@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 {48218, title = {Actin dynamics and competition for myosin monomer govern the sequential amplification of myosin filaments.}, journal = {Nature Cell Biology}, volume = {19}, year = {2017}, month = {2017 Jan 23}, pages = {85-93}, abstract = {

The cellular mechanisms governing non-muscle myosin II (NM2) filament assembly are largely unknown. Using EGFP-NM2A knock-in fibroblasts and multiple super-resolution imaging modalities, we characterized and quantified the sequential amplification of NM2 filaments within lamellae, wherein filaments emanating from single nucleation events continuously partition, forming filament clusters that populate large-scale actomyosin structures deeper in the cell. Individual partitioning events coincide spatially and temporally with the movements of diverging actin fibres, suppression of which inhibits partitioning. These and other data indicate that NM2A filaments are partitioned by the dynamic movements of actin fibres to which they are bound. Finally, we showed that partition frequency and filament growth rate in the lamella depend on MLCK, and that MLCK is competing with centrally active ROCK for a limiting pool of monomer with which to drive lamellar filament assembly. Together, our results provide new insights into the mechanism and spatio-temporal regulation of NM2 filament assembly in cells.

}, issn = {1476-4679}, doi = {10.1038/ncb3463}, author = {Beach, Jordan R and Bruun, Kyle S and Shao, Lin and Li, Dong and Swider, Zac and Remmert, Kirsten and Zhang, Yingfan and Conti, Mary A and Adelstein, Robert S and Rusan, Nasser M and Betzig, Eric and Hammer, John A} } @article {48750, title = {Contractile actomyosin arcs promote the activation of primary mouse T cells in a ligand-dependent manner.}, journal = {PLoS One}, volume = {12}, year = {2017}, month = {2017}, pages = {e0183174}, abstract = {

Mechano-transduction is an emerging but still poorly understood component of T cell activation. Here we investigated the ligand-dependent contribution made by contractile actomyosin arcs populating the peripheral supramolecular activation cluster (pSMAC) region of the immunological synapse (IS) to T cell receptor (TCR) microcluster transport and proximal signaling in primary mouse T cells. Using super resolution microscopy, OT1-CD8+ mouse T cells, and two ovalbumin (OVA) peptides with different affinities for the TCR, we show that the generation of organized actomyosin arcs depends on ligand potency and the ability of myosin 2 to contract actin filaments. While weak ligands induce disorganized actomyosin arcs, strong ligands result in organized actomyosin arcs that correlate well with tension-sensitive CasL phosphorylation and the accumulation of ligands at the IS center. Blocking myosin 2 contractility greatly reduces the difference in the extent of Src and LAT phosphorylation observed between the strong and the weak ligand, arguing that myosin 2-dependent force generation within actin arcs contributes to ligand discrimination. Together, our data are consistent with the idea that actomyosin arcs in the pSMAC region of the IS promote a mechano-chemical feedback mechanism that amplifies the accumulation of critical signaling molecules at the IS.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0183174}, author = {Hong, Jinsung and Murugesan, Sricharan and Betzig, Eric and Hammer, John A} } @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 {47971, title = {V-1 regulates capping protein activity in vivo.}, journal = {Proceedings of the National Academy of Sciences of the United States of America}, volume = {113}, year = {2016}, month = {2016 Oct 25}, pages = {E6610-9}, abstract = {

Capping Protein (CP) plays a central role in the creation of the Arp2/3-generated branched actin networks comprising lamellipodia and pseudopodia by virtue of its ability to cap the actin filament barbed end, which promotes Arp2/3-dependent filament nucleation and optimal branching. The highly conserved protein V-1/Myotrophin binds CP tightly in vitro to render it incapable of binding the barbed end. Here we addressed the physiological significance of this CP antagonist in Dictyostelium, which expresses a V-1 homolog that we show is very similar biochemically to mouse V-1. Consistent with previous studies of CP knockdown, overexpression of V-1 in Dictyostelium reduced the size of pseudopodia and the cortical content of Arp2/3 and induced the formation of filopodia. Importantly, these effects scaled positively with the degree of V-1 overexpression and were not seen with a V-1 mutant that cannot bind CP. V-1 is present in molar excess over CP, suggesting that it suppresses CP activity in the cytoplasm at steady state. Consistently, cells devoid of V-1, like cells overexpressing CP described previously, exhibited a significant decrease in cellular F-actin content. Moreover, V-1-null cells exhibited pronounced defects in macropinocytosis and chemotactic aggregation that were rescued by V-1, but not by the V-1 mutant. Together, these observations demonstrate that V-1 exerts significant influence in vivo on major actin-based processes via its ability to sequester CP. Finally, we present evidence that V-1{\textquoteright}s ability to sequester CP is regulated by phosphorylation, suggesting that cells may manipulate the level of active CP to tune their "actin phenotype."

