excluded volume effects then dictate the resulting chromosome and ribosome spatial distributions. Direct observation of radial contraction of the nucleoids 0-5 min after treatment with either transcription- or translation-halting drugs supports the hypothesis that simultaneous transcription, translation, and insertion of proteins into the membrane ('transertion') exerts an expanding force on the chromosomal DNA. Breaking of the DNA-RNA polymerase-mRNA-ribosome-membrane chain in either of two ways causes similar nucleoid contraction on a similar timescale. We suggest that chromosomal expansion due to transertion enables co-transcriptional translation throughout the nucleoids.

Nonperturbative imaging of nucleoid morphology in live bacterial cells during an antimicrobial peptide attack.

Studies of time-dependent drug and environmental effects on single, live bacterial cells would benefit significantly from a permeable, nonperturbative, long-lived fluorescent stain specific to the nucleoids (chromosomal DNA). The ideal stain would not affect cell growth rate or nucleoid morphology and dynamics, even during laser illumination for hundreds of camera frames. In this study, time-dependent, single-cell fluorescence imaging with laser excitation and a sensitive electron-multiplying charge-coupled-device (EMCCD) camera critically tested the utility of "dead-cell stains" (SYTOX orange and SYTOX green) and "live-cell stains" (DRAQ5 and SYTO 61) and also 4',6-diamidino-2-phenylindole (DAPI). Surprisingly, the dead-cell stains were nearly ideal for imaging live Escherichia coli, while the live-cell stains and DAPI caused nucleoid expansion and, in some cases, cell permeabilization and the halting of growth. SYTOX orange performed well for both the Gram-negative E. coli and the Gram-positive Bacillus subtilis. In an initial application, we used two-color fluorescence imaging to show that the antimicrobial peptide cecropin A destroyed nucleoid-ribosome segregation over 20 min after permeabilization of the E. coli cytoplasmic membrane, reminiscent of the long-term effects of the drug rifampin. In contrast, the human cathelicidin LL-37, while similar to cecropin A in structure, length, charge, and the ability to permeabilize bacterial membranes, had no observable effect on nucleoid-ribosome segregation. Possible underlying causes are suggested.

COPII with ALG2 and ESCRTs control lysosome-dependent microautophagy of ER exit sites.

Endoplasmic reticulum exit sites (ERESs) are tubular outgrowths of endoplasmic reticulum that serve as the earliest station for protein sorting and export into the secretory pathway. How these structures respond to different cellular conditions remains unclear. Here, we report that ERESs undergo lysosome-dependent microautophagy when Ca2+ is released by lysosomes in response to nutrient stressors such as mTOR inhibition or amino acid starvation in mammalian cells. Targeting and uptake of ERESs into lysosomes were observed by super-resolution live-cell imaging and focus ion beam scanning electron microscopy (FIB-SEM). The mechanism was ESCRT dependent and required ubiquitinated SEC31, ALG2, and ALIX, with a knockout of ALG2 or function-blocking mutations of ALIX preventing engulfment of ERESs by lysosomes. In vitro, reconstitution of the pathway was possible using lysosomal lipid-mimicking giant unilamellar vesicles and purified recombinant components. Together, these findings demonstrate a pathway of lysosome-dependent ERES microautophagy mediated by COPII, ALG2, and ESCRTS induced by nutrient stress.

Partitioning of RNA polymerase activity in live Escherichia coli from analysis of single-molecule diffusive trajectories.

