It's complicated: the interplay of Kif1c mRNA localization in cell protrusions, assembly of protein binding partners on the KIF1C protein, and cell migration.

Distinct subcellular localizations of mRNAs have been described across a wide variety of cell types. While common themes emerge for neuronal cells, functional roles of mRNA localization in space and time are much less understood in nonneuronal cells. Emerging areas of interest are cell models with protrusions, often linked with cell mobility in cancer systems. In this issue of Genes & Development, Norris and Mendell (pp. 191-203) systematically investigate a link between mRNA localization to cell protrusions in a mouse melanoma cell system and a mechanistic link to downstream consequences for cell mobility. The study first identifies a model mRNA of interest in an unbiased way that exhibits a set of phenotypes associated with cell mobility. The candidate mRNA that fulfills all requirements is Kif1c mRNA. Further systematic investigation links Kif1c mRNA localization to assembly of a protein-protein network on the KIF1C protein itself. What's clear is that this work will inspire a further mechanistic dissection of the Kif1c mRNA/KIF1C protein interplay in this important nonneuronal model cell system. More broadly, this work suggests that a broad set of model mRNAs should be investigated to understand mRNA dynamics and downstream functional consequences across a variety of cell models.

A Critical and Comparative Review of Fluorescent Tools for Live-Cell Imaging.

Fluorescent tools have revolutionized our ability to probe biological dynamics, particularly at the cellular level. Fluorescent sensors have been developed on several platforms, utilizing either small-molecule dyes or fluorescent proteins, to monitor proteins, RNA, DNA, small molecules, and even cellular properties, such as pH and membrane potential. We briefly summarize the impressive history of tool development for these various applications and then discuss the most recent noteworthy developments in more detail. Particular emphasis is placed on tools suitable for single-cell analysis and especially live-cell imaging applications. Finally, we discuss prominent areas of need in future fluorescent tool development-specifically, advancing our capability to analyze and integrate the plethora of high-content data generated by fluorescence imaging.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

RNAs directly regulate a vast array of cellular processes, emphasizing the need for robust approaches to fluorescently label and track RNAs in living cells. Here, we develop an RNA imaging platform using the cobalamin riboswitch as an RNA tag and a series of probes containing cobalamin as a fluorescence quencher. This highly modular 'Riboglow' platform leverages different colored fluorescent dyes, linkers and riboswitch RNA tags to elicit fluorescence turn-on upon binding RNA. We demonstrate the ability of two different Riboglow probes to track mRNA and small noncoding RNA in live mammalian cells. A side-by-side comparison revealed that Riboglow outperformed the dye-binding aptamer Broccoli and performed on par with the gold standard RNA imaging system, the MS2-fluorescent protein system, while featuring a much smaller RNA tag. Together, the versatility of the Riboglow platform and ability to track diverse RNAs suggest broad applicability for a variety of imaging approaches.

A Multicolor Split-Fluorescent Protein Approach to Visualize Listeria Protein Secretion in Infection.

Listeria monocytogenes is an intracellular food-borne pathogen that has evolved to enter mammalian host cells, survive within them, spread from cell to cell, and disseminate throughout the body. A series of secreted virulence proteins from Listeria are responsible for manipulation of host-cell defense mechanisms and adaptation to the intracellular lifestyle. Identifying when and where these virulence proteins are located in live cells over the course of Listeria infection can provide valuable information on the roles these proteins play in defining the host-pathogen interface. These dynamics and protein levels may vary from cell to cell, as bacterial infection is a heterogeneous process both temporally and spatially. No assay to visualize virulence proteins over time in infection with Listeria or other Gram-positive bacteria has been developed. Therefore, we adapted a live, long-term tagging system to visualize a model Listeria protein by fluorescence microscopy on a single-cell level in infection. This system leverages split-fluorescent proteins, in which the last strand of a fluorescent protein (a 16-amino-acid peptide) is genetically fused to the virulence protein of interest. The remainder of the fluorescent protein is produced in the mammalian host cell. Both individual components are nonfluorescent and will bind together and reconstitute fluorescence upon virulence-protein secretion into the host cell. We demonstrate accumulation and distribution within the host cell of the model virulence protein InlC in infection over time. A modular expression platform for InlC visualization was developed. We visualized InlC by tagging it with red and green split-fluorescent proteins and compared usage of a strong constitutive promoter versus the endogenous promoter for InlC production. This split-fluorescent protein approach is versatile and may be used to investigate other Listeria virulence proteins for unique mechanistic insights in infection progression.

Folding the proteome.

