Nicotinic partial agonists provide an accepted aid for smoking cessation and thus contribute to decreasing tobacco-related disease. Improved drugs constitute a continued area of study. However, there remains no reductionist method to examine the cellular and subcellular pharmacokinetic properties of these compounds in living cells. Here, we developed new intensity-based drug sensing fluorescent reporters ('iDrugSnFRs') for the nicotinic partial agonists dianicline, cytisine, and two cytisine derivatives - 10-fluorocytisine and 9-bromo-10-ethylcytisine. We report the first atomic-scale structures of liganded periplasmic binding protein-based biosensors, accelerating development of iDrugSnFRs and also explaining the activation mechanism. The nicotinic iDrugSnFRs detect their drug partners in solution, as well as at the plasma membrane (PM) and in the endoplasmic reticulum (ER) of cell lines and mouse hippocampal neurons. At the PM, the speed of solution changes limits the growth and decay rates of the fluorescence response in almost all cases. In contrast, we found that rates of membrane crossing differ among these nicotinic drugs by > 30 fold. The new nicotinic iDrugSnFRs provide insight into the real-time pharmacokinetic properties of nicotinic agonists and provide a methodology whereby iDrugSnFRs can inform both pharmaceutical neuroscience and addiction neuroscience.

Visualization of specific molecules and their assembly in real time and space is essential to delineate how cellular dynamics and signaling circuit are orchestrated during cell division cycle. Our recent studies reveal structural insights into human centromere-kinetochore core CCAN complex. Here we introduce a method for optically imaging trimeric and tetrameric protein interactions at nanometer spatial resolution in live cells using fluorescence complementation-based Förster resonance energy transfer (FC-FRET). Complementary fluorescent protein molecules were first used to visualize dimerization followed by FRET measurements. Using FC- FRET, we visualized centromere CENP-SXTW tetramer assembly dynamics in live cells, and dimeric interactions between CENP-TW dimer and kinetochore protein Spc24/25 dimer in dividing cells. We further delineated the interactions of monomeric CENP-T with Spc24/25 dimer in dividing cells. Surprisingly, our analyses revealed critical role of CDK1 kinase activity in the initial recruitment of Spc24/25 by CENP-T. However, interactions between CENP-T and Spc24/25 during chromosome segregation is independent of CDK1. Thus, FC-FRET provides a unique approach to delineate spatiotemporal dynamics of trimerized and tetramerized proteins at nanometer scale and establishes a platform to report the precise regulation of multimeric protein interactions in space and time in live cells.

Fluorescence imaging has revolutionized biomedical research over the past three decades. Its high molecular specificity and unrivalled single-molecule-level sensitivity have enabled breakthroughs in a number of research fields. For in vivo applications its major limitation is its superficial imaging depth, a result of random scattering in biological tissues causing exponential attenuation of the ballistic component of a light wave. Here, we present fluorescence imaging beyond the ballistic regime by combining single-cycle pulsed ultrasound modulation and digital optical phase conjugation. We demonstrate a near-isotropic three-dimensional localized sound–light interaction zone. With the exceptionally high optical gain provided by the digital optical phase conjugation system, we can deliver sufficient optical power to a focus inside highly scattering media for not only fluorescence imaging but also a variety of linear and nonlinear spectroscopy measurements. This technology paves the way for many important applications in both fundamental biology research and clinical studies.

Subcellular pharmacokinetic measurements have informed the study of central nervous system (CNS)-acting drug mechanisms. Recent investigations have been enhanced by the use of genetically encoded fluorescent biosensors for drugs of interest at the plasma membrane and in organelles. We describe screening and validation protocols for identifying hit pairs comprising a drug and biosensor, with each screen including 13-18 candidate biosensors and 44-84 candidate drugs. After a favorable hit pair is identified and validated via these protocols, the biosensor is then optimized, as described in other papers, for sensitivity and selectivity to the drug. We also show sample hit pair data that may lead to future intensity-based drug-sensing fluorescent reporters (iDrugSnFRs). These protocols will assist scientists to use fluorescence responses as criteria in identifying favorable fluorescent biosensor variants for CNS-acting drugs that presently have no corresponding biosensor partner. eLife (2022), DOI: 10.7554/eLife.74648 Graphical abstract.

Fluorescent indicators and actuators provide a means to optically observe and perturb dynamic events in living animals. Although chemistry and protein engineering have contributed many useful tools to observe and perturb cells, an emerging strategy is to use chemigenetics: systems in which a small molecule dye interacts with a genetically encoded protein domain. Here we review chemigenetic strategies that have been successfully employed in living animals as photosensitizers for photoablation experiments, fluorescent cell cycle indicators, and fluorescent indicators for studying dynamic biological signals. Although these strategies at times suffer from challenges, e.g. delivery of the small molecule and assembly of the chemigenetic unit in living animals, the advantages of using small molecules with high brightness, low photobleaching, no chromophore maturation time and expanded color palette, combined with the ability to genetically target them to specific cell types, make chemigenetic fluorescent actuators and indicators an attractive strategy for use in living animals.

