@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 {68167, title = {Super-Resolution Imaging of Fas/CD95 Reorganization Induced by Membrane-Bound Fas Ligand Reveals Nanoscale Clustering Upstream of FADD Recruitment.}, journal = {Cells}, volume = {11}, year = {2022}, month = {2022 Jun 12}, abstract = {

Signaling through the TNF-family receptor Fas/CD95 can trigger apoptosis or non-apoptotic cellular responses and is essential for protection from autoimmunity. Receptor clustering has been observed following interaction with Fas ligand (FasL), but the stoichiometry of Fas, particularly when triggered by membrane-bound FasL, the only form of FasL competent at inducing programmed cell death, is not known. Here we used super-resolution microscopy to study the behavior of single molecules of Fas/CD95 on the plasma membrane after interaction of Fas with FasL on planar lipid bilayers. We observed rapid formation of Fas protein superclusters containing more than 20 receptors after interactions with membrane-bound FasL. Fluorescence correlation imaging demonstrated recruitment of FADD dependent on an intact Fas death domain, with lipid raft association playing a secondary role. Flow-cytometric FRET analysis confirmed these results, and also showed that some Fas clustering can occur in the absence of FADD and caspase-8. Point mutations in the Fas death domain associated with autoimmune lymphoproliferative syndrome (ALPS) completely disrupted Fas reorganization and FADD recruitment, confirming structure-based predictions of the critical role that these residues play in Fas-Fas and Fas-FADD interactions. Finally, we showed that induction of apoptosis correlated with the ability to form superclusters and recruit FADD.

}, keywords = {Apoptosis, Cluster Analysis, Fas Ligand Protein, fas Receptor, Fas-Associated Death Domain Protein, Receptors, Tumor Necrosis Factor}, issn = {2073-4409}, doi = {10.3390/cells11121908}, author = {Frazzette, Nicholas and Cruz, Anthony C and Wu, Xufeng and Hammer, John A and Lippincott-Schwartz, Jennifer and Siegel, Richard M and Sengupta, Prabuddha} } @article {47993, title = {Formin-generated actomyosin arcs propel T cell receptor microcluster movement at the immune synapse.}, journal = {The Journal of Cell Biology}, volume = {215}, year = {2016}, month = {2016 Oct 31}, pages = {383-99}, abstract = {

Actin assembly and inward flow in the plane of the immunological synapse (IS) drives the centralization of T cell receptor microclusters (TCR MCs) and the integrin leukocyte functional antigen 1 (LFA-1). Using structured-illumination microscopy (SIM), we show that actin arcs populating the medial, lamella-like region of the IS arise from linear actin filaments generated by one or more formins present at the IS distal edge. After traversing the outer, Arp2/3-generated, lamellipodia-like region of the IS, these linear filaments are organized by myosin II into antiparallel concentric arcs. Three-dimensional SIM shows that active LFA-1 often aligns with arcs, whereas TCR MCs commonly reside between arcs, and total internal reflection fluorescence SIM shows TCR MCs being swept inward by arcs. Consistently, disrupting actin arc formation via formin inhibition results in less centralized TCR MCs, missegregated integrin clusters, decreased T-B cell adhesion, and diminished TCR signaling. Together, our results define the origin, organization, and functional significance of a major actomyosin contractile structure at the IS that directly propels TCR MC transport.

}, issn = {1540-8140}, doi = {10.1083/jcb.201603080}, author = {Murugesan, Sricharan and Hong, Jinsung and Yi, Jason and Li, Dong and Beach, Jordan R and Shao, Lin and Meinhardt, John and Madison, Grey and Wu, Xufeng and Betzig, Eric and Hammer, John A} } @article {6293, title = {Noninvasive imaging beyond the diffraction limit of 3D dynamics in thickly fluorescent specimens.}, journal = {Cell}, volume = {151}, year = {2012}, month = {2012 Dec 7}, pages = {1370-85}, abstract = {

Optical imaging of the dynamics of living specimens involves tradeoffs between spatial resolution, temporal resolution, and phototoxicity, made more difficult in three dimensions. Here, however, we report that rapid three-dimensional (3D) dynamics can be studied beyond the diffraction limit in thick or densely fluorescent living specimens over many time points by combining ultrathin planar illumination\ produced by scanned Bessel beams with super-resolution structured illumination microscopy. We demonstrate in\ vivo karyotyping of chromosomes during mitosis and identify different dynamics for the actin cytoskeleton at the dorsal and ventral surfaces of fibroblasts. Compared to spinning disk confocal microscopy, we\ demonstrate substantially reduced photodamage when imaging rapid morphological changes in D.\ discoideum cells, as well as improved contrast and resolution at depth within developing C.\ elegans embryos. Bessel beam structured plane illumination thus promises new insights into complex biological phenomena that require 4D subcellular spatiotemporal detail in either a single or multicellular context.

}, keywords = {Animals, Brain, Caenorhabditis elegans, Cell Line, Dermatitis, Dictyostelium, Drosophila melanogaster, Fibroblasts, Fluorescence, Humans, Imaging, Karyotyping, Larva, Microscopy, Mitosis, Optical Imaging, Phototoxic, Three-Dimensional, Tumor}, issn = {1097-4172}, doi = {10.1016/j.cell.2012.10.008}, author = {Gao, Liang and Shao, Lin and Higgins, Christopher D and Poulton, John S and Peifer, Mark and Davidson, Michael W and Wu, Xufeng and Goldstein, Bob and Betzig, Eric} }