HIV-1 Tat transactivation is vital for completion of the viral life cycle and has been implicated in determining proviral latency. We present an extensive experimental/computational study of an HIV-1 model vector (LTR-GFP-IRES-Tat) and show that stochastic fluctuations in Tat influence the viral latency decision. Low GFP/Tat expression was found to generate bifurcating phenotypes with clonal populations derived from single proviral integrations simultaneously exhibiting very high and near zero GFP expression. Although phenotypic bifurcation (PheB) was correlated with distinct genomic integration patterns, neither these patterns nor other extrinsic cellular factors (cell cycle/size, aneuploidy, chromatin silencing, etc.) explained PheB. Stochastic computational modeling successfully accounted for PheB and correctly predicted the dynamics of a Tat mutant that were subsequently confirmed by experiment. Thus, Tat stochastics appear sufficient to generate PheB (and potentially proviral latency), illustrating the importance of stochastic fluctuations in gene expression in a mammalian system.

Summary Phase transitions driven by intrinsically disordered protein regions (IDRs) have emerged as a ubiquitous mechanism for assembling liquid-like RNA/protein (RNP) bodies and other membrane-less organelles. However, a lack of tools to control intracellular phase transitions limits our ability to understand their role in cell physiology and disease. Here, we introduce an optogenetic platform, which uses light to activate IDR-mediated phase transitions in living cells. We use this “optoDroplet” system to study condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1. Above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally-definable liquid optoDroplets. FUS optoDroplet assembly is fully reversible even after multiple activation cycles. However, cells driven deep within the phase boundary form solid-like gels, which undergo aging into irreversible aggregates. This system can thus elucidate not only physiological phase transitions, but also their link to pathological aggregates.

SUMMARY The complex, interconnected architecture of cell-signaling networks makes it challenging to disentangle how cells process extracellular information to make decisions. We have developed an optogenetic approach to selectively activate isolated intracellular signaling nodes with light and use this method to follow the flow of information from the signaling protein Ras. By measuring dose and frequency responses in single cells, we characterize the precision, timing, and efficiency with which signals are transmitted from Ras to Erk. Moreover, we elucidate how a single pathway can specify distinct physiological outcomes: by combining distinct temporal patterns of stimulation with proteomic profiling, we identify signaling programs that differentially respond to Ras dynamics, including a paracrine circuit that activates STAT3 only after persistent (>1 hr) Ras activation. Optogenetic stimulation provides a powerful tool for analyzing the intrinsic transmission properties of pathway modules and identifying how they dynamically encode distinct outcomes.

The optimization of engineered metabolic pathways requires careful control over the levels and timing of metabolic enzyme expression1-4. Optogenetic tools are ideal for achieving such precise control, as light can be applied and removed instantly without complex media changes. Here we show that light-controlled transcription can be used to enhance the biosynthesis of valuable products in engineered Saccharomyces cerevisiae. We introduce new optogenetic circuits to shift cells from a light-induced growth phase to a darkness-induced production phase, which allows us to control fermentation purely with light. Furthermore, optogenetic control of engineered pathways enables a new mode of bioreactor operation using periodic light pulses to tune enzyme expression during the production phase of fermentation to increase yields. Using these advances, we control the mitochondrial isobutanol pathway to produce up to 8.49 ± 0.31 g/L of isobutanol and 2.38 ± 0.06 g/L of 2-methyl-1-butanol micro-aerobically from glucose. These results make a compelling case for the application of optogenetics to metabolic engineering for valuable products.

Summary Recent studies show that liquid-liquid phase separation plays a key role in the assembly of diverse intracellular structures. However, the biophysical principles by which phase separation can be precisely localized within subregions of the cell are still largely unclear, particularly for low-abundance proteins. Here we introduce an oligomerizing biomimetic system, “Corelets”, and utilize its rapid and quantitative light-controlled tunability to map full intracellular phase diagrams, which dictate the concentrations at which phase separation occurs, and the mode of phase separation. Surprisingly, both experiments and simulations show that while intracellular concentrations may be insufficient for global phase separation, sequestering protein ligands to slowly diffusing nucleation centers can move the cell into a different region of the phase diagram, resulting in localized phase separation. This diffusive capture mechanism liberates the cell from the constraints of global protein abundance and is likely exploited to pattern condensates associated with diverse biological processes.