Protein folding is an essential prerequisite for protein function and hence cell function. Kinetic and thermodynamic studies of small proteins that refold reversibly were essential for developing our current understanding of the fundamentals of protein folding mechanisms. However, we still lack sufficient understanding to accurately predict protein structures from sequences, or the effects of disease-causing mutations. To date, model proteins selected for folding studies represent only a small fraction of the complexity of the proteome and are unlikely to exhibit the breadth of folding mechanisms used in vivo. We are in urgent need of new methods - both theoretical and experimental - that can quantify the folding behavior of a truly broad set of proteins under in vivo conditions. Such a shift in focus will provide a more comprehensive framework from which to understand the connections between protein folding, the molecular basis of disease, and cell function and evolution.

Multiple driving forces required for efficient secretion of autotransporter virulence proteins.

Autotransporter (AT) proteins are a broad class of virulence proteins from Gram-negative bacterial pathogens that require their own C-terminal transmembrane domain to translocate their N-terminal passenger across the bacterial outer membrane (OM). But given the unavailability of ATP or a proton gradient across the OM, it is unknown what energy source(s) drives this process. Here we used a combination of computational and experimental approaches to quantitatively compare proposed AT OM translocation mechanisms. We show directly for the first time that when translocation was blocked an AT passenger remained unfolded in the periplasm. We demonstrate that AT secretion is a kinetically controlled, non-equilibrium process coupled to folding of the passenger and propose a model connecting passenger conformation to secretion kinetics. These results reconcile seemingly contradictory reports regarding the importance of passenger folding as a driving force for OM translocation but also reveal that another energy source is required to initiate translocation.

Of linkers and autochaperones: an unambiguous nomenclature to identify common and uncommon themes for autotransporter secretion.

Autotransporter (AT) proteins provide a diverse array of important virulence functions to Gram-negative bacterial pathogens, and have also been adapted for protein surface display applications. The 'autotransporter' moniker refers to early models that depicted these proteins facilitating their own translocation across the bacterial outer membrane. Although translocation is less autonomous than originally proposed, AT protein segments upstream of the C-terminal transmembrane ß-barrel have nevertheless consistently been found to contribute to efficient translocation and/or folding of the N-terminal virulence region (the 'passenger'). However, defining the precise secretion functions of these AT regions has been complicated by the use of multiple overlapping and ambiguous terms to define AT sequence, structural, and functional features, including 'autochaperone', 'linker' and 'junction'. Moreover, the precise definitions and boundaries of these features vary among ATs and even among research groups, leading to an overall murky picture of the contributions of specific features to translocation. Here we propose a unified, unambiguous nomenclature for AT structural, functional and conserved sequence features, based on explicit criteria. Applied to 16 well-studied AT proteins, this nomenclature reveals new commonalities for translocation but also highlights that the autochaperone function is less closely associated with a conserved sequence element than previously believed.

Evaluating Riboglow-FLIM probes for RNA sensing.

We recently developed Riboglow-FLIM, where we genetically tag and track RNA molecules in live cells through measuring the fluorescence lifetime of a small molecule probe that binds the RNA tag. Here, we systematically and quantitatively evaluated key elements of Riboglow-FLIM that may serve as the foundation for Riboglow-FLIM applications and further tool development efforts. Our investigation focused on measuring changes in fluorescence lifetime of representative Riboglow-FLIM probes with different linkers and fluorophores in different environments. In vitro measurements revealed distinct lifetime differences among the probe variants as a result of different linker designs and fluorophore selections. To expand on the platform's versatility, probes in a wide variety of mammalian cell types were examined using fluorescence lifetime imaging microscopy (FLIM), and possible effects on cell physiology were evaluated by metabolomics. The results demonstrated that variations in lifetime were dependent on both probe and cell type. Interestingly, distinct differences in lifetime values were observed between cell lines, while no overall change in cell health was measured. These findings underscore the importance of probe selection and cellular environment when employing Riboglow-FLIM for RNA detection, serving as a foundation for future tool development and applications across diverse fields and biological systems.

Visualizing orthogonal RNAs simultaneously in live mammalian cells by fluorescence lifetime imaging microscopy (FLIM).

Visualization of RNAs in live cells is critical to understand biology of RNA dynamics and function in the complex cellular environment. Detection of RNAs with a fluorescent marker frequently involves genetically fusing an RNA aptamer tag to the RNA of interest, which binds to small molecules that are added to live cells and have fluorescent properties. Engineering efforts aim to improve performance and add versatile features. Current efforts focus on adding multiplexing capabilities to tag and visualize multiple RNAs simultaneously in the same cell. Here, we present the fluorescence lifetime-based platform Riboglow-FLIM. Our system requires a smaller tag and has superior cell contrast when compared with intensity-based detection. Because our RNA tags are derived from a large bacterial riboswitch sequence family, the riboswitch variants add versatility for using multiple tags simultaneously. Indeed, we demonstrate visualization of two RNAs simultaneously with orthogonal lifetime-based tags.

