Rv0547c, a functional oxidoreductase, supports Mycobacterium tuberculosis persistence by reprogramming host mitochondrial fatty acid metabolism.

Mycobacterium tuberculosis (Mtb) successfully thrives in the host by adjusting its metabolism and manipulating the host environment. In this study, we investigated the role of Rv0547c, a protein that carries mitochondria-targeting sequence (MTS), in mycobacterial persistence. We show that Rv0547c is a functional oxidoreductase that targets host-cell mitochondria. Interestingly, the localization of Rv0547c to mitochondria was independent of the predicted MTS but depended on specific arginine residues at the N- and C-terminals. As compared to the mitochondria-localization defective mutant, Rv0547c-2SDM, wild-type Rv0547c increased mitochondrial membrane fluidity and spare respiratory capacity. To comprehend the possible reason, comparative lipidomics was performed that revealed a reduced variability of long-chain and very long-chain fatty acids as well as altered levels of phosphatidylcholine and phosphatidylinositol class of lipids upon expression of Rv0547c, explaining the increased membrane fluidity. Additionally, the over representation of propionate metabolism and ß-oxidation intermediates in Rv0547c-targeted mitochondrial fractions indicated altered fatty acid metabolism, which corroborated with changes in oxygen consumption rate (OCR) upon etomoxir treatment in HEK293T cells transiently expressing Rv0547c, resulting in enhanced mitochondrial fatty acid oxidation capacity. Furthermore, Mycobacterium smegmatis over expressing Rv0547c showed increased persistence during infection of THP-1 macrophages, which correlated with its increased expression in Mtb during oxidative and nutrient starvation stresses. This study identified for the first time an Mtb protein that alters mitochondrial metabolism and aids in survival in host macrophages by altering fatty acid metabolism to its benefit and, at the same time increases mitochondrial spare respiratory capacity to mitigate infection stresses and maintain cell viability.

A molecular rotor FLIM probe reveals dynamic coupling between mitochondrial inner membrane fluidity and cellular respiration.

The inner mitochondrial membrane (IMM), housing components of the electron transport chain (ETC), is the site for respiration. The ETC relies on mobile carriers; therefore, it has long been argued that the fluidity of the densely packed IMM can potentially influence ETC flux and cell physiology. However, it is unclear if cells temporally modulate IMM fluidity upon metabolic or other stimulation. Using a photostable, red-shifted, cell-permeable molecular-rotor, Mitorotor-1, we present a multiplexed approach for quantitatively mapping IMM fluidity in living cells. This reveals IMM fluidity to be linked to cellular-respiration and responsive to stimuli. Multiple approaches combining in vitro experiments and live-cell fluorescence (FLIM) lifetime imaging microscopy (FLIM) show Mitorotor-1 to robustly report IMM 'microviscosity'/fluidity through changes in molecular free volume. Interestingly, external osmotic stimuli cause controlled swelling/compaction of mitochondria, thereby revealing a graded Mitorotor-1 response to IMM microviscosity. Lateral diffusion measurements of IMM correlate with microviscosity reported via Mitorotor-1 FLIM-lifetime, showing convergence of independent approaches for measuring IMM local-order. Mitorotor-1 FLIM reveals mitochondrial heterogeneity in IMM fluidity; between-and-within cells and across single mitochondrion. Multiplexed FLIM lifetime imaging of Mitorotor-1 and NADH autofluorescence reveals that IMM fluidity positively correlates with respiration, across individual cells. Remarkably, we find that stimulating respiration, through nutrient deprivation or chemically, also leads to increase in IMM fluidity. These data suggest that modulating IMM fluidity supports enhanced respiratory flux. Our study presents a robust method for measuring IMM fluidity and suggests a dynamic regulatory paradigm of modulating IMM local order on changing metabolic demand.

S. mediterranea ETS-1 regulates the function of cathepsin-positive cells and the epidermal lineage landscape via basement membrane remodeling.

