Dynamin-2 Regulates Fusion Pore Expansion and Quantal Release through a Mechanism that Involves Actin Dynamics in Neuroendocrine Chromaffin Cells

Agustín D Martínez,Valerie Calco,Ana M Cárdenas,Natalia Barraza,Sébastien Houy,Arlek M González-Jamett,María José Guerra,Eduardo Couve,Stéphane Ory,Ximena Báez-Matus,Stéphane Gasman,Fanny Momboisse,Alan Neely

doi:10.1371/journal.pone.0070638

Abstract

Over the past years, dynamin has been implicated in tuning the amount and nature of transmitter released during exocytosis. However, the mechanism involved remains poorly understood. Here, using bovine adrenal chromaffin cells, we investigated whether this mechanism rely on dynamin’s ability to remodel actin cytoskeleton. According to this idea, inhibition of dynamin GTPase activity suppressed the calcium-dependent de novo cortical actin and altered the cortical actin network. Similarly, expression of a small interfering RNA directed against dynamin-2, an isoform highly expressed in chromaffin cells, changed the cortical actin network pattern. Disruption of dynamin-2 function, as well as the pharmacological inhibition of actin polymerization with cytochalasine-D, slowed down fusion pore expansion and increased the quantal size of individual exocytotic events. The effects of cytochalasine-D and dynamin-2 disruption were not additive indicating that dynamin-2 and F-actin regulate the late steps of exocytosis by a common mechanism. Together our data support a model in which dynamin-2 directs actin polymerization at the exocytosis site where both, in concert, adjust the hormone quantal release to efficiently respond to physiological demands.

Highlights

Dynamin is a mechano-GTPase encoded by three distinct genes (DNM1, DNM2 and DNM3) that generates membrane deformation and triggers membrane fission [1]
Dynamin participates in several cellular processes that are dependent on the actin cytoskeleton dynamics, some of them are actin comet [6], [7] lamellipodia formation [8], T cell activation [9], phagocytosis [10] and different types of endocytosis [11,12,13,14]
As these results indicate that dynamin-2 is highly expressed in bovine chromaffin cells (BCC), we investigated the role of this isoform in the exocytosis

Summary

Introduction

Dynamin is a mechano-GTPase encoded by three distinct genes (DNM1, DNM2 and DNM3) that generates membrane deformation and triggers membrane fission [1]. Studies in knockout animals show that only dynamin-2 is critical during early embryonic development [4] and that the absence of dynamin-1 or -3 can be compensated by the other isoforms [5]. From these findings arises the idea that the different dynamin isoforms have overlapping roles and their relative contribution to endocytosis in a given tissue is mostly determined by their abundance rather than on structural specialization [5]. Dynamin participates in several cellular processes that are dependent on the actin cytoskeleton dynamics, some of them are actin comet [6], [7] lamellipodia formation [8], T cell activation [9], phagocytosis [10] and different types of endocytosis [11,12,13,14]

Methods

Results

Conclusion