Lou, J. — Page 4 | bioRxiv Science

bioRxiv2019

FcγRIIB I232T polymorphic change allosterically suppresses ligand binding

Fc{gamma}RIIB bindings to its ligand suppress immune cell activation. A single-nucleotide polymorphic (SNP) change, I232T, in the transmembrane (TM) domain of Fc{gamma}RIIB loses its suppression function, which clinically associates with systemic lupus erythematosus (SLE). Previously, we reported that I232T tilts Fc{gamma}RIIBs TM domain. In this study, combining with molecular dynamics simulations and single-cell FRET assay, we further revealed that such tilting by I232T unexpectedly bends the Fc{gamma}RIIBs ectodomain towards plasma membrane to allosterically impede Fc{gamma}RIIBs ligand association. We then used single-cell biomechanical assay to further find out that I232T also reduces two-dimensional in-situ binding affinities and association rates of Fc{gamma}RIIB interacting with its ligands by three-folds. This allosteric regulation by a SNP provides an intrinsic molecular mechanism for functional loss of Fc{gamma}RIIB-I232T in SLE patients.

immunology↗

bioRxiv2018

Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.

To investigate how chromatin architecture is spatiotemporally organised at a double strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image correlation spectroscopy (ICS) of histone FLIM-FRET microscopy data acquired in live cells co-expressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that when coupled with laser micro-irradiation induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ATM and RNF8 regulate rapid chromatin decompaction at DSBs and formation of a compact chromatin ring surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.\n\nSIGNIFICANCE STATEMENTChromatin dynamics play a central role in the DNA damage response (DDR). A long-standing obstacle in the DDR field was the lack of technology capable of visualising chromatin dynamics at double strand break (DSB) sites. Here we describe novel biophysical methods that quantify spatiotemporal chromatin compaction dynamics in living cells. Using these novel tools, we identify how chromatin architecture is reorganised at a DSB locus to enable repair factor access and demarcate the lesion from the surrounding nuclear environment. Further, we identify novel regulatory roles for key DDR enzymes in this process. Finally, we demonstrate method utility with physical, pharmacological and genetic manipulation of the chromatin environment, identifying method potential for use in future studies of chromatin biology.

biophysics↗