3 research outputs found
Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy
The specific and
covalent labeling of the protein HaloTag with
fluorescent probes in living cells makes it a powerful tool for bioimaging.
However, the irreversible attachment of the probe to HaloTag precludes
imaging applications that require transient binding of the probe and
comes with the risk of irreversible photobleaching. Here, we introduce
exchangeable ligands for fluorescence labeling of HaloTag (xHTLs)
that reversibly bind to HaloTag and that can be coupled to rhodamines
of different colors. In stimulated emission depletion (STED) microscopy,
probe exchange of xHTLs allows imaging with reduced photobleaching
as compared to covalent HaloTag labeling. Transient binding of fluorogenic
xHTLs to HaloTag fusion proteins enables points accumulation for imaging
in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore
introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT
and STED microscopy. xHTLs thus open up new possibilities in imaging
across microscopy platforms for a widely used labeling approach
Exchangeable HaloTag Ligands for Super-Resolution Fluorescence Microscopy
The specific and
covalent labeling of the protein HaloTag with
fluorescent probes in living cells makes it a powerful tool for bioimaging.
However, the irreversible attachment of the probe to HaloTag precludes
imaging applications that require transient binding of the probe and
comes with the risk of irreversible photobleaching. Here, we introduce
exchangeable ligands for fluorescence labeling of HaloTag (xHTLs)
that reversibly bind to HaloTag and that can be coupled to rhodamines
of different colors. In stimulated emission depletion (STED) microscopy,
probe exchange of xHTLs allows imaging with reduced photobleaching
as compared to covalent HaloTag labeling. Transient binding of fluorogenic
xHTLs to HaloTag fusion proteins enables points accumulation for imaging
in nanoscale topography (PAINT) and MINFLUX microscopy. We furthermore
introduce pairs of xHTLs and HaloTag mutants for dual-color PAINT
and STED microscopy. xHTLs thus open up new possibilities in imaging
across microscopy platforms for a widely used labeling approach
EHD2-mediated restriction of caveolar dynamics regulates cellular fatty acid uptake
Eps15-homology domain containing protein 2 (EHD2) is a dynamin-related ATPase located at the neck of caveolae, but its physiological function has remained unclear. Here, we found that global genetic ablation of EHD2 in mice leads to increased lipid droplet size in fat tissue. This organismic phenotype was paralleled at the cellular level by increased fatty acid uptake via a caveolae- and CD36-dependent pathway that also involves dynamin. Concomitantly, elevated numbers of detached caveolae were found in brown and white adipose tissue lacking EHD2, and increased caveolar mobility in mouse embryonic fibroblasts. EHD2 expression itself was down-regulated in the visceral fat of two obese mouse models and obese patients. Our data suggest that EHD2 controls a cell-autonomous, caveolae-dependent fatty acid uptake pathway and imply that low EHD2 expression levels are linked to obesity