Extracellular matrix (ECM) is an important component of stem cell niche. Remodeling of ECM mediated by ECM regulators, such as matrix metalloproteinases (MMPs) plays a vital role in stem cell function. However, the mechanisms that modulate the function of ECM regulators in the stem cell niche are understudied. Here, we explored the role of the transcription factor (TF) ETS-1, which is expressed in the cathepsin-positive cell population, in regulating the expression of the ECM regulator, mt-mmpA, thereby modulating basement membrane thickness. In planarians, the basement membrane around the gut/inner parenchyma is thought to act as a niche for pluripotent stem cells. It has been shown that the early epidermal progenitors migrate outwards from this region and progressively differentiate to maintain the terminal epidermis. Our data shows that thickening of the basement membrane in the absence of ets-1 results in defective migration of stem cell progeny. Furthermore, the absence of ets-1 leads to a defective epidermal progenitor landscape, despite its lack of expression in those cell types. Together, our results demonstrate the active role of ECM remodeling in regulating tissue homeostasis and regeneration in the planarian Schmidtea mediterranea. This article has an associated First Person interview with one of the co-first authors of the paper.

Snail maintains the stem/progenitor state of skin epithelial cells and carcinomas through the autocrine effect of matricellular protein Mindin.

Preservation of a small population of cancer stem cells (CSCs) within a heterogeneous carcinoma serves as a paradigm to understand how select cells in a tissue maintain their undifferentiated status. In both embryogenesis and cancer, Snail has been correlated with stemness, but the molecular underpinning of this phenomenon remains largely ill-defined. In models of cutaneous squamous cell carcinoma (cSCC), we discovered a non-epithelial-mesenchymal transition function for the transcription factor Snail in maintaining the stemness of epidermal keratinocytes. Snail-expressing cells secrete the matricellular protein Mindin, which functions in an autocrine fashion to activate a Src-STAT3 pathway to reinforce their stem/progenitor phenotype. This pathway is activated by the engagement of Mindin with the leukocyte-specific integrin, CD11b (ITGAM), which is also unexpectedly expressed by epidermal keratinocytes. Interestingly, disruption of this signaling module in human cSCC attenuates tumorigenesis, suggesting that targeting Mindin would be a promising therapeutic approach to hinder cancer recurrence.

Molecular and functional characterization of buffalo nasal epithelial odorant binding proteins and their structural insights by in silico and biochemical approaches.

The olfactory system is capable of detecting and distinguishing thousands of environmental odorants that play a key role in reproduction, social behaviours including pheromones influenced classical events. Membrane secretary odorant binding proteins (OBPs) are soluble lipocalins, localized in the nasal membrane of mammals. They bind and carry odorants within the nasal epithelium to putative olfactory transmembrane receptors (ORs). OBP has not yet been exploited to develop a suitable technique to detect oestrus which is being reported as a difficult task in buffalo. In the present study, using molecular biology and protein engineering approaches, we have cloned six novel OBP isoforms from buffalo nasal epithelium odorant-binding proteins (bnOBPs). Furthermore, 3 D models were developed and molecular-docking, dynamics experiments were performed by in silico approaches. In particular, we found four residues (Phe104, Phe134, Phe69 and Asn118) in OBP1a, which contributed to favourable interactions towards two sex pheromones, specifically oleic acid and p-cresol. We expressed this protein in Escherichia coli from female buffalo urine and validated through fluorescence quenching studies to show similar strong binding affinities of OBP1a to oleic acid and p-cresol. By using structural data, the binding specificity was also verified by site-directed mutagenesis of the four residues followed by in vitro binding assays. Our results enable us to better understand the functions of different nasal epithelium OBP isoforms in buffaloes. They also lead to improved understanding of the interaction between olfactory proteins and odorants to develop highly selective biosensing devices for non-invasive detection of oestrus in buffaloes. Communicated by Ramaswamy H. Sarma.

