Rhodamine dyes are excellent scaffolds for developing a broad range of fluorescent probes. A key property of rhodamines is their equilibrium between a colorless lactone and fluorescent zwitterion. Tuning the lactone–zwitterion equilibrium constant (KL–Z) can optimize dye properties for specific biological applications. Here, we use known and novel organic chemistry to prepare a comprehensive collection of rhodamine dyes to elucidate the structure–activity relationships that govern KL–Z. We discovered that the auxochrome substituent strongly affects the lactone–zwitterion equilibrium, providing a roadmap for the rational design of improved rhodamine dyes. Electron-donating auxochromes, such as julolidine, work in tandem with fluorinated pendant phenyl rings to yield bright, red-shifted fluorophores for live-cell single-particle tracking (SPT) and multicolor imaging. The N-aryl auxochrome combined with fluorination yields red-shifted Förster resonance energy transfer (FRET) quencher dyes useful for creating a new semisynthetic indicator to sense cAMP using fluorescence lifetime imaging microscopy (FLIM). Together, this work expands the synthetic methods available for rhodamine synthesis, generates new reagents for advanced fluorescence imaging experiments, and describes structure–activity relationships that will guide the design of future probes.

We describe the development and application of methods for high-throughput neuroanatomy in Drosophila using light microscopy. These tools enable efficient multicolor stochastic labeling of neurons at both low and high densities. Expression of multiple membrane-targeted and distinct epitope-tagged proteins is controlled both by a transcriptional driver and by stochastic, recombinase-mediated excision of transcription-terminating cassettes. This MultiColor FlpOut (MCFO) approach can be used to reveal cell shapes and relative cell positions and to track the progeny of precursor cells through development. Using two different recombinases, the number of cells labeled and the number of color combinations observed in those cells can be controlled separately. We demonstrate the utility of MCFO in a detailed study of diversity and variability of Distal medulla (Dm) neurons, multicolumnar local interneurons in the adult visual system. Similar to many brain regions, the medulla has a repetitive columnar structure that supports parallel information processing together with orthogonal layers of cell processes that enable communication between columns. We find that, within a medulla layer, processes of the cells of a given Dm neuron type form distinct patterns that reflect both the morphology of individual cells and the relative positions of their arbors. These stereotyped cell arrangements differ between cell types and can even differ for the processes of the same cell type in different medulla layers. This unexpected diversity of coverage patterns provides multiple independent ways of integrating visual information across the retinotopic columns and implies the existence of multiple developmental mechanisms that generate these distinct patterns.

Light-inducible dimerization protein modules enable precise temporal and spatial control of biological processes in non-invasive fashion. Among them, Magnets are small modules engineered from the photoreceptor Vivid by orthogonalizing the homodimerization interface into complementary heterodimers. Both Magnets components, which are well-tolerated as protein fusion partners, are photoreceptors requiring simultaneous photoactivation to interact, enabling high spatiotemporal confinement of dimerization with a single-excitation wavelength. However, Magnets require concatemerization for efficient responses and cell preincubation at 28C to be functional. Here we overcome these limitations by engineering an optimized Magnets pair requiring neither concatemerization nor low temperature preincubation. We validated these 'enhanced' Magnets (eMags) by using them to rapidly and reversibly recruit proteins to subcellular organelles, to induce organelle contacts, and to reconstitute OSBP-VAP ER-Golgi tethering implicated in phosphatidylinositol-4-phosphate transport and metabolism. eMags represent a very effective tool to optogenetically manipulate physiological processes over whole cells or in small subcellular volumes.

Enzyme-based self-labeling tags enable the covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing cell-permeable multifunctional ligands that contain fluorophores in combination with affinity tags or pharmacological agents. This is challenging since attachment of additional chemical moieties onto fluorescent ligands can adversely affect membrane permeability. To address this problem, we examined the chemical properties of rhodamine-based self-labeling tag ligands through the lens of medicinal chemistry. We found that the lactone-zwitterion equilibrium constant () of rhodamines inversely correlates with their distribution coefficients (log), suggesting that ligands based on dyes exhibiting low and high log values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 within the nucleus. We found that translocation of BRD4 to constitutive heterochromatin in cells leads to apparent increases in transcriptional activity. These fluorescent reagents enable affinity capture and translocation of intracellular proteins in living cells, and our general design concepts will facilitate the design of multifunctional chemical tools for biology.