The ability to apply precise inputs to signaling species in live cells would be transformative for interrogating and understanding complex cell signaling systems. Here, we report a method for applying custom signaling inputs using feedback control of an optogenetic protein-protein interaction. We apply this strategy for perturbation of protein localization and phosphoinositide 3-kinase activity, generating time-varying signals and clamping signals to buffer against cell-to-cell variability or pathway activation changes. KeywordsOptogenetics; feedback control; systems biology; single cell methods To dissect how cell signaling networks sense, encode and process information, we need not only a parts list but also an understanding of how their constituent molecular components vary over time in response to diverse input signals. One powerful set of approaches for interrogating cellular circuits combines controlled, time-varying perturbations with live cell signaling activity readouts 1-3 . This strategy can be used to analyze how a pathway maps diverse inputs to outputs and to distinguish the nature, timescale, and strength of feedback connections acting within a biological network.Genetically encoded light-gated proteins (optogenetics) represent a promising technology for delivering precise intracellular inputs to individual cells. However, their broad HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript application faces three challenges. First, cells vary in their expression level of optogenetic components, so the same light input will drive different activity levels across a population of cells. Second, even within an individual cell the relationship between light and activity can be complex and nonlinear. Thus, it is very difficult to identify how light levels should be varied to drive a defined timecourse of intracellular activation. Finally, many signaling pathways incorporate regulatory connections whose strength varies over time. Even delivering a constant level of pathway activity can require light inputs that compensate for the timing and strength of intracellular feedback.In this study, we address each of these challenges by coupling a tunable optogenetic module with automated control of its light input (Fig. 1a). Using live-cell measurements of intracellular activation to update light levels in real time, we implemented a computational feedback controller to act as a 'concentration clamp,' analogous to voltage clamping for neuron excitation 4 or positional clamping 5 for molecular mechanical systems. Our controller can drive precise time-varying activity levels, automatically identifying the light input required to correct for a nonlinear light-activity relationship. It can deliver custom light levels to each cell within a population to compensate for cell-to-cell variability in optogenetic component expression. Finally, we show the controller can be used to clamp a downstream signaling node at a defined level, even when the node is affected by additional regu...

Summary Cell signaling networks coordinate specific patterns of protein expression in response to external cues. Yet the logic by which signaling pathway activity determines the eventual abundance of target proteins is complex and poorly understood. Here, we describe an approach for simultaneously controlling the Ras/Erk pathway while monitoring a target gene’s transcription and protein accumulation in single live cells. We apply our approach to dissect how Erk activity is decoded by immediate-early genes (IEGs). We find that IEG transcription decodes Erk dynamics through a shared band-pass filtering circuit: repeated Erk pulses transcribe IEGs more efficiently than sustained Erk inputs. However, despite highly similar transcriptional responses, each IEG exhibits dramatically different protein-level accumulation, demonstrating a high degree of post-transcriptional regulation by combinations of multiple pathways. Our results demonstrate that the Ras/Erk pathway is decoded both by dynamic filters and logic gates to shape target gene responses in a context-specific manner.

Optogenetic modules offer cell biologists unprecedented new ways to poke and prod cells. The combination of these precision perturbative tools with observational tools, such as fluorescent proteins, may dramatically accelerate our ability to understand the inner workings of the cell.Biology has always been primarily an observational science, and in the modern era, the development of genetically encoded fluorescent proteins such as GFP has given us the unprecedented ability to peer into the living cell and to observe its inner workings. We can now study individual cells in culture or in the context of a whole organism and directly observe where proteins are localized, their dynamics and their variability in expression level. More than ever, we now appreciate that the cell is not a bag of molecules but an anisotropic structure with highly complex spatial organization. We can see examples of how this organization shifts in dynamic processes, ranging from cell-shape changes to signal transduction propagated from the plasma membrane to the nucleus.But what are the mechanisms that underlie and orchestrate these complex behaviors? Sadly, our ability to systematically perturb and interrogate the intracellular networks that control cell behavior (and thus our ability to understand their mechanism) has lagged behind our ability to observe these behaviors. The standard genetic perturbation techniquesknockdown, overexpression and mutation-are extremely effective at identifying the proteins involved in a phenotype, but are less effective at extracting mechanism. These perturbations are slow in timescale and broad in effect and, except in lucky circumstances, are more likely to destroy rather than modulate specific spatiotemporal features of the network's response. Pharmacological perturbations have been extremely useful tools-small molecules that target or block specific molecules give the investigator the ability to rapidly switch off the function of a target protein. But these approaches do not allow spatial control, and in most cases good fortune or considerable engineering 1 is required to obtain highly specific inhibitors. Light-gated protein modules provide a potentially transformative solution to the problem of dissecting cellular network function. There is currently an explosion of new light-controlled modules that can, in principle, be used to control the function and localization of diverse proteins. Such general new tools could usher in a new era of perturbative biology that would transform our ability to interrogate, dissect and understand the mechanisms of complex biological systems. Such light-gated modules might serve as the workhorse perturbative tool that complements GFP as an analytical tool. Here we briefly review optogenetic tools that have emerged over the last few years and discuss how they may be applied to cell biology in the near future.