}, issn = {1091-6490}, doi = {10.1073/pnas.1605350113}, author = {Jung, Goeh and Alexander, Christopher J and Wu, Xufeng S and Piszczek, Grzegorz and Chen, Bi-Chang and Betzig, Eric and Hammer, John A} } @article {47020, title = {Extended-resolution structured illumination imaging of endocytic and cytoskeletal dynamics.}, journal = {Science (New York, N.Y.)}, volume = {349}, year = {2015}, month = {2015 Aug 28}, abstract = {

Super-resolution fluorescence microscopy is distinct among nanoscale imaging tools in its ability to image protein dynamics in living cells. Structured illumination microscopy (SIM) stands out in this regard because of its high speed and low illumination intensities, but typically offers only a twofold resolution gain. We extended the resolution of live-cell SIM through two approaches: ultrahigh numerical aperture SIM at 84-nanometer lateral resolution for more than 100 multicolor frames, and nonlinear SIM with patterned activation at 45- to 62-nanometer resolution for approximately 20 to 40 frames. We applied these approaches to image dynamics near the plasma membrane of spatially resolved assemblies of clathrin and caveolin, Rab5a in early endosomes, and α-actinin, often in relationship to cortical actin. In addition, we examined mitochondria, actin, and the Golgi apparatus dynamics in three dimensions.

}, issn = {1095-9203}, doi = {10.1126/science.aab3500}, author = {Li, Dong and Shao, Lin and Chen, Bi-Chang and Zhang, Xi and Zhang, Mingshu and Moses, Brian and Milkie, Daniel E and Beach, Jordan R and Hammer, John A and Pasham, Mithun and Kirchhausen, Tomas and Baird, Michelle A and Davidson, Michael W and Xu, Pingyong and Betzig, Eric} } @article {46527, title = {Myosin 18A coassembles with nonmuscle myosin 2 to form mixed bipolar filaments.}, journal = {Current Biology}, volume = {25}, year = {2015}, month = {2015 Mar 30}, pages = {942-8}, abstract = {

Class-18 myosins are most closely related to conventional class-2 nonmuscle myosins (NM2). Surprisingly, the purified head domains of Drosophila, mouse, and human myosin 18A (M18A) lack actin-activated ATPase activity and the ability to translocate actin filaments, suggesting that the functions of M18A in\ vivo do not depend on intrinsic motor activity. M18A has the longest coiled coil of any myosin outside of the class-2 myosins, suggesting that it might form bipolar filaments similar to conventional myosins. To address this possibility, we expressed and purified full-length mouse M18A using the baculovirus/Sf9 system. M18A did not form large bipolar filaments under any of the conditions tested. Instead, M18A formed an \~{}65-nm-long bipolar structure with two heads at each end. Importantly, when NM2 was polymerized in the presence of M18A, the two myosins formed mixed bipolar filaments, as evidenced by cosedimentation, electron microscopy, and single-molecule imaging. Moreover, super-resolution imaging of NM2 and M18A using fluorescently tagged proteins and immunostaining of endogenous proteins showed that NM2 and M18A are present together within individual filaments inside living cells. Together, our in\ vitro and live-cell imaging data argue strongly that M18A coassembles with NM2 into mixed bipolar filaments. M18A could regulate the biophysical properties of these filaments and, by virtue of its extra N- and C-terminal domains, determine the localization and/or molecular interactions of the filaments. Given the numerous, fundamental cellular and developmental roles attributed to NM2, our results have far-reaching biological implications.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2015.02.012}, author = {Billington, Neil and Beach, Jordan R and Heissler, Sarah M and Remmert, Kirsten and Guzik-Lendrum, Stephanie and Nagy, Attila and Takagi, Yasuharu and Shao, Lin and Li, Dong and Yang, Yi and Zhang, Yingfan and Barzik, Melanie and Betzig, Eric and Hammer, John A and Sellers, James R} } @article {45617, title = {Lattice light-sheet microscopy: imaging molecules to embryos at high spatiotemporal resolution.}, journal = {Science}, volume = {346}, year = {2014}, month = {2014 Oct 24}, pages = {1257998}, abstract = {

Although fluorescence microscopy provides a crucial window into the physiology of living specimens, many biological processes are too fragile, are too small, or occur too rapidly to see clearly with existing tools. We crafted ultrathin light sheets from two-dimensional optical lattices that allowed us to image three-dimensional (3D) dynamics for hundreds of volumes, often at subsecond intervals, at the diffraction limit and beyond. We applied this to systems spanning four orders of magnitude in space and time, including the diffusion of single transcription factor molecules in stem cell spheroids, the dynamic instability of mitotic microtubules, the immunological synapse, neutrophil motility in a 3D matrix, and embryogenesis in Caenorhabditis elegans and Drosophila melanogaster. The results provide a visceral reminder of the beauty and the complexity of living systems.

