Evidence for P-Glycoprotein Involvement in Cell Volume Regulation Using Coulter Sizing in Flow Cytometry.

Jennifer Pasquier,Jessica Hoarau-Véchot,Matthieu Marin,Frank Foll,Damien Rioult,Nadine Abu-Kaoud

doi:10.3390/ijms160714318

Abstract

The regulation of cell volume is an essential function that is coupled to a variety of physiological processes such as receptor recycling, excitability and contraction, cell proliferation, migration, and programmed cell death. Under stress, cells undergo emergency swelling and respond to such a phenomenon with a regulatory volume decrease (RVD) where they release cellular ions, and other osmolytes as well as a concomitant loss of water. The link between P-glycoprotein, a transmembrane transporter, and cell volume regulation is controversial, and changes in cells volume are measured using microscopy or electrophysiology. For instance, by using the patch-clamp method, our team demonstrated that chloride currents activated in the RVD were more intense and rapid in a breast cancer cell line overexpressing the P-glycoprotein (P-gp). The Cell Lab Quanta SC is a flow cytometry system that simultaneously measures electronic volume, side scatter and three fluorescent colors; altogether this provides unsurpassed population resolution and accurate cell counting. Therefore, here we propose a novel method to follow cellular volume. By using the Coulter-type channel of the cytometer Cell Lab Quanta SC MPL (multi-platform loading), we demonstrated a role for the P-gp during different osmotic treatments, but also a differential activity of the P-gp through the cell cycle. Altogether, our data strongly suggests a role of P-gp in cell volume regulation.

Highlights

Each living cell type seems to have a form and volume that is well defined, and determined by the size of their cytoplasmic membrane as well as their cytosolic content
We develop an original method based on flow cytometry analysis to study the role of the P-gp in the regulatory volume decrease (RVD) in response to hypo-osmotic shocks
Using Hoffman modulation contrast imaging on a freshly plated 50:50 co-culture, we revealed a clear morphological difference between MCF7 and MCF7/Doxo (Figure 1B top panel)

Summary

Introduction

Each living cell type seems to have a form and volume that is well defined, and determined by the size of their cytoplasmic membrane as well as their cytosolic content. Variations of osmotic pressures lead to cell volume regulation [1,2]. In the hypotonic or hypertonic context, the ability of cells to regulate their volume in a short period of time (around a minute) in order to avoid swelling or shrinkage is a fundamental mechanism [3]. Under hypo-osmotic conditions, cells are able to escape a burst after swelling by activation of a mechanism known as regulatory volume decrease (RVD) [4]. Even under a regular osmotic pressure, the cell volume is fluctuating in response to cell events such as cell proliferation, migration, glycolysis or cell death [5,6,7,8].

Results

Discussion

Conclusion