In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5β1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.

Quantifying Polarized Extracellular Matrix Secretion in Cultured Endothelial Cells

Clapero F.
Co-first
;
Tortarolo D.
Co-first
;
Valdembri D.
Co-last
;
Serini G.
Co-last
2021-01-01

Abstract

In endothelial cells (ECs), the onset of apicobasal polarity is primarily regulated by the interaction of integrins with the surrounding extracellular matrix (ECM). ECs secrete and polymerize fibronectin (FN), a unique, permissive substrate that allows for vascular morphogenesis and lumen formation. We previously identified a signaling pathway that, under the control of the adhesion site adaptor protein PPFIA1, integrates the polarized secretion of freshly synthesized FN with the recycling of conformationally active α5β1 integrin, the main FN receptor in ECs. To characterize the functional role of PPFIA1-dependent signaling in ECs, we set up a Transwell-based assay to quantify the polarized secretion of ECM proteins. To this aim, we allowed ECs to form a confluent monolayer on the Transwell membrane and checked its integrity by measuring transendothelial electric resistance and controlling the stability of tight junctions over time by fluorescent confocal microscope analysis. Finally, we quantified apical and basolateral FN secretion in control and PPFIA1-silenced EC culture medium by western blot analysis coupled to spike-in normalization.
Methods in Molecular Biology
Humana Press Inc.
2217
301
311
978-1-0716-0961-3
978-1-0716-0962-0
Cell polarity; Fibronectin; Golgi; Integrins; Secretion; Tight junctions
Clapero F.; Tortarolo D.; Valdembri D.; Serini G.
File in questo prodotto:
File Dimensione Formato  
17 Serini EDITED with Figures.pdf

Accesso aperto

Tipo di file: PREPRINT (PRIMA BOZZA)
Dimensione 617.76 kB
Formato Adobe PDF
617.76 kB Adobe PDF Visualizza/Apri

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2318/1764765
Citazioni
  • ???jsp.display-item.citation.pmc??? ND
  • Scopus 1
  • ???jsp.display-item.citation.isi??? ND
social impact