Superresolution fluorescence microscopy is used to locate single copies of RNA polymerase (RNAP) in live Escherichia coli and track their diffusive motion. On a timescale of 0.1-1 s, most copies separate remarkably cleanly into two diffusive states. The "slow" RNAPs, which move indistinguishably from DNA loci, are assigned to specifically bound copies (with fractional population ftrxn) that are initiating transcription, elongating, pausing, or awaiting termination. The "mixed-state" RNAP copies, with effective diffusion constant Dmixed = 0.21 µm(2) s(-1), are assigned as a rapidly exchanging mixture of nonspecifically bound copies (fns) and copies undergoing free, three-dimensional diffusion within the nucleoids (ffree). Longer trajectories of 7-s duration reveal transitions between the slow and mixed states, corroborating the assignments. Short trajectories of 20-ms duration enable direct observation of the freely diffusing RNAP copies, yielding Dfree = 0.7 µm(2) s(-1). Analysis of single-particle trajectories provides quantitative estimates of the partitioning of RNAP into different states of activity: ftrxn = 0.54 ± 0.07, fns = 0.28 ± 0.05, ffree = 0.12 ± 0.03, and fnb = 0.06 ± 0.05 (fraction unable to bind to DNA on a 1-s timescale). These fractions disagree with earlier estimates.

Phosphorylation alters FMRP granules and determines their transport or protein synthesis abilities.

Fragile X messenger ribonucleoprotein (FMRP) is an RNA-binding protein implicated in autism that suppresses translation and forms granules. While FMRP function has been well-studied, how phosphorylation regulates granule binding and function remains limited. Here, we found that Fragile X patient-derived I304N mutant FMRP could not stably bind granules, underscoring the essential nature of FMRP granule association for function. Next, phosphorylation on serine 499 (S499) led to differences in puncta size, intensity, contrast, and transport as shown by phospho-deficient (S499A) and phospho-mimic (S499D) mutant FMRP granules. Additionally, S499D exchanged slowly on granules relative to S499A, suggesting that phosphorylated FMRP can attenuate translation. Furthermore, the S499A mutant enhanced translation in presynaptic boutons of the mouse hippocampus. Thus, the phospho-state of FMRP altered the structure of individual granules with changes in transport and translation to achieve spatiotemporal regulation of local protein synthesis. Teaser: The phosphorylation-state of S499 on FMRP can change FMRP granule structure and function to facilitate processive transport or local protein synthesis.

Lysosomal release of amino acids at ER three-way junctions regulates transmembrane and secretory protein mRNA translation.

One-third of the mammalian proteome is comprised of transmembrane and secretory proteins that are synthesized on endoplasmic reticulum (ER). Here, we investigate the spatial distribution and regulation of mRNAs encoding these membrane and secretory proteins (termed "secretome" mRNAs) through live cell, single molecule tracking to directly monitor the position and translation states of secretome mRNAs on ER and their relationship to other organelles. Notably, translation of secretome mRNAs occurred preferentially near lysosomes on ER marked by the ER junction-associated protein, Lunapark. Knockdown of Lunapark reduced the extent of secretome mRNA translation without affecting translation of other mRNAs. Less secretome mRNA translation also occurred when lysosome function was perturbed by raising lysosomal pH or inhibiting lysosomal proteases. Secretome mRNA translation near lysosomes was enhanced during amino acid deprivation. Addition of the integrated stress response inhibitor, ISRIB, reversed the translation inhibition seen in Lunapark knockdown cells, implying an eIF2 dependency. Altogether, these findings uncover a novel coordination between ER and lysosomes, in which local release of amino acids and other factors from ER-associated lysosomes patterns and regulates translation of mRNAs encoding secretory and membrane proteins.

Quartz crystal microbalance with dissipation monitoring and surface plasmon resonance studies of carboxymethyl cellulose adsorption onto regenerated cellulose surfaces.

