Dynamic shuttling of TIA-1 accompanies the recruitment of mRNA to mammalian stress granules.

Mammalian stress granules (SGs) harbor untranslated mRNAs that accumulate in cells exposed to environmental stress. Drugs that stabilize polysomes (emetine) inhibit the assembly of SGs, whereas drugs that destabilize polysomes (puromycin) promote the assembly of SGs. Moreover, emetine dissolves preformed SGs as it promotes the assembly of polysomes, suggesting that these mRNP species (i.e., SGs and polysomes) exist in equilibrium. We used green flourescent protein-tagged SG-associated RNA-binding proteins (specifically, TIA-1 and poly[A] binding protein [PABP-I]) to monitor SG assembly, disassembly, and turnover in live cells. Fluorescence recovery after photobleaching shows that both TIA-1 and PABP-I rapidly and continuously shuttle in and out of SGs, indicating that the assembly of SGs is a highly dynamic process. This unexpected result leads us to propose that mammalian SGs are sites at which untranslated mRNAs are sorted and processed for either reinitiation, degradation, or packaging into stable nonpolysomal mRNP complexes. A truncation mutant of TIA-1 (TIA-1DeltaRRM), which acts as a transdominant inhibitor of SG assembly, promotes the expression of cotransfected reporter genes in COS transfectants, suggesting that this process of mRNA triage might, directly or indirectly, influence protein expression.

Expression of ryanodine receptors in human embryonic kidney (HEK293) cells.

It has been shown previously that mobilization of caffeine-sensitive intracellular calcium (Ca2+i) stores increased the release of amyloid beta-peptide (Abeta) from transfected human embryonic kidney cells (HEK293) [Querfurth, Jiang, Geiger and Selkoe (1997) J. Neurochem. 69, 1580-1591]. The present study was to test the hypothesis that the caffeine/Abeta responses were due to interactions with specific subtypes of ryanodine receptors (RyR) using [3H]ryanodine receptor binding, epifluorescence imaging of Ca2+i, immunocytofluorescence, immunoprecipitation and PCR techniques. [3H]Ryanodine bound to a single class of high-affinity caffeine-sensitive sites (Kd=9.9+/-1.6 nM, Bmax=25+/-4 fmol/mg of protein). RyRs were immuno-decorated in a punctate reticulo-linear pattern. Results from SDS/PAGE and reverse transcriptase-PCR demonstrated endogenous expression of type 1 (skeletal) and type 2 (cardiac) RyRs. HEK293 cell RyRs were functionally active, because (i) [Ca2+]i increased 2.8-fold over baseline following applications of 5-15 mM caffeine, (ii) repetitive spiked increases in [Ca2+]i were observed, and (iii) evidence for a use-dependent block was obtained. Some of these findings were extended to include HeLa and human fibroblast cell lines, suggesting a broader applicability to cells of epithelioid lineage. Implications for the processing of the beta-amyloid precursor protein in Alzheimer's disease and for calcium channel research using transfected HEK293 cells are discussed.

Red cell membrane remodeling in sickle cell anemia. Sequestration of membrane lipids and proteins in Heinz bodies.

In red cells from patients with sickle cell anemia, hemoglobin S denatures and forms Heinz bodies. Binding of Heinz bodies to the inner surface of the sickle cell membrane promotes clustering and colocalization of the membrane protein band 3, outer surface-bound autologous IgG and, to some extent, the membrane proteins glycophorin and ankyrin. Loss of transbilayer lipid asymmetry is also found in certain populations of sickle red cells. The lateral distribution of sickle cell membrane lipids has not been examined, however. In this report, we examine by fluorescence microscopy the incorporation and distribution of the fluorescent phospholipid analogues 7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-phosphatidylserine and NBD-phosphatidylcholine in sickle red cells. Both phospholipid analogues are observed to accumulate prominently at sites of Heinz bodies. Accumulation at sites of Heinz bodies is also shown by 1,'1-dihexadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, a fluorescent lipid analogue that readily crosses membranes, but not by fluorescein-phosphatidylethanolamine, an analogue that is localized to the outer leaflet of the membrane. Double labeling and confocal microscopy techniques show that NBD-lipids, band 3 protein, protein 4.1, ankyrin, and spectrin are all sequestered within sickle red cells and colocalized at sites of Heinz bodies. We propose that Heinz bodies provide a hydrophobic surface on which sickle red cell membrane lipids and proteins are sequestered.

