TY  - JOUR
A1  - Markert, Sebastian Matthias
A1  - Britz, Sebastian
A1  - Proppert, Sven
A1  - Lang, Marietta
A1  - Witvliet, Daniel
A1  - Mulcahy, Ben
A1  - Sauer, Markus
A1  - Zhen, Mei
A1  - Bessereau, Jean-Louis
A1  - Stigloher, Christian
T1  - Filling the gap: adding super-resolution to array tomography for correlated ultrastructural and molecular identification of electrical synapses at the C. elegans connectome
JF  - Neurophotonics
N2  - Correlating molecular labeling at the ultrastructural level with high confidence remains challenging. Array tomography (AT) allows for a combination of fluorescence and electron microscopy (EM) to visualize subcellular protein localization on serial EM sections. Here, we describe an application for AT that combines near-native tissue preservation via high-pressure freezing and freeze substitution with super-resolution light microscopy and high-resolution scanning electron microscopy (SEM) analysis on the same section. We established protocols that combine SEM with structured illumination microscopy (SIM) and direct stochastic optical reconstruction microscopy (dSTORM). We devised a method for easy, precise, and unbiased correlation of EM images and super-resolution imaging data using endogenous cellular landmarks and freely available image processing software. We demonstrate that these methods allow us to identify and label gap junctions in Caenorhabditis elegans with precision and confidence, and imaging of even smaller structures is feasible. With the emergence of connectomics, these methods will allow us to fill in the gap-acquiring the correlated ultrastructural and molecular identity of electrical synapses.
KW  - caenorhabditis elegans
KW  - localization micoscopy
KW  - fluorescent-probes
KW  - junction proteins
KW  - resolution limit
KW  - direct stochasticoptical reconstruction microscopy
KW  - structured illumination microscopy
KW  - correlative light and electron microscopy
KW  - gap junction
KW  - neural circuits
KW  - nervous-system
KW  - image data
KW  - reconstruction
KW  - innexins
KW  - super-resolution microscopy
Y1  - 2016
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-187292
VL  - 3
IS  - 4
ER  - 
TY  - JOUR
A1  - Panzer, Sabine
A1  - Brych, Annika
A1  - Batschauer, Alfred
A1  - Terpitz, Ulrich
T1  - Opsin 1 and Opsin 2 of the corn smut fungus ustilago maydis are green light-driven proton pumps
JF  - Frontiers in Microbiology
N2  - In fungi, green light is absorbed by rhodopsins, opsin proteins carrying a retinal molecule as chromophore. The basidiomycete Ustilago maydis, a fungal pathogen that infects corn plants, encodes three putative photoactive opsins, called ops1 (UMAG_02629), ops2 (UMAG_00371), and ops3 (UMAG_04125). UmOps1 and UmOps2 are expressed during the whole life cycle, in axenic cultures as well as in planta, whereas UmOps3 was recently shown to be absent in axenic cultures but highly expressed during plant infection. Here we show that expression of UmOps1 and UmOps2 is induced by blue light under control of white collar 1 (Wco1). UmOps1 is mainly localized in the plasma membrane, both when expressed in HEK cells and U. maydis sporidia. In contrast, UmOps2 was mostly found intracellularly in the membranes of vacuoles. Patch-clamp studies demonstrated that both rhodopsins are green light-driven outward rectifying proton pumps. UmOps1 revealed an extraordinary pH dependency with increased activity in more acidic environment. Also, UmOps1 showed a pronounced, concentration-dependent enhancement of pump current caused by weak organic acids (WOAs), especially by acetic acid and indole-3-acetic acid (IAA). In contrast, UmOps2 showed the typical behavior of light-driven, outwardly directed proton pumps, whereas UmOps3 did not exhibit any electrogenity. With this work, insights were gained into the localization and molecular function of two U. maydis rhodopsins, paving the way for further studies on the biological role of these rhodopsins in the life cycle of U. maydis.
KW  - Ustilago maydis
KW  - patch-clamp
KW  - fungal rhodopsins
KW  - microbial rhodopsins
KW  - acetate
KW  - indole-3-acetic acid
KW  - structured illumination microscopy
KW  - sporidia
Y1  - 2019
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-201453
VL  - 10
ER  -