Adsorption of anionic polyelectrolytes, sodium salts of carboxymethyl celluloses (CMCs) with different degrees of substitution (DS = 0.9 and 1.2), from aqueous electrolyte solutions onto regenerated cellulose surfaces was studied using quartz crystal microbalance with dissipation monitoring (QCM-D) and surface plasmon resonance (SPR) experiments. The influence of both calcium chloride (CaCl(2)) and sodium chloride (NaCl) on CMC adsorption was examined. The QCM-D results demonstrated that CaCl(2) (divalent cation) caused significantly greater CMC adsorption onto regenerated cellulose surfaces than NaCl (monovalent cation) at the same ionic strength. The CMC layers adsorbed onto regenerated cellulose surfaces from CaCl(2) solutions exhibited greater stability upon exposure to flowing water than layers adsorbed from NaCl solutions. Both QCM-D and SPR results showed that CMC adsorption onto regenerated cellulose surfaces from CaCl(2) solutions increased with increasing CaCl(2) concentration up to the solubility limit (10 mM). Voigt-based viscoelastic modeling of the QCM-D data indicated that the CMC layers adsorbed onto regenerated cellulose surfaces had shear viscosities of η(f) ≈ 10(-3) N·s·m(-2) and elastic shear moduli of µ(f) ≈ 10(5) N·m(-2). Furthermore, the combination of SPR spectroscopy and QCM-D showed that the CMC layers contained 90-95% water. Adsorption isotherms for CMCs in CaCl(2) solutions were also obtained from QCM-D and were fit by Freundlich isotherms. This study demonstrated that CMC adsorption from CaCl(2) solutions is useful for the modification of cellulose surfaces.

FLIM Imaging for Metabolic Studies in Live Cells.

Fluorescent biochemical sensors allow probing metabolic states in a living cell with high spatiotemporal dynamics. This chapter describes a method for the in situ detection of changes in NAD+ level in living cells using fluorescence lifetime imaging (FLIM).

A General Method to Improve Fluorophores Using Deuterated Auxochromes.

Fluorescence microscopy relies on dyes that absorb and then emit photons. In addition to fluorescence, fluorophores can undergo photochemical processes that decrease quantum yield or result in spectral shifts and irreversible photobleaching. Chemical strategies that suppress these undesirable pathways-thereby increasing the brightness and photostability of fluorophores-are crucial for advancing the frontier of bioimaging. Here, we describe a general method to improve small-molecule fluorophores by incorporating deuterium into the alkylamino auxochromes of rhodamines and other dyes. This strategy increases fluorescence quantum yield, inhibits photochemically induced spectral shifts, and slows irreparable photobleaching, yielding next-generation labels with improved performance in cellular imaging experiments.

De novo design of tunable, pH-driven conformational changes.

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.

Single-Cell, Time-Lapse Reactive Oxygen Species Detection in E. coli.

Detection of reactive oxygen species (ROS) in bacteria has been limited to bulk biochemical assays. Although they are powerful and quantitative tools to understand the overall production of ROS in E. coli, such assays provide limited spatial and temporal information when correlating cellular phenotype with perturbations such as antibiotics or other treatments. We have developed single-cell, time-lapse assays to detect ROS in live E. coli. The assays utilize flow systems on a fluorescence microscope to correlate symptoms aroused from biological or chemical perturbations with the in situ detection of ROS. ROS is detected by fluorogenic dyes that accumulate inside the cell, allowing detection of ROS in single cells in both homogeneous and heterogeneous samples using CellROX Green and Amplex® Red/APEX2. © 2018 by John Wiley & Sons, Inc.

mTOR-dependent phosphorylation controls TFEB nuclear export.

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.

Spatial Distribution and Ribosome-Binding Dynamics of EF-P in Live Escherichia coli.

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-PK34A 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 τfree ≈ 16 ms. The typical residence time of an EF-P on the ribosome is very short, τbound ≈ 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.

Lights, Camera, Action! Antimicrobial Peptide Mechanisms Imaged in Space and Time.

Deeper understanding of the bacteriostatic and bactericidal mechanisms of antimicrobial peptides (AMPs) should help in the design of new antibacterial agents. Over several decades, a variety of biochemical assays have been applied to bulk bacterial cultures. While some of these bulk assays provide time resolution of the order of 1min, they do not capture faster mechanistic events. Nor can they provide subcellular spatial information or discern cell-to-cell heterogeneity within the bacterial population. Single-cell, time-resolved imaging assays bring a completely new spatiotemporal dimension to AMP mechanistic studies. We review recent work that provides new insights into the timing, sequence, and spatial distribution of AMP-induced effects on bacterial cells.