TY  - JOUR
A1  - Lukeš, Tomáš
A1  - Glatzová, Daniela
A1  - Kvíčalová, Zuzana
A1  - Levet, Florian
A1  - Benda, Aleš
A1  - Letschert, Sebastian
A1  - Sauer, Markus
A1  - Brdička, Tomáš
A1  - Lasser, Theo
A1  - Cebecauer, Marek
T1  - Quantifying protein densities on cell membranes using super-resolution optical fluctuation imaging
JF  - Nature Communications
N2  - Quantitative approaches for characterizing molecular organization of cell membrane molecules under physiological and pathological conditions profit from recently developed super-resolution imaging techniques. Current tools employ statistical algorithms to determine clusters of molecules based on single-molecule localization microscopy (SMLM) data. These approaches are limited by the ability of SMLM techniques to identify and localize molecules in densely populated areas and experimental conditions of sample preparation and image acquisition. We have developed a robust, model-free, quantitative clustering analysis to determine the distribution of membrane molecules that excels in densely labeled areas and is tolerant to various experimental conditions, i.e. multiple-blinking or high blinking rates. The method is based on a TIRF microscope followed by a super-resolution optical fluctuation imaging (SOFI) analysis. The effectiveness and robustness of the method is validated using simulated and experimental data investigating nanoscale distribution of CD4 glycoprotein mutants in the plasma membrane of T cells.
KW  - biology
KW  - fluorescence imaging
KW  - imaging the immune system
KW  - super-resolution microscopy
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-172993
VL  - 8
ER  - 
TY  - JOUR
A1  - Fischer, Annette
A1  - Harrison, Kelly S
A1  - Ramirez, Yesid
A1  - Auer, Daniela
A1  - Chowdhury, Suvagata Roy
A1  - Prusty, Bhupesh K
A1  - Sauer, Florian
A1  - Dimond, Zoe
A1  - Kisker, Caroline
A1  - Hefty, P Scott
A1  - Rudel, Thomas
T1  - Chlamydia trachomatis-containing vacuole serves as deubiquitination platform to stabilize Mcl-1 and to interfere with host defense
JF  - eLife
N2  - Obligate intracellular Chlamydia trachomatis replicate in a membrane-bound vacuole called inclusion, which serves as a signaling interface with the host cell. Here, we show that the chlamydial deubiquitinating enzyme (Cdu) 1 localizes in the inclusion membrane and faces the cytosol with the active deubiquitinating enzyme domain. The structure of this domain revealed high similarity to mammalian deubiquitinases with a unique α-helix close to the substrate-binding pocket. We identified the apoptosis regulator Mcl-1 as a target that interacts with Cdu1 and is stabilized by deubiquitination at the chlamydial inclusion. A chlamydial transposon insertion mutant in the Cdu1-encoding gene exhibited increased Mcl-1 and inclusion ubiquitination and reduced Mcl-1 stabilization. Additionally, inactivation of Cdu1 led to increased sensitivity of C. trachomatis for IFNγ and impaired infection in mice. Thus, the chlamydial inclusion serves as an enriched site for a deubiquitinating activity exerting a function in selective stabilization of host proteins and protection from host defense.
KW  - cell-autonomous defense
KW  - Chlamydia trachomatis
KW  - deubiquitinase
KW  - Mcl-1
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-171073
VL  - 6
IS  - e21465
ER  - 
TY  - JOUR
A1  - Kaiser, Sebastian
A1  - Sauer, Florian
A1  - Kisker, Caroline
T1  - The structural and functional characterization of human RecQ4 reveals insights into its helicase mechanism
JF  - Nature Communications
N2  - RecQ4 is a member of the RecQ helicase family, an evolutionarily conserved class of enzymes, dedicated to preserving genomic integrity by operating in telomere maintenance, DNA repair and replication. While reduced RecQ4 activity is associated with cancer predisposition and premature aging, RecQ4 upregulation is related to carcinogenesis and metastasis. Within the RecQ family, RecQ4 assumes an exceptional position, lacking several characteristic RecQ domains. Here we present the crystal structure of human RecQ4, encompassing the conserved ATPase core and a novel C-terminal domain that lacks resemblance to the RQC domain observed in other RecQ helicases. The new domain features a zinc-binding site and two distinct types of winged-helix domains, which are not involved in canonical DNA binding or helicase activity. Based on our structural and functional analysis, we propose that RecQ4 exerts a helicase mechanism, which may be more closely related to bacterial RecQ helicases than to its human family members.
KW  - x-ray crystallography
KW  - enzymes
KW  - RecQ4
KW  - humans
Y1  - 2017
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-170769
VL  - 8
IS  - 15907
ER  -