Establishing Riboglow-FLIM to visualize noncoding RNAs inside live zebrafish embryos.

The central role of RNAs in health and disease calls for robust tools to visualize RNAs in living systems through fluorescence microscopy. Live zebrafish embryos are a popular system to investigate multicellular complexity as disease models. However, RNA visualization approaches in whole organisms are notably underdeveloped. Here, we establish our RNA tagging and imaging platform Riboglow-FLIM for complex cellular imaging applications by systematically evaluating FLIM capabilities. We use adherent mammalian cells as models for RNA visualization. Additional complexity of analyzing RNAs in whole mammalian animals is achieved by injecting these cells into a zebrafish embryo system for cell-by-cell analysis in this model of multicellularity. We first evaluate all variable elements of Riboglow-FLIM quantitatively before assessing optimal use in whole animals. In this way, we demonstrate that a model noncoding RNA can be detected robustly and quantitatively inside live zebrafish embryos using a far-red Cy5-based variant of the Riboglow platform. We can clearly resolve cell-to-cell heterogeneity of different RNA populations by this methodology, promising applicability in diverse fields.

A multicolor riboswitch-based platform for imaging of RNA in live mammalian cells.

A key approach to investigating RNA species in live mammalian cells is the ability to label them with fluorescent tags and track their dynamics in the complex cellular environment. The growing appreciation for the diversity of RNAs in nature, especially the roles of small, non-coding RNAs for cell function, calls for development of orthogonal RNA tagging systems. We previously developed Riboglow, a new RNA tagging system that features modular elements and hence the possibility to customize features for each application of choice. Riboglow consists of an RNA tag that is genetically fused to the RNA of interest and a small molecule that binds the RNA tag and elicits a fluorescence light up signal. Here, we present an overview of the Riboglow platform and compare and contrast the system with existing RNA tagging systems. Two step by step protocols for implementation of RNA imaging with Riboglow in live mammalian cells are presented, with special emphasis on guidelines that drive choices for modular elements in the Riboglow platform. Such modular elements include the RNA tag sequence and size, the number of RNA tag repeats per tagged RNA, the fluorescent color of the probe, the identity of the chemical linker in the probe, and the concentration of the probe used in live cells. Together, Riboglow is a new RNA tagging platform that enables robust live cell imaging of RNA dynamics, and this detailed protocol and guidelines for implementation will enable broad usage of Riboglow.

Illuminating RNA Biology: Tools for Imaging RNA in Live Mammalian Cells.

The central dogma teaches us that DNA makes RNA, which in turn makes proteins, the main building blocks of the cell. But this over simplified linear transmission of information overlooks the vast majority of the genome produces RNAs that do not encode proteins and the myriad ways that RNA regulates cellular functions. Historically, one of the challenges in illuminating RNA biology has been the lack of tools for visualizing RNA in live cells. But clever approaches for exploiting RNA binding proteins, in vitro RNA evolution, and chemical biology have resulted in significant advances in RNA visualization tools in recent years. This review provides an overview of current tools for tagging RNA with fluorescent probes and tracking their dynamics, localization andfunction in live mammalian cells.

DegP Chaperone Suppresses Toxic Inner Membrane Translocation Intermediates.

The periplasm of Gram-negative bacteria includes a variety of molecular chaperones that shepherd the folding and targeting of secreted proteins. A central player of this quality control network is DegP, a protease also suggested to have a chaperone function. We serendipitously discovered that production of the Bordetella pertussis autotransporter virulence protein pertactin is lethal in Escherichia coli ΔdegP strains. We investigated specific contributions of DegP to secretion of pertactin as a model system to test the functions of DegP in vivo. The DegP chaperone activity was sufficient to restore growth during pertactin production. This chaperone dependency could be relieved by changing the pertactin signal sequence: an E. coli signal sequence leading to co-translational inner membrane (IM) translocation was sufficient to suppress lethality in the absence of DegP, whereas an E. coli post-translational signal sequence was sufficient to recapitulate the lethal phenotype. These results identify a novel connection between the DegP chaperone and the mechanism used to translocate a protein across the IM. Lethality coincided with loss of periplasmic proteins, soluble σE, and proteins regulated by this essential stress response. These results suggest post-translational IM translocation can lead to the formation of toxic periplasmic folding intermediates, which DegP can suppress.