}, issn = {1095-9203}, doi = {10.1126/science.1257998}, author = {Chen, Bi-Chang and Legant, Wesley R and Wang, Kai and Shao, Lin and Milkie, Daniel E and Davidson, Michael W and Janetopoulos, Chris and Wu, Xufeng S and Hammer, John A and Liu, Zhe and English, Brian P and Mimori-Kiyosue, Yuko and Romero, Daniel P and Ritter, Alex T and Lippincott-Schwartz, Jennifer and Fritz-Laylin, Lillian and Mullins, R Dyche and Mitchell, Diana M and Bembenek, Joshua N and Reymann, Anne-Cecile and B{\"o}hme, Ralph and Grill, Stephan W and Wang, Jennifer T and Seydoux, Geraldine and Tulu, U Serdar and Kiehart, Daniel P and Betzig, Eric} } @article {45152, title = {Nonmuscle myosin II isoforms coassemble in living cells.}, journal = {Current Biology}, volume = {24}, year = {2014}, month = {2014 May 19}, pages = {1160-6}, abstract = {

Nonmuscle myosin II (NM II) powers myriad developmental and cellular processes, including embryogenesis, cell migration, and cytokinesis [1]. To exert its functions, monomers of NM II assemble into bipolar filaments that produce a contractile force on the actin cytoskeleton. Mammalian cells express up to three isoforms of NM II (NM IIA, IIB, and IIC), each of which possesses distinct biophysical properties and supports unique as well as redundant cellular functions [2-8]. Despite previous efforts [9-13], it remains unclear whether NM II isoforms assemble in living cells to produce mixed (heterotypic) bipolar filaments or whether filaments consist entirely of a single isoform (homotypic). We addressed this question using fluorescently tagged versions of NM IIA, IIB, and IIC, isoform-specific immunostaining of the endogenous proteins, and two-color total internal reflection fluorescence structured-illumination microscopy, or TIRF-SIM, to visualize individual myosin II bipolar filaments inside cells. We show that NM II isoforms coassemble into heterotypic filaments in a variety of settings, including various types of stress fibers, individual filaments throughout the cell, and the contractile ring. We also show that the differential distribution of NM IIA and NM IIB typically seen in confocal micrographs of well-polarized cells is reflected in the composition of individual bipolar filaments. Interestingly, this differential distribution is less pronounced in freshly spread cells, arguing for the existence of a sorting mechanism acting over time. Together, our work argues that individual NM II isoforms are potentially performing both isoform-specific and isoform-redundant functions while coassembled with other NM II isoforms.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2014.03.071}, author = {Beach, Jordan R and Shao, Lin and Remmert, Kirsten and Li, Dong and Betzig, Eric and Hammer, John A} } @article {47647, title = {Rab10 and myosin-Va mediate insulin-stimulated GLUT4 storage vesicle translocation in adipocytes.}, journal = {The Journal of cell biology}, volume = {198}, year = {2012}, month = {2012 Aug 20}, pages = {545-60}, abstract = {

Rab proteins are important regulators of insulin-stimulated GLUT4 translocation to the plasma membrane (PM), but the precise steps in GLUT4 trafficking modulated by particular Rab proteins remain unclear. Here, we systematically investigate the involvement of Rab proteins in GLUT4 trafficking, focusing on Rab proteins directly mediating GLUT4 storage vesicle (GSV) delivery to the PM. Using dual-color total internal reflection fluorescence (TIRF) microscopy and an insulin-responsive aminopeptidase (IRAP)-pHluorin fusion assay, we demonstrated that Rab10 directly facilitated GSV translocation to and docking at the PM. Rab14 mediated GLUT4 delivery to the PM via endosomal compartments containing transferrin receptor (TfR), whereas Rab4A, Rab4B, and Rab8A recycled GLUT4 through the endosomal system. Myosin-Va associated with GSVs by interacting with Rab10, positioning peripherally recruited GSVs for ultimate fusion. Thus, multiple Rab proteins regulate the trafficking of GLUT4, with Rab10 coordinating with myosin-Va to mediate the final steps of insulin-stimulated GSV translocation to the PM.

}, keywords = {3T3-L1 Cells, Adipocytes, Animals, Cell Membrane, Cytoplasmic Vesicles, Glucose Transporter Type 4, Insulin, Mice, Myosin Heavy Chains, Myosin Type V, Protein Transport, rab GTP-Binding Proteins}, issn = {1540-8140}, doi = {10.1083/jcb.201111091}, author = {Chen, Yu and Wang, Yan and Zhang, Jinzhong and Deng, Yongqiang and Jiang, Li and Song, Eli and Wu, Xufeng S and Hammer, John A and Xu, Tao and Lippincott-Schwartz, Jennifer} }