The characterization of the nuclear dynamics of syntenin‐2, a PIP₂ binding PDZ protein

Abstract

Cellular signaling is largely dependent on the presence, that is, assembly/disassembly, of supramolecular complexes. Postsynaptic density protein, Discs-large, Zona occludens (PDZ) domains play important roles in the assembly of various signaling complexes. Syntenin-2 (S2) is a PDZ protein that interacts with nuclear phosphatidylinositol 4,5-bisphosphate (PIP 2 ). Although nuclear lipids emerge as key players in nuclear processes, the global significance of nuclear phosphoinositide-protein interactions is still poorly understood. Those phosphoinositide-protein interactions that have been studied in detail appear to have profound physiological effects. To our knowledge none of these were investigated by dynamic studies such as Fluorescence Correlation Spectroscopy (FCS), Fluorescence Cross-Correlation Spectroscopy (FCCS), or Fluorescence Recovery After Photobleaching (FRAP). Although the exact function of S2 is unknown, siRNA experiments suggest that this PDZ protein plays a role in the organization of nuclear PIP 2 , cell division, and cell survival. As a consequence of its PIP 2 interaction, its reported self-association in a yeast two-hybrid study and its speculated interaction with many, yet unidentified, proteins one can hypothesize that S2 plays an important role in cell signaling. Therefore, we studied the dynamics of S2 using FCS, FCCS, and FRAP, utilizing an active truncated form deleted for the first 94 amino acids (S2-DN). We showed that S2-DN self-associates and is distributed in three groups. One immobile group, one slow diffusing group, which interacts with the nuclear environment and one fast diffusing group, which is not incorporated in high molecular weight complexes. In addition, our FCS and FRAP measurements on S2-DN mutants affected in their PIP 2 binding showed that PIP 2 plays an important role in the distribution of S2-DN among these groups, and favors the enrichment of S2-DN in the slow diffusing and immobile group. This work indicates that S2 relies on nuclear PIP 2 to interact with practically immobile structures, possibly chromatin. ' 2013 International Society for Advancement of Cytometry Key terms syntenin-2; PDZ; PIP 2 ; nucleus; fluorescence correlation spectroscopy; fluorescence cross-correlation spectroscopy; fluorescence recovery after photobleaching CELLULAR signaling is largely dependent on the presence, that is, assembly/disassembly, of supramolecular complexes. In the dynamic process of (re)arranging multimeric protein complexes, the actions of a broad variety of protein modules are crucial. PDZ domains are among the most common protein domains and are present in organisms as diverse as bacteria, yeast, plants, invertebrates, and vertebrates (1). Their name refers to the first three proteins in which they were originally identified (PSD-95/SAP90, Discs-large and Zona occludens 1) (2).In the canonical mode PDZ domains recognize motifs of 3-7 amino acids that are present at the C-terminal end of membrane proteins (3). In 2005 it was shown that the PDZ domains of synteni...

Targeting tumor invasion: the roles of MDA-9/Syntenin

“…Syntenin 2α shares > 70% homology in the PDZ domains as MDA-9/Syntenin, whereas syntenin 2β is a shorter isoform of 2α that lacks 85 residues at the N-terminal [19]. To date, relatively few studies have focused on this isoform, yet like MDA-9/Syntenin, it has also been shown to interact with PIP2 [52]. One study found lower expression of syntenin-2 in colorectal cancer as compared to normal tissue but was not found to be a significant prognostic marker [53].…”

“…In other work, syntenin-2 was found in higher amounts in bile from biliary stenosis related to malignant causes, such as pancreatic adenocarcinoma or cholangiocarcinoma, compared to nonmalignant samples such as chronic pancreatitis or biliary stones [54]. Initial studies suggest roles for syntenin-2 in cell division, nuclear PIP2 organization and cell survival [52], although these interesting areas remain relatively unexplored.…”

Recent applications of fluorescence correlation spectroscopy in live systems

“…An alternative FCS approach to detecting oligomerization is based on cross-correlation between two populations of the same molecule labelled by distinct fluorophores; a positive cross-correlation indicates presence of complexes containing each at least two molecules of interest and the ratio between cross-correlation and autocorrelation amplitudes gives the fraction of molecules in oligomers [91,[119][120][121] (number 5 in Fig. 1).…”

“…1). To estimate the size of the oligomers, the authors used FCS brightness analysis [120]. To estimate the size of the oligomers, the authors used FCS brightness analysis [120].…”

“…For example Geeraets et al found by FCCS that syntenin-2 self-associates in the cell nucleus. To estimate the size of the oligomers, the authors used FCS brightness analysis [120]. The study of Ridelis et al contains an interesting example of applications of FCCS to characterise the oligomerisation state of photoconvertible proteins; the cross-correlation between the two forms of protein measured at the 50% photoconversion level is an indication of oligomerisation of the protein [121].…”