Oral Presentation Australasian Extracellular Vesicles Conference 2018

Sarco/endoplasmic reticulum ATPase inhibition activates calcium signalling pathways for microvesicle biogenesis (#11)

Jack D Taylor 1 , Michael Johnson 2 , Gregory Monteith 3 , Mary Bebawy 1
  1. Graduate School of Health, University of Technology Sydney, Sydney, NSW, Australia
  2. School of Life Sciences, The University of Technology Sydney, Ultimo, New South Wales, Australia
  3. The School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia

An increase in intracellular Ca2+ is a key initiator of microvesicle (MV) biogenesis. The Ca2+-signalling pathway(s) implicated in this are currently unknown. This study aims to elucidate the Ca2+ pathways involved in MV biogenesis in malignant and non-malignant cells in an attempt to identify selective drug targets for vesicle inhibition.

Interrogation of the Ca2+ signalling pathway was done using the SERCA inhibitor, thapsigargin (TG), the Calpain inhibitor II (ALLM) and the inhibitor of Store Operated Ca2+ entry (YM58483).  AFM was used to study cell surface topography, and flow cytometry was used to quantitate vesiculation changes in response to inhibitors in HBEC-D3, MCF-7, and MCF-7/Dx cells. Real-time deconvolution (DeltaVision personalVD, Elite) microscopy was used for live cell imaging using CellLight Plasma Membrane-RFP, Bacmam 2.0®.

ALLM selectively inhibited vesiculation in malignant cells confirming a basal Ca2+-calpain dominant pathway. This was not observed for non-maligant cells confirming an alternative vesiculation pathway independent of calpain. Depletion of endoplasmic reticulum (ER) Ca2+ stores by TG alone resulted in a slight and significant increase in vesiculation in malignant and non-malignant cells respectively. Treatment with YM58483 had no effect. Treatment with both TG and YM58483 resulted in inhibition of vesiculation in both cell types, suggesting calpain is responsible for differences in vesiculation observed in malignant vs non-malignant cells.

We show for the first time the involvement of SERCA/SOCE Ca2+ signalling in MV vesiculation. Differences in basal vesiculation in malignant and non-malignant cells are at the level of calpain rather than the SERCA/SOCE pathway.