Preprint: https://doi.org/10.1101/2022.07.02.498544 
Preprint: https://doi.org/10.32388/0xcyuc 

We demonstrate a meaningful prospective power analysis for an (admittedly idealized) illustrative connectome inference task. Modeling neurons as vertices and synapses as edges in a simple random graph model, we optimize the trade-off between the number of (putative) edges identified and the accuracy of the edge identification procedure. We conclude that explicit analysis of the quantity/quality trade-off is imperative for optimal neuroscientific experimental design. In particular, identifying edges faster/more cheaply, but with more error, can yield superior inferential performance.

Nociception generally evokes rapid withdrawal behavior in order to protect the tissue from harmful insults. Most nociceptive neurons responding to mechanical insults display highly branched dendrites, an anatomy shared by Caenorhabditis elegans FLP and PVD neurons, which mediate harsh touch responses. Although several primary molecular nociceptive sensors have been characterized, less is known about modulation and amplification of noxious signals within nociceptor neurons. First, we analyzed the FLP/PVD network by optogenetics and studied integration of signals from these cells in downstream interneurons. Second, we investigated which genes modulate PVD function, based on prior single-neuron mRNA profiling of PVD.

To expand the range of experiments that are accessible with optogenetics, we developed a photocleavable protein (PhoCl) that spontaneously dissociates into two fragments after violet-light-induced cleavage of a specific bond in the protein backbone. We demonstrated that PhoCl can be used to engineer light-activatable Cre recombinase, Gal4 transcription factor, and a viral protease that in turn was used to activate opening of the large-pore ion channel Pannexin-1.

In most animals, the brain makes behavioral decisions that are transmitted by descending neurons to the nerve cord circuitry that produces behaviors. In insects, only a few descending neurons have been associated with specific behaviors. To explore how descending neurons control an insect's movements, we developed a novel method to systematically assay the behavioral effects of activating individual neurons on freely behaving terrestrial D. melanogaster. We calculated a two-dimensional representation of the entire behavior space explored by these flies and we associated descending neurons with specific behaviors by identifying regions of this space that were visited with increased frequency during optogenetic activation. Applying this approach across a large collection of descending neurons, we found that (1) activation of most of the descending neurons drove stereotyped behaviors, (2) in many cases multiple descending neurons activated similar behaviors, and (3) optogenetically-activated behaviors were often dependent on the behavioral state prior to activation.

Intracellular signaling processes are frequently based on direct interactions between proteins and organelles. A fundamental strategy to elucidate the physiological significance of such interactions is to utilize optical dimerization tools. These tools are based on the use of small proteins or domains that interact with each other upon light illumination. Optical dimerizers are particularly suitable for reproducing and interrogating a given protein‐protein interaction and for investigating a protein's intracellular role in a spatially and temporally precise manner. Described in this article are genetic engineering strategies for the generation of modular light‐activatable protein dimerization units and instructions for the preparation of optogenetic applications in mammalian cells. Detailed protocols are provided for the use of light‐tunable switches to regulate protein recruitment to intracellular compartments, induce intracellular organellar membrane tethering, and reconstitute protein function using enhanced Magnets (eMags), a recently engineered optical dimerization system. © 2021 Wiley Periodicals LLC.

To truly understand biological systems, one must possess the ability to selectively manipulate their parts and observe the outcome. (For purposes of this review, we refer mostly to targets of neuroscience; however, the principles covered here largely extend to myriad samples from microbes to plants to the intestine, etc.).