Engineered RNA biosensors enable ultrasensitive SARS-CoV-2 detection in a simple color and luminescence assay.

The continued resurgence of the COVID-19 pandemic with multiple variants underlines the need for diagnostics that are adaptable to the virus. We have developed toehold RNA-based sensors across the SARS-CoV-2 genome for direct and ultrasensitive detection of the virus and its prominent variants. Here, isothermal amplification of a fragment of SARS-CoV-2 RNA coupled with activation of our biosensors leads to a conformational switch in the sensor. This leads to translation of a reporter protein, for example, LacZ or nano-lantern that is easily detected using color/luminescence. By optimizing RNA amplification and biosensor design, we have generated a highly sensitive diagnostic assay that is capable of detecting as low as 100 copies of viral RNA with development of bright color. This is easily visualized by the human eye and quantifiable using spectrophotometry. Finally, this PHAsed NASBA-Translation Optical Method (PHANTOM) using our engineered RNA biosensors efficiently detects viral RNA in patient samples. This work presents a powerful and universally accessible strategy for detecting COVID-19 and variants. This strategy is adaptable to further viral evolution and brings RNA bioengineering center-stage.

Discovery of a body-wide photosensory array that matures in an adult-like animal and mediates eye-brain-independent movement and arousal.

The ability to respond to light has profoundly shaped life. Animals with eyes overwhelmingly rely on their visual circuits for mediating light-induced coordinated movements. Building on previously reported behaviors, we report the discovery of an organized, eye-independent (extraocular), body-wide photosensory framework that allows even a head-removed animal to move like an intact animal. Despite possessing sensitive cerebral eyes and a centralized brain that controls most behaviors, head-removed planarians show acute, coordinated ultraviolet-A (UV-A) aversive phototaxis. We find this eye-brain-independent phototaxis is mediated by two noncanonical rhabdomeric opsins, the first known function for this newly classified opsin-clade. We uncover a unique array of dual-opsin-expressing photoreceptor cells that line the periphery of animal body, are proximal to a body-wide nerve net, and mediate UV-A phototaxis by engaging multiple modes of locomotion. Unlike embryonically developing cerebral eyes that are functional when animals hatch, the body-wide photosensory array matures postembryonically in "adult-like animals." Notably, apart from head-removed phototaxis, the body-wide, extraocular sensory organization also impacts physiology of intact animals. Low-dose UV-A, but not visible light (ocular-stimulus), is able to arouse intact worms that have naturally cycled to an inactive/rest-like state. This wavelength selective, low-light arousal of resting animals is noncanonical-opsin dependent but eye independent. Our discovery of an autonomous, multifunctional, late-maturing, organized body-wide photosensory system establishes a paradigm in sensory biology and evolution of light sensing.

A Tunable Palette of Molecular Rotors Allows Multicolor, Ratiometric Fluorescence Imaging and Direct Mapping of Mitochondrial Heterogeneity.

Environment-sensitive molecular probes offer the potential for a comprehensive mapping of the complex cellular milieu. We present here a radically new strategy of multiplexing highly sensitive, spectrally tuned fluorescent dyes for sensing cellular microenvironment. To achieve this multicolor, ratiometric cellular imaging, we first developed a series of highly sensitive, tunable molecular rotors for mitochondrial imaging, with emission wavelengths spanning the visible spectrum. These fluorogenic merocyanine dyes are all sensitive to solvent viscosity despite distinctive photophysical features. Our results show that merocyanine dyes can show a rotor-like behavior despite significant changes to the conventional donor-acceptor or push-pull scaffolds, thereby revealing conserved features of rotor dye chemistry. Developing closely related but spectrally separated dyes that have distinct response functions allows us to do ″two-color, two-dye″ imaging of the mitochondrial microenvironment. Our results with multidye, combinatorial imaging provide a direct visualization of the intrinsic heterogeneity of the mitochondrial microenvironment. The overall mitochondrial microenvironment (including contributions from local membrane order) as reported through two-color fluorescence ″ratio″ changes of multiplexed rotor dyes shows dynamic heterogeneity with distinct spatiotemporal signatures that evolve over time and respond to chemical perturbations. Our results offer a powerful illustration of how multiplexed dye imaging allows the quantitative imaging of mitochondrial membrane order and cellular microenvironment.

