HIGH-RESOLUTION SPATIOTEMPORAL ANALYSIS OF SOMATOSTATIN RECEPTOR TYPE 2 (SSTR2) - FILAMIN A (FLNA) INTERACTION BY SINGLE-MOLECULE IMAGING
Tesi di Dottorato
Data di Pubblicazione:
2017
Citazione:
HIGH-RESOLUTION SPATIOTEMPORAL ANALYSIS OF SOMATOSTATIN RECEPTOR TYPE 2 (SSTR2) - FILAMIN A (FLNA) INTERACTION BY SINGLE-MOLECULE IMAGING / D. Treppiedi ; tutor: G. Mantovani. Università degli Studi di Milano, 2017 Apr 21. 29. ciclo, Anno Accademico 2016. [10.13130/treppiedi-donatella_phd2017-04-21].
Abstract:
DOTTORATO DI RICERCA IN MEDICINA CLINICA E SPERIMENTALE (XXIX ciclo)
Abstract ENG
Cognome: TREPPIEDI Nome: DONATELLA Matricola n.: R10592
Title: "High-resolution spatiotemporal analysis of Somatostatin Receptor Type 2 (SSTR2) – Filamin A (FLNA) interaction by single-molecule imaging"
Background: SSTR2 is the main pharmacological target to treat acromegalic patients harboring GH-secreting pituitary adenomas. However, about 30% of patients displays resistance to somatostatin analogues (SSAs). Recent published data from our group demonstrated that the cytoskeletal protein FLNA plays an essential role in tumor responsiveness by regulating SSTR2 signaling and stability after prolonged stimulation. To date, there are no evidence in the literature describing the dynamic of SSTR2-FLNA interaction at the plasma membrane in vivo.
Aim: Aim of my PhD project was to follow the spatiotemporal behavior of SSTR2-FLNA complexes in real time by high resolution strategy. In particular I wanted to investigate the presence of a spatial distribution of SSTR2-FLNA complexes at the plasma membrane, estimate SSTR2 lateral mobility and elucidate a possible involvement of FLNA in regulating this biological phenomenon. A further goal was to evaluate the impact of FLNA-SSTR2 binding on ligand-induced SSTR2 clusters organization and internalization.
Materials and Methods: To characterize the dynamics of SSTR2-FLNA complexes in living CHO cells we used the single-molecule imaging approach, a method that combines the labelling of SNAP/CLIP-tagged proteins (SNAP-tagged SSTR2 and CLIP-tagged FLNA) with small organic fluorophores and the use of a total internal reflection fluoresence (TIRF) microscope. To calculate SSTR2 lateral mobility, mean square displacement (MSD) analysis were performed using the u-track algorithm implemented in Matlab. Confocal microscopy was used to evaluate the receptor surface distribution, agonist-mediated clusterization and alignment with actin filaments. Immunofluoresce experiments were assessed to evaluate the colocalization between SSTR2 clusters and AP-2, one of the early endocytosis marker, whereas the overall SSTR2 internalization process was analyzed by both imaging (confocal microscopy) and biochemical (biotinylation assay) strategies. All these SSTR2 aspects were evaluated in the presence or the absence of FLNA interaction. In particular, the overexpression of the dominant negative mutant FLNA 19-20 was used to abolish SSTR2-FLNA binding.
Results: First, the motion of freely diffusing SSTR2 particles was observed to slow down in CHO cells treated with 100nM BIM23120 for 5-10min (mean diffusion coefficients from 0,123µm2*s-1 to 0,101µm2*s-1). MSD analysis showed a significant increase in the SSTR2 fraction with diffusion coefficient values ≤ 0.05μm2*s-1 with respect to unstimulated cells (28,1% vs 14,4%, 100nM BIM23120 vs control, respectively, expressed as fraction of total particles, P < 0.05). The presence of the FLNA truncated mutant, that selectively prevents SSTR2-FLNA binding (FLNA 19-20), did not influence the SSTR2 agonist effect on receptor mobility. Such data was further confirmed in melanoma cell lines A7 (FLNA-expressing cells), M2 (FLNA-lacking cells). Then, we described the nature of the interactions between SSTR2 particles and FLNA fibers as extremely dynamic and transient under resting condition, whereas they resulted long-lasting and more stable after100nM BIM23120 exposure. Interestingly, when both FLNA and SSTR2 were expressed at single molecule level, SSTR2-FLNA complexes formation was seen to occur preferentially along actin filaments, in stimulated cells only. Furthermore, when overexpressed and activated by the agonist, SSTR2 was observed to undergo clust
Tipologia IRIS:
Tesi di dottorato
Elenco autori:
D. Treppiedi
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