Microdomain Formation Controls Spatiotemporal Dynamics of Cell‐Surface Glycoproteins

The effect of galectin‐mediated microdomain formation on the spatiotemporal dynamics of glycosylated membrane proteins in human microvascular endothelial cells (HMEC‐1) was studied qualitatively and quantitatively by high‐resolution fluorescence microscopy and artificially mimicked by metabolic glycoprotein engineering. Two types of membrane proteins, sialic acid‐bearing proteins (SABPs) and mucin‐type proteins (MTPs), were investigated. For visualization they were metabolically labeled with azido sugars and then coupled to a cyclooctyne‐conjugated fluorescent dye by click chemistry. Both spatial (diffusion) and temporal (residence time) dynamics of SABPs and MTPs on the membrane were investigated after treatment with exogenous galectin‐1 or ‐3. Strong effects of galectin‐mediated lattice formation were observed for MTPs (decreased spatial mobility), but not for SABPs. Lattice formation also strongly decreased the turnover of MTPs (increased residence time on the cell membrane). The effects of galectin‐mediated crosslinking was accurately mimicked by streptavidin‐mediated crosslinking of biotin‐tagged glycoproteins and verified by single‐molecule tracking. This technique allows the induction of crosslinking of membrane proteins under precisely controlled conditions, thereby influencing membrane residence time and the spatial dynamics of glycans on the cell membrane in a controlled way.     

Symposium der Arbeitsgruppe Wissenschafts-geschichte zur Geschichte der Kaiser-Wilhelm-/Max-Planck-Gesellschaft

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A cytochrome b5 is required for full activity of flavonoid 3', 5'-hydroxylase, a cytochrome P450 involved in the formation of blue flower colors

The substitution pattern of anthocyanin pigments is a main determinant of flower color. Flavonoid 3',5'-hydroxylase (F3'5'H) is a cytochrome P450 enzyme (Cyt P450) that catalyzes the 3', 5'-hydroxylation of dihydroflavonols, the precursors of purple anthocyanins. Species such as rose and carnation lack F3'5'H activity and are, therefore, unable to generate purple or blue flowers. Petunia, on the other hand, contains two loci, termed hf1 and hf2, that encode a Cyt P450 with F3'5'H activity. Here we report the identification of an additional petunia gene that is required for 3',5' substitution of anthocyanins and purple flower colors. It encodes a cytochrome b5 and is expressed exclusively in the flower. Inactivation of the gene by targeted transposon mutagenesis reduced F3'5'H enzyme activity and the accumulation of 5'-substituted anthocyanins, resulting in an altered flower color. However, no phenotypic effect on the activity of other Cyt P450s, involved in the synthesis of hormones or general phenylpropanoids, was observed. These data provide in vivo evidence for the regulation of the activity of specific Cyt P450s by a cytochrome b5.