A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

Dynamic expression of tRNA-derived small RNAs define cellular states.

Transfer RNA (tRNA)-derived small RNAs (tsRNAs) have recently emerged as important regulators of protein translation and shown to have diverse biological functions. However, the underlying cellular and molecular mechanisms of tsRNA function in the context of dynamic cell-state transitions remain unclear. Expression analysis of tsRNAs in distinct heterologous cell and tissue models of stem vs. differentiated states revealed a differentiation-dependent enrichment of 5'-tsRNAs. We report the identification of a set of 5'-tsRNAs that is upregulated in differentiating mouse embryonic stem cells (mESCs). Notably, interactome studies with differentially enriched 5'-tsRNAs revealed a switch in their association with "effector" RNPs and "target" mRNAs in different cell states. We demonstrate that specific 5'-tsRNAs can preferentially interact with the RNA-binding protein, Igf2bp1, in the RA-induced differentiated state. This association influences the transcript stability and thereby translation of the pluripotency-promoting factor, c-Myc, thus providing a mechanistic basis for how 5'-tsRNAs can modulate stem cell states in mESCs. Together our study highlights the role of 5'-tsRNAs in defining distinct cell states.

Membrane-Permeant, Environment-Sensitive Dyes Generate Biosensors within Living Cells.

Dyes with environment-sensitive fluorescence have proven useful to study the spatiotemporal dynamics of protein activity in living cells. When attached to proteins, their fluorescence can reflect protein conformational changes, post-translational modifications, or protein interactions. However, the utility of such dye-protein conjugates has been limited because it is difficult to load them into cells. They usually must be introduced using techniques that perturb cell physiology, limit throughput, or generate fluorescent vesicles (e.g., electroporation, microinjection, or membrane transduction peptides). Here we circumvent these problems by modifying a proven, environment-sensitive biosensor fluorophore so that it can pass through cell membranes without staining intracellular compartments and can be attached to proteins within living cells using unnatural amino acid (UAA) mutagenesis. Reactive groups were incorporated for attachment to UAAs or small molecules (mero166, azide; mero167, alkyne; mero76, carboxylic acid). These dyes are bright and fluoresce at long wavelengths (reaching ε = 100 000 M-1 cm-1, ϕ = 0.24, with excitation 565 nm and emission 594 nm). The utility of mero166 was demonstrated by in-cell labeling of a UAA to generate a biosensor for the small GTPase Cdc42. In addition, conjugation of mero166 to a small molecule produced a membrane-permeable probe that reported the localization of the DNA methyltransferase G9a in cells. This approach provides a strategy to access biosensors for many targets and to more practically harness the varied environmental sensitivities of synthetic dyes.

KMT1 family methyltransferases regulate heterochromatin-nuclear periphery tethering via histone and non-histone protein methylation.

Euchromatic histone methyltransferases (EHMTs), members of the KMT1 family, methylate histone and non-histone proteins. Here, we uncover a novel role for EHMTs in regulating heterochromatin anchorage to the nuclear periphery (NP) via non-histone methylation. We show that EHMTs methylate and stabilize LaminB1 (LMNB1), which associates with the H3K9me2-marked peripheral heterochromatin. Loss of LMNB1 methylation or EHMTs abrogates heterochromatin anchorage at the NP We further demonstrate that the loss of EHMTs induces many hallmarks of aging including global reduction of H3K27methyl marks and altered nuclear morphology. Consistent with this, we observe a gradual depletion of EHMTs, which correlates with loss of methylated LMNB1 and peripheral heterochromatin in aging human fibroblasts. Restoration of EHMT expression reverts peripheral heterochromatin defects in aged cells. Collectively, our work elucidates a new mechanism by which EHMTs regulate heterochromatin domain organization and reveals their impact on fundamental changes associated with the intrinsic aging process.