Drugs are the most commonly employed way of introducing such perturbations, but they act on endogenous proteins that frequently exist in multiple cell types, complicating the interpretation of experiments. Whatever the applied stimulus, it is best to introduce optimized exogenous reagents into the systems under studydenabling manipulations to be targeted to speci!c cells and pathways. (It is also possible to target manipulations through other means, such as drugs that acquire cell-type speci!city through targeting via antibodies and/or cell surface receptor ligands, but as far as we are aware, existing reagents fall short in terms of necessary speci!city.) Many types of perturbations are useful in living systems and can be divided into rough categories such as the following: depolarize or hyperpolarize cells, induce or repress the activity of a speci!c pathway, induce or inhibit expression of a particular gene, activate or repress a speci!c protein, degrade a speci!c protein, etc. User-supplied triggers for such manipulations to occur include the following: addition of a small molecule (“chemogenetics”dideally inert on endogenous proteins) [1], sound waves (“sonogenetics”) [2], alteration of temperature (“thermogenetics”d almost exclusively used for small invertebrates) [3], and light (“optogenetics”). There are reports of using magnetic !elds (“magnetogenetics”) [4], but there is no evidence that such effects are reproducible or even physically possible [5,6]. Of these, the most commonly used, for multiple reasons, is light.

Many factors make light an ideal user-controlled stimulus for the manipulation of samples. Light is quickly delivered, and most light-sensitive proteins and other molecules respond quickly to light stimuli, making many optogenetic systems relatively rapid in comparison to, for instance, drug-modulated systems. Light is also quite easy to deliver in localized patterns, allowing for targeted stimulation. Multiple wavelengths can be delivered separately to distinct (or overlapping) regions, potentially allowing combinatorial control of diverse components. Finally, light can be delivered to shallow brain regions (and peripheral sites) relatively noninvasively, and to deeper brain regions with some effort.

However, there are also a number of shortcomings of using light for control. Robust and uniform penetration of light into the sample is the most signi!cant concern. For systems requiring modulation of many cells, particularly at depth, the use of systems controlled by small molecule drugs would generally be recommended instead of optogenetic approaches. When light is delivered through the use of !bers, lenses, or other optical devices, such interventions can produce signi!- cant cellular death, scar formation, and biofouling. The foreign-body response of tissue to objects triggers substantial molecular alterations, the implications of which are incompletely de!ned, but can involve reactive astrogliosis, oxidative stress, and perturbed vascularization. Head-mounted lightdelivery devices can be heavy and/or restrictive, and thus perturb behavior, particularly for small animals (e.g., mouse behavior is much more disrupted than rat behavior). More generally, all light causes tissue heating, which can have dramatic effects on cell health, physiology, and animal behavior. This is most concerning for tiny animals such as "ies. Light itself also damages tissue, most obviously through photochemistry (e.g., oxidation and radicalization) and photobleaching of critical endogenousmolecules. Furthermore, of course, light is ubiquitous, meaning that the sample is never completely unstimulated, despite precautions. Light passes through the eyes into the brain with surprising ease, and even through the skull with modest ef!cacy [7]dwhich can disrupt animal behavior (as can the converse: stimulating light in the brain perceived as a visual stimulus through the back of the eyes.) Light-responsive proteins exist in all samples, particularly in the eyes but to some extent in all tissuesdnotably, deep-brain photoreceptors [8].

The use of optogenetic tools has accelerated research on many fronts in disparate !elds. Additional, perhaps most, limitations on the utility of optogenetics must, however, be placed squarely on the shortcomings of the current suite of tools (and potential inherent limits in their performance.) The vast majority of optogenetic effectors are gated by blue light, which has signi!cant penetration issues and can be phototoxic under high intensity; redder wavelengths would in general be preferred. Furthermore, multiplexing requires tools making use of other parts of the visible spectrum (and redder wavelengths). A related issue is that most chromophores for optogenetic reagents have very broad action spectra (w250 nm bandwidth for retinal; w200 nm bandwidth for "avin), complicating both multiplexing and their use alongside many optical imaging reagentsdnarrower action spectra would be preferred for effectors in most situations. More generally, the current classes of optogenetic effectors are few, mostly limited to (1) channels and pumps (most with poor ion selectivity), (2) dimerizers, and (3) a handful of enzymes. The number of optogenetic tools that perform a very speci!c function in cells is small. Although progress has undeniably been made, much additional research and engineering will be required to dramatically expand the optogenetic toolkit.

Rather than providing a survey of research !ndings, this review covers general considerations of optogenetics experiments, and then focuses largely on molecular tools: the existing suite, their features and limitations, and goals for the creation and validation of additional reagents.