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.

Optogenetic tools can be used to manipulate neuronal activity in a reversible and specific manner. In recent years, such methods have been applied to uncover causal relationships between activity in specified neuronal circuits and behavior in the larval zebrafish. In this small, transparent, genetic model organism, noninvasive manipulation and monitoring of neuronal activity with light is possible throughout the nervous system. Here we review recent work in which these new tools have been applied to zebrafish, and discuss some of the existing challenges of these approaches.

Optogenetics combines optics and genetics to control neuronal activity with cell-type specificity and millisecond temporal precision. Its use in model organisms such as rodents, Drosophila, and Caenorhabditis elegans is now well-established. However, application of this technology in nonhuman primates (NHPs) has been slow to develop. One key challenge has been the delivery of viruses and light to the brain through the thick dura mater of NHPs, which can only be penetrated with large-diameter devices that damage the brain. The opacity of the NHP dura prevents visualization of the underlying cortex, limiting the spatial precision of virus injections, electrophysiological recordings, and photostimulation. Here, we describe a new optogenetics approach in which the native dura is replaced with an optically transparent artificial dura. This artificial dura can be penetrated with fine glass micropipettes, enabling precisely targeted injections of virus into brain tissue with minimal damage to cortex. The expression of optogenetic agents can be monitored visually over time. Most critically, this optical window permits targeted, noninvasive photostimulation and concomitant measurements of neuronal activity via intrinsic signal imaging and electrophysiological recordings. We present results from both anesthetized-paralyzed (optical imaging) and awake-behaving NHPs (electrophysiology). The improvements over current methods made possible by the artificial dura should enable the widespread use of optogenetic tools in NHP research, a key step toward the development of therapies for neuropsychiatric and neurological diseases in humans.

Research on the biology of addiction has advanced significantly over the last 50 years expanding our understanding of the brain mechanisms underlying reward, reinforcement and craving. Novel experimental approaches and techniques have provided an ever increasing armory of tools to dissect behavioral processes, neural networks and molecular mechanisms. The ultimate goal is to reintegrate this knowledge into a coherent, mechanistic framework of addiction to help identify new treatment. This can be greatly facilitated by using tools that allow, with great spatial and temporal specificity, to link molecular changes with altered activation of neural circuits and behavior. Such specificity can now be achieved by using optogenetic tools. Our review describes the general principles of optogenetics and its use to understand the links between neural activity and behavior. We also provide an overview of recent studies using optogenetic tools in addiction and consider some outstanding questions of addiction research that are particularly amenable for optogenetic approaches.

Lysosomes are active sites to integrate cellular metabolism and signal transduction. A collection of proteins enriched at lysosomes mediate these metabolic and signaling functions. Both lysosomal metabolism and lysosomal signaling have been linked with longevity regulation; however, how lysosomes adjust their protein composition to accommodate this regulation remains unclear. Using large-scale proteomic profiling, we systemically profiled lysosome- enriched proteomes in association with different longevity mechanisms. We further discovered the lysosomal recruitment of AMPK and nucleoporin proteins and their requirements for longevity in response to increased lysosomal lipolysis. Through comparative proteomic analyses of lysosomes from different tissues and labeled with different markers, we discovered lysosomal heterogeneity across tissues as well as the specific enrichment of the Ragulator complex on Cystinosin positive lysosomes. Together, this work uncovers lysosomal proteome heterogeneity at different levels and provides resources for understanding the contribution of lysosomal proteome dynamics in modulating signal transduction, organelle crosstalk and organism longevity.

The visual system of Drosophila is an excellent model for determining the interactions that direct the differentiation of the nervous system’s many unique cell types. Glia are essential not only in the development of the nervous system, but also in the function of those neurons with which they become associated in the adult. Given their role in visual system development and adult function we need to both accurately and reliably identify the different subtypes of glia, and to relate the glial subtypes in the larval brain to those previously described for the adult. We viewed driver expression in subsets of larval eye disc glia through the earliest stages of pupal development to reveal the counterparts of these cells in the adult. Two populations of glia exist in the lamina, the first neuropil of the adult optic lobe: those that arise from precursors in the eye-disc/optic stalk and those that arise from precursors in the brain. In both cases, a single larval source gives rise to at least three different types of adult glia. Furthermore, analysis of glial cell types in the second neuropil, the medulla, has identified at least four types of astrocyte-like (reticular) glia. Our clarification of the lamina’s adult glia and identification of their larval origins, particularly the respective eye disc and larval brain contributions, begin to define developmental interactions which establish the different subtypes of glia.

Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adult Drosophila central brain, we identify five different types of glia based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement of gcm (glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in a gcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in the Drosophila brain.

Natural behaviors are a coordinated symphony of motor acts which drive self-induced or reafferent sensory activation. Single sensors only signal presence and magnitude of a sensory cue; they cannot disambiguate exafferent (externally-induced) from reafferent sources. Nevertheless, animals readily differentiate between these sources of sensory signals to make appropriate decisions and initiate adaptive behavioral outcomes. This is mediated by predictive motor signaling mechanisms, which emanate from motor control pathways to sensory processing pathways, but how predictive motor signaling circuits function at the cellular and synaptic level is poorly understood. We use a variety of techniques, including connectomics from both male and female electron microscopy volumes, transcriptomics, neuroanatomical, physiological and behavioral approaches to resolve the network architecture of two pairs of ascending histaminergic neurons (AHNs), which putatively provide predictive motor signals to several sensory and motor neuropil. Both AHN pairs receive input primarily from an overlapping population of descending neurons, many of which drive wing motor output. The two AHN pairs target almost exclusively non-overlapping downstream neural networks including those that process visual, auditory and mechanosensory information as well as networks coordinating wing, haltere, and leg motor output. These results support the conclusion that the AHN pairs multi-task, integrating a large amount of common input, then tile their output in the brain, providing predictive motor signals to non-overlapping sensory networks affecting motor control both directly and indirectly.