Highly Responsive Fluorescent Assemblies Allow for Unique, Multiparametric Sensing of the Phospholipid Membrane Environment.

Despite decades-long extensive research, probes that provide a comprehensive description of the lipid membrane microenvironment are still lacking. Here, a "smart" pyrene-terpyridine probe for multiparametric sensing of lipid membranes is reported. The complexity of the associated local microenvironment can be described by the distinct features of the probe fluorescence. The self-assembly of the probe molecules in phospholipid bilayers was sensitive to membrane order and phase state. The self-assembled probes showed a unique emission, influenced by dye-dye interactions and excited-state charge transfer. Moreover, this emission was sensitive to interfacial hydration, with very specific changes in emission wavelengths and fluorescence lifetimes upon variation of lipid compositions and properties. In parallel, changes in the lipid order and hydration affected the ground-state interactions in the dye aggregates and, thus, could be measured through ratiometric changes in the excitation and emission readouts. In addition, fluorescence anisotropy measurements provided another way to study the nature of dye aggregates in lipid bilayers. Overall, this report demonstrates how multiple aspects of the membrane microenvironment can be sensed through the unique fluorescence signatures of this "smart" probe in lipid membranes, and it establishes a new paradigm in lipid-membrane sensing.

FMRP Interacts with C/D Box snoRNA in the Nucleus and Regulates Ribosomal RNA Methylation.

FMRP is an RNA-binding protein that is known to localize in the cytoplasm and in the nucleus. Here, we have identified an interaction of FMRP with a specific set of C/D box snoRNAs in the nucleus. C/D box snoRNAs guide 2'O methylations of ribosomal RNA (rRNA) on defined sites, and this modification regulates rRNA folding and assembly of ribosomes. 2'O methylation of rRNA is partial on several sites in human embryonic stem cells, which results in ribosomes with differential methylation patterns. FMRP-snoRNA interaction affects rRNA methylation on several of these sites, and in the absence of FMRP, differential methylation pattern of rRNA is significantly altered. We found that FMRP recognizes ribosomes carrying specific methylation patterns on rRNA and the recognition of methylation pattern by FMRP may potentially determine the translation status of its target mRNAs. Thus, FMRP integrates its function in the nucleus and in the cytoplasm.

A unique self-assembly-driven probe for sensing a lipid bilayer: ratiometric probing of vesicle to micelle transition.

An amphiphilic pyrene-terpyridine (Pytpy) probe forms novel, fluorescent nanoaggregates in phospholipid membranes. This unique membrane-driven self-assembly of Pytpy shows large Stokes shifts and long-lived fluorescent states and efficiently reports on vesicle-micelle transition through ratiometric changes. Strikingly, Pytpy can even distinguish between bilayer-like domains and more-dynamic micelle-like 'rim' phases that co-exist in mixed assemblies (bicelles).

Tunable Emission from Fluorescent Organic Nanoparticles in Water: Insight into the Nature of Self-Assembly and Photoswitching.

Excitation-dependent tuning of the emission behavior of fluorescent organic nanoparticles (FONs) with two simple luminescent pyrenyl-pyridyl conjugates as model systems is demonstrated. In the case of the compound with a flexible bis-picolyl moiety, the simultaneous presence of multiple ground-state species with distinct absorption and emission characteristics can be observed. The relative ratios of these species can easily be modulated, and it is possible to selectively stimulate any one of them individually by choosing an appropriate excitation channel. Moreover, at high concentration, a drastic change in the nature of the self-assembly is observed, which shifts from donor-acceptor-type self-assembly to exciplex-type self-agglomeration. On the contrary, the compound containing a rigid terpyridine unit has only a single ground state and shows no such tunable emission. However, it can exhibit multiple emission bands in water, whereby the positions of their emission maxima depend on the extent of aggregation-induced planarization of the probe molecules. Overall, this work demonstrates multimodal modulation of FON emission and a gives insight into how molecular order can translate into complete switching of nanoparticle self-assembly and photophysics.