Development of an Optical Zn2+ Probe Based on a Single Fluorescent Protein.

Various fluorescent probes have been developed to reveal the biological functions of intracellular labile Zn2+. Here, we present Green Zinc Probe (GZnP), a novel genetically encoded Zn2+ sensor design based on a single fluorescent protein (single-FP). The GZnP sensor is generated by attaching two zinc fingers (ZF) of the transcription factor Zap1 (ZF1 and ZF2) to the two ends of a circularly permuted green fluorescent protein (cpGFP). Formation of ZF folds induces interaction between the two ZFs, which induces a change in the cpGFP conformation, leading to an increase in fluorescence. A small sensor library is created to include mutations in the ZFs, cpGFP and linkers between ZF and cpGFP to improve signal stability, sensor brightness and dynamic range based on rational protein engineering, and computational design by Rosetta. Using a cell-based library screen, we identify sensor GZnP1, which demonstrates a stable maximum signal, decent brightness (QY = 0.42 at apo state), as well as specific and sensitive response to Zn2+ in HeLa cells (Fmax/Fmin = 2.6, Kd = 58 pM, pH 7.4). The subcellular localizing sensors mito-GZnP1 (in mitochondria matrix) and Lck-GZnP1 (on plasma membrane) display sensitivity to Zn2+ (Fmax/Fmin = 2.2). This sensor design provides freedom to be used in combination with other optical indicators and optogenetic tools for simultaneous imaging and advancing our understanding of cellular Zn2+ function.

Dynamics and Energy Contributions for Transport of Unfolded Pertactin through a Protein Nanopore.

To evaluate the physical parameters governing translocation of an unfolded protein across a lipid bilayer, we studied protein transport through aerolysin, a passive protein channel, at the single-molecule level. The protein model used was the passenger domain of pertactin, an autotransporter virulence protein. Transport of pertactin through the aerolysin nanopore was detected as transient partial current blockades as the unfolded protein partially occluded the aerolysin channel. We compared the dynamics of entry and transport for unfolded pertactin and a covalent end-to-end dimer of the same protein. For both the monomer and the dimer, the event frequency of current blockades increased exponentially with the applied voltage, while the duration of each event decreased exponentially as a function of the electrical potential. The blockade time was twice as long for the dimer as for the monomer. The calculated activation free energy includes a main enthalpic component that we attribute to electrostatic interactions between pertactin and the aerolysin nanopore (despite the low Debye length), plus an entropic component due to confinement of the unfolded chain within the narrow pore. Comparing our experimental results to previous studies and theory suggests that unfolded proteins cross the membrane by passing through the nanopore in a somewhat compact conformation according to the "blob" model of Daoud and de Gennes.

Autotransporters: The Cellular Environment Reshapes a Folding Mechanism to Promote Protein Transport.

We know very little about how the cellular environment affects protein folding mechanisms. Here, we focus on one unique aspect of that environment that is difficult to recapitulate in the test tube: the effect of a folding vector. When protein folding is initiated at one end of the polypeptide chain, folding starts from a much smaller ensemble of conformations than during refolding of a full-length polypeptide chain. But to what extent can vectorial folding affect protein folding kinetics and the conformations of folding intermediates? We focus on recent studies of autotransporter proteins, the largest class of virulence proteins from pathogenic Gram-negative bacteria. Autotransporter proteins are secreted across the bacterial inner membrane from N→C-terminus, which, like refolding in vitro, retards folding. But in contrast, upon C→N-terminal secretion across the outer membrane autotransporter folding proceeds orders of magnitude faster. The potential impact of vectorial folding on the folding mechanisms of other proteins is also discussed.

ATP-independent control of autotransporter virulence protein transport via the folding properties of the secreted protein.

Autotransporter (AT) proteins are the largest class of extracellular virulence proteins secreted from Gram-negative bacteria. The mechanism by which AT proteins cross the bacterial outer membrane (OM), in the absence of ATP or another external energy source, is unknown. Here we demonstrate a linear correlation between localized regions of stability (ΔG(folding)) in the mature virulence protein (the AT "passenger") and OM secretion efficiency. Destabilizing the C-terminal ß-helical domain of a passenger reduced secretion efficiency. In contrast, destabilizing the globular N-terminal domain of a passenger produced a linearly correlated increase in secretion efficiency. Thus, C-terminal passenger stability facilitates OM secretion, whereas N-terminal stability hinders it. The contributions of regional passenger stability to OM secretion demonstrate a crucial role for the passenger itself in directing its secretion, suggesting a novel type of ATP-independent, folding-driven transporter.