a-fluorescence-fluctuation-spectroscopy-assay-protein-protein

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

University of Potsdam

The amyloid precursor-like protein 1 (APLP1) is a type I transmembrane protein that plays a role in synaptic adhesion and synaptogenesis. Past investigations indicated that APLP1 is involved in the formation of protein-protein complexes that bridge the junctions between neighboring cells. Nevertheless, APLP1-APLP1  interactions have never been directly observed in higher eukaryotic cells. Here, we investigated APLP1 interactions and dynamics directly in living human embryonic kidney cells using fluorescence fluctuation spectroscopy techniques, namely cross-correlation scanning fluorescence correlation spectroscopy and number and brightness analysis. Our results show that APLP1 forms homotypic  complexes at cell-cell contacts. In the presence of zinc ions, the protein forms macroscopic clusters, exhibiting an even higher degree of  binding and strongly reduced dynamics. Further evidence from giant plasma membrane vesicles suggests that the presence of an intact cortical cytoskeleton is required for zinc-induced  multimerization. Subsequently, large adhesion platforms bridging interacting cells are formed through APLP1-APLP1  interactions. Taken together, our results provide direct evidence that APLP1 functions as a neuronal zinc-dependent adhesion protein and allow a more detailed understanding of the molecular mechanisms driving the formation of APLP1 adhesion platforms.

Direct evidence of amyloid precursor-like protein 1  interactions in cell-cell adhesion platforms investigated via fluorescence fluctuation spectroscopy.

Fluorescence fluctuation spectroscopy has become a popular toolbox for non-disruptive analysis of molecular interactions in living cells. The quantification of protein oligomerization in the native cellular environment is highly relevant for a detailed understanding of complex biological processes. An important parameter in this context is the molecular brightness, which serves as a direct measure of oligomerization and can be easily extracted from temporal or spatial fluorescence fluctuations. However, fluorescent proteins (FPs) typically used in such studies suffer from complex photophysical transitions and limited maturation, inducing non-fluorescent states. Here, we show how these processes strongly affect molecular brightness measurements. We perform a systematic characterization of non-fluorescent states for commonly used FPs and provide a simple guideline for accurate, unbiased oligomerization measurements in living cells. Further, we focus on novel red FPs and demonstrate that mCherry2, an mCherry variant, possesses superior properties with regards to precise quantification of oligomerization.

Salvatore Chiantia

Institute for Biochemistry and Biology,
Cell Membrane Biophysics Group,
Institute for Biochemistry and Biology, Cell Membrane Biophysics Group

University of Potsdam

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Salvatore Chiantia

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Institute for Biochemistry and Biology,Cell Membrane Biophysics Group,Institute for Biochemistry and Biology, Cell Membrane Biophysics Group

University of Potsdam

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Institute for Biochemistry and Biology,
Cell Membrane Biophysics Group,
Institute for Biochemistry and Biology, Cell Membrane Biophysics Group