@article {69472, title = {The physical and cellular mechanism of structural color change in zebrafish.}, journal = {Proc Natl Acad Sci U S A}, volume = {121}, year = {2024}, month = {2024 Jun 04}, pages = {e2308531121}, abstract = {

Many animals exhibit remarkable colors that are produced by the constructive interference of light reflected from arrays of intracellular guanine crystals. These animals can fine-tune their crystal-based structural colors to communicate with each other, regulate body temperature, and create camouflage. While it is known that these changes in color are caused by changes in the angle of the crystal arrays relative to incident light, the cellular machinery that drives color change is not understood. Here, using a combination of 3D focused ion beam scanning electron microscopy (FIB-SEM), micro-focused X-ray diffraction, superresolution fluorescence light microscopy, and pharmacological perturbations, we characterized the dynamics and 3D cellular reorganization of crystal arrays within zebrafish iridophores during norepinephrine (NE)-induced color change. We found that color change results from a coordinated 20{\textdegree} tilting of the intracellular crystals, which alters both crystal packing and the angle at which impinging light hits the crystals. Importantly, addition of the dynein inhibitor dynapyrazole-a completely blocked this NE-induced red shift by hindering crystal dynamics upon NE addition. FIB-SEM and microtubule organizing center (MTOC) mapping showed that microtubules arise from two MTOCs located near the poles of the iridophore and run parallel to, and in between, individual crystals. This suggests that dynein drives crystal angle change in response to NE by binding to the limiting membrane surrounding individual crystals and walking toward microtubule minus ends. Finally, we found that intracellular cAMP regulates the color change process. Together, our results provide mechanistic insight into the cellular machinery that drives structural color change.

}, keywords = {Animals, Color, Microscopy, Electron, Scanning, Norepinephrine, Pigmentation, Zebrafish, Zebrafish Proteins}, issn = {1091-6490}, doi = {10.1073/pnas.2308531121}, author = {Gur, Dvir and Moore, Andrew S and Deis, Rachael and Song, Pang and Wu, Xufeng and Pinkas, Iddo and Deo, Claire and Iyer, Nirmala and Hess, Harald F and Hammer, John A and Lippincott-Schwartz, Jennifer} } @article {67896, title = {The complexin C-terminal amphipathic helix stabilizes the fusion pore open state by sculpting membranes.}, journal = {Nature Structural \& Molecular Biology}, volume = {29}, year = {2022}, month = {2022 Feb 07}, pages = {97-107}, abstract = {

Neurotransmitter release is mediated by proteins that drive synaptic vesicle fusion with the presynaptic plasma membrane. While soluble N-ethylmaleimide sensitive factor attachment protein receptors (SNAREs) form the core of the fusion apparatus, additional proteins play key roles in the fusion pathway. Here, we report that the C-terminal amphipathic helix of the mammalian accessory protein, complexin (Cpx), exerts profound effects on membranes, including the formation of pores and the efficient budding and fission of vesicles. Using nanodisc-black lipid membrane electrophysiology, we demonstrate that the membrane remodeling activity of Cpx modulates the structure and stability of recombinant exocytic fusion pores. Cpx had particularly strong effects on pores formed by small numbers of SNAREs. Under these conditions, Cpx increased the current through individual pores 3.5-fold, and increased the open time fraction from roughly 0.1 to 1.0. We propose that the membrane sculpting activity of Cpx contributes to the phospholipid rearrangements that underlie fusion by stabilizing highly curved membrane fusion intermediates.

}, issn = {1545-9985}, doi = {10.1038/s41594-021-00716-0}, author = {Courtney, Kevin C and Wu, Lanxi and Mandal, Taraknath and Swift, Mark and Zhang, Zhao and Alaghemandi, Mohammad and Wu, Zhenyong and Bradberry, Mazdak M and Deo, Claire and Lavis, Luke D and Volkmann, Niels and Hanein, Dorit and Cui, Qiang and Bao, Huan and Chapman, Edwin R} } @article {67197, title = {The HaloTag as a general scaffold for far-red tunable chemigenetic indicators.}, journal = {Nature Chemical Biology}, year = {2021}, month = {2021 Apr 01}, abstract = {

Functional imaging using fluorescent indicators has revolutionized biology, but additional sensor scaffolds are needed to access properties such as bright, far-red emission. Here, we introduce a new platform for {\textquoteright}chemigenetic{\textquoteright} fluorescent indicators, utilizing the self-labeling HaloTag protein conjugated to environmentally sensitive synthetic fluorophores. We solve a crystal structure of HaloTag bound to a rhodamine dye ligand to guide engineering efforts to modulate the dye environment. We show that fusion of HaloTag with protein sensor domains that undergo conformational changes near the bound dye results in large and rapid changes in fluorescence output. This generalizable approach affords bright, far-red calcium and voltage sensors with highly tunable photophysical and chemical properties, which can reliably detect single action potentials in cultured neurons.