Hierarchies in light sensing and dynamic interactions between ocular and extraocular sensory networks in a flatworm.

Light sensing has independently evolved multiple times under diverse selective pressures but has been examined only in a handful among the millions of light-responsive organisms. Unsurprisingly, mechanistic insights into how differential light processing can cause distinct behavioral outputs are limited. We show how an organism can achieve complex light processing with a simple "eye" while also having independent but mutually interacting light sensing networks. Although planarian flatworms lack wavelength-specific eye photoreceptors, a 25 nm change in light wavelength is sufficient to completely switch their phototactic behavior. Quantitative photoassays, eye-brain confocal imaging, and RNA interference/knockdown studies reveal that flatworms are able to compare small differences in the amounts of light absorbed at the eyes through a single eye opsin and convert them into binary behavioral outputs. Because planarians can fully regenerate, eye-brain injury-regeneration studies showed that this acute light intensity sensing and processing are layered on simple light detection. Unlike intact worms, partially regenerated animals with eyes can sense light but cannot sense finer gradients. Planarians also show a "reflex-like," eye-independent (extraocular/whole-body) response to low ultraviolet A light, apart from the "processive" eye-brain-mediated (ocular) response. Competition experiments between ocular and extraocular sensory systems reveal dynamic interchanging hierarchies. In intact worms, cerebral ocular response can override the reflex-like extraocular response. However, injury-regeneration again offers a time window wherein both responses coexist, but the dominance of the ocular response is reversed. Overall, we demonstrate acute light intensity-based behavioral switching and two evolutionarily distinct but interacting light sensing networks in a regenerating organism.

Cytoplasmic poly (A)-binding protein critically regulates epidermal maintenance and turnover in the planarian Schmidtea mediterranea.

Identifying key cellular events that facilitate stem cell function and tissue organization is crucial for understanding the process of regeneration. Planarians are powerful model system to study regeneration and stem cell (neoblast) function. Here, using planaria, we show that the initial events of regeneration, such as epithelialization and epidermal organization are critically regulated by a novel cytoplasmic poly A-binding protein, SMED-PABPC2. Knockdown of smed-pabpc2 leads to defects in epidermal lineage specification, disorganization of epidermis and ECM, and deregulated wound healing, resulting in the selective failure of neoblast proliferation near the wound region. Polysome profiling suggests that epidermal lineage transcripts, including zfp-1, are translationally regulated by SMED-PABPC2. Together, our results uncover a novel role for SMED-PABPC2 in the maintenance of epidermal and ECM integrity, critical for wound healing and subsequent processes for regeneration.

A biosensor generated via high-throughput screening quantifies cell edge Src dynamics.

Fluorescent biosensors for living cells currently require laborious optimization and a unique design for each target. They are limited by the availability of naturally occurring ligands with appropriate target specificity. Here we describe a biosensor based on an engineered fibronectin monobody scaffold that can be tailored to bind different targets via high-throughput screening. We made this Src-family kinase (SFK) biosensor by derivatizing a monobody specific for activated SFKs with a bright dye whose fluorescence increases upon target binding. We identified sites for dye attachment and changes to eliminate vesiculation in living cells, providing a generalizable scaffold for biosensor production. This approach minimizes cell perturbation because it senses endogenous, unmodified target, and because sensitivity is enhanced by direct dye excitation. Automated correlation of cell velocities and SFK activity revealed that SFKs are activated specifically during protrusion. Activity correlates with velocity, and peaks 1-2 µm from the leading edge.