Cellular esterases catalyze many essential biological functions by performing hydrolysis reactions on diverse substrates. The promiscuity of esterases complicates assignment of their substrate preferences and biological functions. To identify universal factors controlling esterase substrate recognition, we designed a 32-member structure-activity relationship (SAR) library of fluorogenic ester substrates and used this library to systematically interrogate esterase preference for chain length, branching patterns, and polarity to differentiate common classes of esterase substrates. Two structurally homologous bacterial esterases were screened against this library, refining their previously broad overlapping substrate specificity. esterase ybfF displayed a preference for γ-position thioethers and ethers, whereas Rv0045c from displayed a preference for branched substrates with and without thioethers. We determined that this substrate differentiation was partially controlled by individual substrate selectivity residues Tyr119 in ybfF and His187 in Rv0045c; reciprocal substitution of these residues shifted each esterase's substrate preference. This work demonstrates that the selectivity of esterases is tuned based on transition state stabilization, identifies thioethers as an underutilized functional group for esterase substrates, and provides a rapid method for differentiating structural isozymes. This SAR library could have multi-faceted future applications including in vivo imaging, biocatalyst screening, molecular fingerprinting, and inhibitor design.

Flies and other insects use vision to regulate their groundspeed in flight, enabling them to fly in varying wind conditions. Compared with mechanosensory modalities, however, vision requires a long processing delay ( 100 ms) that might introduce instability if operated at high gain. Flies also sense air motion with their antennae, but how this is used in flight control is unknown. We manipulated the antennal function of fruit flies by ablating their aristae, forcing them to rely on vision alone to regulate groundspeed. Arista-ablated flies in flight exhibited significantly greater groundspeed variability than intact flies. We then subjected them to a series of controlled impulsive wind gusts delivered by an air piston and experimentally manipulated antennae and visual feedback. The results show that an antenna-mediated response alters wing motion to cause flies to accelerate in the same direction as the gust. This response opposes flying into a headwind, but flies regularly fly upwind. To resolve this discrepancy, we obtained a dynamic model of the fly’s velocity regulator by fitting parameters of candidate models to our experimental data. The model suggests that the groundspeed variability of arista-ablated flies is the result of unstable feedback oscillations caused by the delay and high gain of visual feedback. The antenna response drives active damping with a shorter delay ( 20 ms) to stabilize this regulator, in exchange for increasing the effect of rapid wind disturbances. This provides insight into flies’ multimodal sensory feedback architecture and constitutes a previously unknown role for the antennae.

Rapidly and selectively modulating the activity of defined neurons in unrestrained animals is a powerful approach in investigating the circuit mechanisms that shape behavior. In Drosophila melanogaster, temperature-sensitive silencers and activators are widely used to control the activities of genetically defined neuronal cell types. A limitation of these thermogenetic approaches, however, has been their poor temporal resolution. Here we introduce FlyMAD (the fly mind-altering device), which allows thermogenetic silencing or activation within seconds or even fractions of a second. Using computer vision, FlyMAD targets an infrared laser to freely walking flies. As a proof of principle, we demonstrated the rapid silencing and activation of neurons involved in locomotion, vision and courtship. The spatial resolution of the focused beam enabled preferential targeting of neurons in the brain or ventral nerve cord. Moreover, the high temporal resolution of FlyMAD allowed us to discover distinct timing relationships for two neuronal cell types previously linked to courtship song.

Filopodia are peripheral F-actin-rich structures that enable cell sensing of the microenvironment. Fascin is an F-actin-bundling protein that plays a key role in stabilizing filopodia to support efficient adhesion and migration. Fascin is also highly up-regulated in human cancers, where it increases invasive cell behavior and correlates with poor patient prognosis. Previous studies have shown that fascin phosphorylation can regulate F-actin bundling, and that this modification can contribute to subcellular fascin localization and function. However, the factors that regulate fascin dynamics within filopodia remain poorly understood. In the current study, we used advanced live-cell imaging techniques and a fascin biosensor to demonstrate that fascin phosphorylation, localization, and binding to F-actin are highly dynamic and dependent on local cytoskeletal architecture in cells in both 2D and 3D environments. Fascin dynamics within filopodia are under the control of formins, and in particular FMNL2, that binds directly to dephosphorylated fascin. Our data provide new insight into control of fascin dynamics at the nanoscale and into the mechanisms governing rapid cytoskeletal adaptation to environmental changes. This filopodia-driven exploration stage may represent an essential regulatory step in the transition from static to migrating cancer cells.

Focal adhesions (FAs) connect inner workings of cell to the extracellular matrix to control cell adhesion, migration and mechanosensing. Previous studies demonstrated that FAs contain three vertical layers, which connect extracellular matrix to the cytoskeleton. By using super-resolution iPALM microscopy, we identify two additional nanoscale layers within FAs, specified by actin filaments bound to tropomyosin isoforms Tpm1.6 and Tpm3.2. The Tpm1.6-actin filaments, beneath the previously identified α-actinin cross-linked actin filaments, appear critical for adhesion maturation and controlled cell motility, whereas the adjacent Tpm3.2-actin filament layer beneath seems to facilitate adhesion disassembly. Mechanistically, Tpm3.2 stabilizes ACF-7/MACF1 and KANK-family proteins at adhesions, and hence targets microtubule plus-ends to FAs to catalyse their disassembly. Tpm3.2 depletion leads to disorganized microtubule network, abnormally stable FAs, and defects in tail retraction during migration. Thus, FAs are composed of distinct actin filament layers, and each may have specific roles in coupling adhesions to the cytoskeleton, or in controlling adhesion dynamics.