}, issn = {1552-4469}, doi = {10.1038/s41589-021-00775-w}, author = {Deo, Claire and Abdelfattah, Ahmed S and Bhargava, Hersh K and Berro, Adam J and Falco, Natalie and Farrants, Helen and Moeyaert, Benjamien and Chupanova, Mariam and Lavis, Luke D and Schreiter, Eric R} } @article {65276, title = {Accurate measurement of fast endocytic recycling kinetics in real time.}, journal = {Journal of Cell Science}, volume = {133}, year = {2020}, month = {2020 Jan 22}, abstract = {

The fast turnover of membrane components through endocytosis and recycling allows precise control of the composition of the plasma membrane. Endocytic recycling can be rapid with some molecules returning to the plasma membrane with a \<5 minutes. Existing methods to study these trafficking pathways utilize chemical, radioactive, or fluorescent labeling of cell surface receptors in pulse-chase experiments, which require tedious washing steps and manual collection of samples. Here, we introduce a live-cell endocytic recycling assay, based on a newly designed cell-impermeable, fluorogenic ligand for HaloTag: {\textquoteright}Janelia Fluor 635i{\textquoteright} (JFi; i=impermeant) which allows real-time detection of membrane receptor recycling at steady state. We used this method to study the effect of iron depletion on transferrin receptor (TfR) recycling using the chelator desferrioxamine. We found this perturbation significantly increases the TfR recycling rate. The high temporal resolution and simplicity of this assay provides a clear advantage over extant methods and makes it ideal for large scale cellular imaging studies. This assay can be adapted to examine other cellular kinetic parameters such as protein turnover and biosynthetic trafficking.

}, issn = {1477-9137}, doi = {10.1242/jcs.231225}, author = {Jonker, Caspar T H and Deo, Claire and Zager, Patrick J and Tkachuk, Ariana N and Weinstein, Alan M and Rodriguez-Boulan, Enrique and Lavis, Luke D and Schreiner, Ryan} } @article {65274, title = {Bright and tunable far-red chemigenetic indicators.}, journal = {bioRxiv}, year = {2020}, month = {2020 Jan 9}, author = {Deo, Claire and Abdelfattah, Ahmed S and Bhargava, Hersh K. and Berro, Adam J. and Falco, Natalie and Moeyaert, Benjamien and Chupanova,Mariam and Lavis, Luke D and Schreiter, Eric R} } @article {65150, title = {Isomeric tuning yields bright and targetable red Ca indicators.}, journal = {Journal of the American Chemical Society}, volume = {141}, year = {2019}, month = {2019 Sep 04}, pages = {13734-13738}, abstract = {

Targeting small-molecule fluorescent indicators using genetically encoded protein tags yields new hybrid sensors for biological imaging. Optimization of such systems requires redesign of the synthetic indicator to allow cell-specific targeting without compromising the photophysical properties or cellular performance of the small-molecule probe. We developed a bright and sensitive Ca indicator by systematically exploring the relative configuration of dye and chelator, which can be targeted using the HaloTag self-labeling tag system. Our "isomeric tuning" approach is generalizable, yielding a far-red targetable indicator to visualize Ca fluxes in the primary cilium.

}, issn = {1520-5126}, doi = {10.1021/jacs.9b06092}, author = {Deo, Claire and Sheu, Shu-Hsien and Seo, Jinyoung and Clapham, David E and Lavis, Luke D} } @article {49050, title = {Synthetic and genetically encoded fluorescent neural activity indicators.}, journal = {Current Opinion in Neurobiology}, volume = {50}, year = {2018}, month = {2018 Feb 14}, pages = {101-108}, abstract = {

The ultimate goal of neuroscience is to relate the complex activity of cells and cell-networks to behavior and cognition. This requires tools and techniques to visualize neuronal activity. Fluorescence microscopy is an ideal tool to measure activity of cells in the brain due to the high sensitivity of the technique and the growing portfolio of optical hardware and fluorescent sensors. Here, we give a chemist{\textquoteright}s perspective on the recent progress of fluorescent activity indicators that enable the measurement of cellular events in the living brain. We discuss advances in both chemical and genetically encoded sensors and look forward to hybrid indicators, which incorporate synthetic organic dyes into genetically encoded protein constructs.

}, issn = {1873-6882}, doi = {10.1016/j.conb.2018.01.003}, author = {Deo, Claire and Lavis, Luke D} }