The precise control of many T cell functions relies on cytoso-

No abstract

BackgroundOlfactory discrimination depends on the large numbers of odorant receptor genes and differential ligand-receptor signaling among neurons expressing different receptors. In this study, we describe an in vitro system that enables the expression of exogenous odorant receptors in cultured olfactory sensory neurons. Olfactory sensory neurons in the culture express characteristic signaling molecules and, therefore, provide a system to study receptor function within its intrinsic cellular environment.ResultsWe demonstrate that cultured olfactory sensory neurons express endogenous odorant receptors. Lentiviral vector-mediated gene transfer enables successful ectopic expression of odorant receptors. We show that the ectopically expressed mouse I7 is functional in the cultured olfactory sensory neurons. When two different odorant receptors are ectopically expressed simultaneously, both receptor proteins co-localized in the same olfactory sensory neurons up to 10 days in vitro.ConclusionThis culture technique provided an efficient method to culture olfactory sensory neurons whose morphology, molecular characteristics and maturation progression resembled those observed in vivo. Using this system, regulation of odorant receptor expression and its ligand specificity can be studied in its intrinsic cellular environment.

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...

A jumonji domain containing gene 6 (Jmjd6), previously referred to as phosphatidylserine receptor (PSR) gene, plays an important role in cell differentiation and development of multiple organs, although mechanisms of its action are not known. The Jmjd6 gene product was initially identified as a membrane protein that participates in phagocytosis. However, the later findings that recombinant Jmjd6 in expression systems was targeted to the nucleus challenged the role of Jmjd6 as a membrane receptor. Using immunocytochemistry approach we studied the subcellular distribution of endogenous Jmjd6 protein in THP-1 cells activated with phorbol 12-myristate 13 acetate (PMA). We found that treatment with PMA stimulated Jmjd6 expression in the cytosol of activated cells. Furthermore, Jmjd6 initially appeared at the cell surface of immature phagocytes (1-2 days after activation) but then translocated into the nucleus of differentiated macrophage-like cells (5-9 days after activation). Anti-Jmjd6 antibodies suppressed the engulfment of dead cell corpses by THP-1 cells expressing the Jmjd6 at the cell surface. These data indicate that Jmjd6 serves as a membrane-associated receptor that regulates phagocytosis in immature macrophages but is dispensable for phagocytosis and has other functions when it is expressed in the cytosol and nucleus of mature macrophage-like cells.