@article {65854, title = {Improved genetically encoded near-infrared fluorescent calcium ion indicators for in vivo imaging.}, journal = {PLoS Biology}, volume = {18}, year = {2020}, month = {2020 Nov 24}, pages = {e3000965}, abstract = {

Near-infrared (NIR) genetically encoded calcium ion (Ca2+) indicators (GECIs) can provide advantages over visible wavelength fluorescent GECIs in terms of reduced phototoxicity, minimal spectral cross talk with visible light excitable optogenetic tools and fluorescent probes, and decreased scattering and absorption in mammalian tissues. Our previously reported NIR GECI, NIR-GECO1, has these advantages but also has several disadvantages including lower brightness and limited fluorescence response compared to state-of-the-art visible wavelength GECIs, when used for imaging of neuronal activity. Here, we report 2 improved NIR GECI variants, designated NIR-GECO2 and NIR-GECO2G, derived from NIR-GECO1. We characterized the performance of the new NIR GECIs in cultured cells, acute mouse brain slices, and Caenorhabditis elegans and Xenopus laevis in vivo. Our results demonstrate that NIR-GECO2 and NIR-GECO2G provide substantial improvements over NIR-GECO1 for imaging of neuronal Ca2+ dynamics.

}, issn = {1545-7885}, doi = {10.1371/journal.pbio.3000965}, author = {Qian, Yong and Cosio, Danielle M Orozco and Piatkevich, Kiryl D and Aufmkolk, Sarah and Su, Wan-Chi and Celiker, Orhan T and Schohl, Anne and Murdock, Mitchell H and Abhi Aggarwal and Chang, Yu-Fen and Wiseman, Paul W and Ruthazer, Edward S and Boyden, Edward S and Campbell, Robert E} } @article {48434, title = {Live-cell super-resolution reveals F-Actin and plasma membrane dynamics at the T Cell synapse.}, journal = {Biophysical Journal}, volume = {112}, year = {2017}, month = {2017 Apr 25}, pages = {1703-13}, abstract = {

The cortical actin cytoskeleton has been shown to be critical for the reorganization and heterogeneity of plasma membrane components of many cells, including T\ cells. Building on previous studies at the T\ cell immunological synapse, we quantitatively assess the structure and dynamics of this meshwork using live-cell superresolution fluorescence microscopy and spatio-temporal image correlation spectroscopy. We show for the first time, to our knowledge, that not only does the dense actin cortex flow in a retrograde fashion toward the synapse center, but the plasma membrane itself shows similar behavior. Furthermore, using two-color, live-cell superresolution cross-correlation spectroscopy, we demonstrate that the two flows are correlated and, in addition, we show that coupling may extend to the outer leaflet of the plasma membrane by examining the flow of GPI-anchored proteins. Finally, we demonstrate that the actin flow is correlated with a third component,\ α-actinin, which upon CRISPR knockout led to reduced plasma membrane flow directionality despite increased actin flow velocity. We hypothesize that this apparent cytoskeletal-membrane coupling could provide a mechanism for driving the observed retrograde flow of signaling molecules such as the TCR, Lck, ZAP70, LAT, and SLP76.

}, doi = {10.1016/j.bpj.2017.01.038}, author = {Ashdown, George W and Burn, Garth L and Williamson, David J and Pandzic, Elvis and Peters, Ruby and Holden, Michael and Ewers, Helge and Shao, Lin and Wiseman, Paul W and Owen, Dylan M} }