Intracellular Ca2+ Release Triggers Translocation of Membrane Marker FM1–43 from the Extracellular Leaflet of Plasma Membrane into Endoplasmic Reticulum in T Lymphocytes

Sepehr Dadsetan,Vyacheslav Shishkin,Alla F Fomina

doi:10.1074/jbc.m501202200

Abstract

Stimulation of T cell receptor in lymphocytes enhances Ca(2+) signaling and accelerates membrane trafficking. The relationships between these processes are not well understood. We employed membrane-impermeable lipid marker FM1-43 to explore membrane trafficking upon mobilization of intracellular Ca(2+) in Jurkat T cells. We established that liberation of intracellular Ca(2+) with T cell receptor agonist phytohemagglutinin P or with Ca(2+)-mobilizing agents ionomycin or thapsigargin induced accumulation of FM1-43 within the lumen of the endoplasmic reticulum (ER), nuclear envelope (NE), and Golgi. FM1-43 loading into ER-NE and Golgi was not mediated via the cytosol because other organelles such as mitochondria and multivesicular bodies located in close proximity to the FM1-43-containing ER were free of dye. Intralumenal FM1-43 accumulation was observed even when Ca(2+) signaling in the cytosol was abolished by the removal of extracellular Ca(2+). Our findings strongly suggest that release of intracellular Ca(2+) may create continuity between the extracellular leaflet of the plasma membrane and the lumenal membrane leaflet of the ER by a mechanism that does not require global cytosolic Ca(2+) elevation.

Highlights

Membrane trafficking plays an important role in shaping T cell responses
We found that stimulation of Jurkat T cells with TCR agonist, phytohemagglutinin P (PHA), or with the Ca2ϩ-mobilizing agents, Tg or Iono, produced FM1– 43 translocation from the PM into the lumen of peripheral endoplasmic reticulum (ER) followed by the dye spreading into nuclear envelope (NE) and Golgi
Our study demonstrated that in Jurkat T lymphocytes, activation of intracellular Ca2ϩ release by stimulation of membrane receptors or by Ca2ϩ-mobilizing agents produced a fast loading of membrane-bound lipid marker FM1– 43 from the extracellular leaflet of the PM into the lumen of intracellular organelles: ER, NE, and Golgi

Summary

Introduction

Membrane trafficking plays an important role in shaping T cell responses. For example, it is well established that engagement of TCR1 with an antigen triggers fast trafficking of the receptor between the PM and endocytic organelles, resulting in serial TCR engagements and an amplification of intracellular signaling [1]. An EM study performed previously in the Xenopus larvae hair cells revealed the presence of FM1– 43 within nonendocytic organelles such as ER, NE, and mitochondria [6] These results led to a suggestion that FM1– 43 can penetrate the PM and diffuse through the cytosol. We found that stimulation of Jurkat T cells with TCR agonist, phytohemagglutinin P (PHA), or with the Ca2ϩ-mobilizing agents, Tg or Iono, produced FM1– 43 translocation from the PM into the lumen of peripheral ER followed by the dye spreading into NE and Golgi. This effect did not require global elevation in [Ca2ϩ]i since it was observed in the absence of extracellular Ca2ϩ. Our findings suggest that intracellular Ca2ϩ release may generate continuity between the ER and the PM, resulting in direct membrane exchange between those organelles

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Conclusion