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

Abstract

Stimulation of T cell receptor in lymphocytes enhances Ca2؉ 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.Membrane trafficking plays an important role in shaping T cell responses. For example, it is well established that engagement of TCR 1 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). TCR stimulation is followed by a fast liberation of Ca 2ϩ from Ca 2ϩ -storing organelles, such as ER and Golgi (2, 3). Alteration of Ca 2ϩ homeostasis induces a variety of cellular responses ranging from changes in cell shape and motility to activation of transcription factors (2). However, it is not clear whether changes in intracellular Ca 2ϩ dynamics affect membrane trafficking in T lymphocytes.Styryl dye FM1-43 is a fluorescent membrane marker that reversibly partitions into but does not penetrate biological membranes (4). We have shown previously that FM1-43 was readily internalized from the PM into the endocytic compartments of human T cells by constitutive endocytosis (5). We also demonstrated that Ca 2ϩ ionophore ionomycin (Iono) or thapsigargin (Tg), a sarco-endoplasmic reticulum calcium ATPase pumps blocker, facilitated FM1-43 internalization (5). These findings suggested that elevation of cytosolic Ca 2ϩ concentration ([Ca 2ϩ ] i ) might accelerate the rate of endocytosis in T cells. However, 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. To further investigate the nature of enhanced FM1-43 internalization following liberation of intracellular Ca 2ϩ in T cells, we studied subcellular distribution of FM1-43 using correlative fluorescence and electron microscopy.We found that sti...

Some assembly required: Constructing the elementary units of store-operated Ca2+ entry

“…Following TG treatment, however, dense HRP reaction product is seen in tubules juxtaposed to the PM, with reduced labeling of the cytoplasmic tubules. Importantly, these structures, found within 10-25 nm of the PM, resemble the junctional ER seen in Jurkat cells expressing ER-targeted HRP [41] and described previously in a variety of cells from oocytes to yeast [63][64][65][66]. Thus, these results suggest that upon store depletion, STIM1 migrates within the ER membrane to a subset of ER tubules juxtaposed to the PM.…”

Death receptor ligation triggers membrane scrambling between Golgi and mitochondria

“…12 Lipid trafficking thus provides an underlying platform for a limited or transient association of Golgi and mitochondrial membranes. 5 The death receptor-induced increase in membrane traffic, partially driven by endocytic events, [3][4][5]19,24,35 may abnormally alter the direction and intensity of inter-organelle contacts, distorting the normal flow of membrane interactions. This would provide a simple explanation for the increased frequency of local intermixing of Golgi and mitochondrial membranes that we have documented here (Figures 1-3).…”

“…35 For instance, Fas signalling may impact on secretory routes linking ER with proximal Golgi by early modification of BAP31 13 or similar traffic regulators that retain selected membrane proteins within the ER. 29 Functional alteration of these ER retention proteins via caspase cleavage could induce a dispersal and surface exposure of specific membrane proteins, 29,30 that may include glycoproteins reacting with HPA.…”

“…15,38 Organelle staining. Mitochondria and other organelles were specifically stained either in solution (for intensified video microscopy (IVM) analysis 37 ) or after attachment to polylysine-coated coverslips 35 with Mitotrackers (MTR green or red) and fluorescent lectins. 21 After 10-60 min incubation, cells were fixed with 4% paraformaldehyde (w/v in PBS), permeabilized with Triton-X100, blocked, incubated with monoclonal antibodies for 1 h, washed again and then incubated for 45-60 min with anti-mouse secondary antibodies conjugated with Alexa Fluor 488 or Rhodamine X.…”

“…21 Measurements were carried out in a plate reader with appropriate filters (Fluoroskan Ascent, Thermo, Basingstoke, UK). Endocytosis was also measured following the fluorescence changes of FM1-43 35 in a plate reader, using 5 Â 10 5 cells/ml in a modified Ringer buffer (RB, containing 145 mM NaCl, 4.5 mM KCl, 2 mM MgCl 2 , 1 mM CaCl 2 , 5 mM K-HEPES, pH 7.4, and 10 mM glucose) and 2-4 mM FM1-43. The activity of cathepsin L was measured using the substrate rhodamine110-FR-bisamide.…”