Immune interferon gamma inhibits translational mobility of a plasma membrane protein in preimplantation stage mouse embryos: a T-helper 1 mechanism for immunologic reproductive failure.

OBJECTIVE: Immune interferon gamma adversely affects mouse embryo development and has been proposed as a mediator of reproductive failure involving T-helper 1 immunity. We hypothesized that one mechanism by which interferon gamma could exert an adverse effect on embryos was by altering plasma membrane organization and transmembrane protein mobility. STUDY DESIGN: The fluorescence photobleaching recovery technique was used to measure the effect of the T-helper 1 cytokine interferon gamma on the translational mobility of a specific embryonic surface glycoprotein recognized by the monoclonal antibody S75. RESULTS: Compared with controls interferon gamma significantly decreased the fractional mobility of fluorescein isothiocyanate S75 in one- and two-cell mouse embryos. CONCLUSION: Interferon gamma may alter plasma membrane domains or cytoskeletal organization in early-stage embryos. By restricting plasma membrane protein mobility interferon gamma could effect T-helper 1-mediated reproductive failure.

Use of the translational mobility of a plasma membrane protein to assess fertilization of mouse oocytes and viability of mouse zygotes and two-cell embryos.

The fluorescence photobleaching recovery (FPR) technique was used to measure the translational mobility of a glycoprotein recognized by the monoclonal antibody (mAb) S75 in plasma membranes of mouse oocytes, zygotes, and two-cell embryos. Glycoprotein fractional mobility (f) was significantly decreased in membranes of unfertilized oocytes compared to zygotes or two-cell embryos (f values, 46 +/- 2 and 65 +/- 2%, respectively; p < 0.0001). Reduced apparent glycoprotein mobility was also observed in morphologically degenerated zygotes and two-cell embryos compared to viable zygotes and two-cell embryos (f values, 8 +/- 1 and 60 +/- 3%, respectively; p < 0.0001). These results indicate that the FPR technique can be used to assess oocyte fertilization and preimplantation embryonic viability. This method may be useful in the evaluation of embryonic viability following in vitro fertilization and in the detection of toxic effects of novel compounds on embryonic development.

Mechanism of spontaneous intracellular calcium fluctuations in single GH4C1 rat pituitary cells.

Individual unstimulated GH4C1 cells exhibited spontaneous dynamic fluctuations in cytosolic free Ca2+ concentration ([Ca2+]i). Either chelation of extracellular Ca2+ with EGTA or treatment with nifedipine inhibited spontaneous [Ca2+]i fluctuations, indicating that the [Ca2+]i profile was dependent on the entry of extracellular Ca2+ via voltage-operated Ca2+ channels (VOCC). Spontaneous [Ca2+]i fluctuations did not resume immediately after exposure of EGTA-pretreated cells to extracellular Ca2+, supporting the hypothesis that the complex [Ca2+]i profiles observed in unstimulated cells required filling of an intracellular Ca2+ pool. BAY K 8644 elicited large rapid oscillations in [Ca2+]i. After chelation of extracellular Ca2+, however, re-addition of Ca2+ plus BAY K 8644 did not result in [Ca2+]i oscillations. The intracellular Ca2+ pool necessary for BAY K-induced oscillations was not the same Ins(1,4,5)P3-sensitive pool stimulated by thyrotropin-releasing hormone (TRH), because the TRH-stimulated Ins(1,4,5)P3-induced [Ca2+]i spike and the BAY K 8644-induced oscillations were differentially sensitive to chelation of extracellular Ca2+ and thapsigargin. Caffeine caused an increase in [Ca2+]i fluctuations in quiescent cells, supporting a role for Ca(2+)-induced Ca2+ release (CICR) in the generation of spontaneous [Ca2+]i fluctuations. In conclusion, the complex spontaneous changes in [Ca2+]i observed in single GH4C1 cells depend on both the influx of extracellular Ca2+ through VOCC and the action of an intracellular Ca2+ pool that increases [Ca2+]i through a CICR-like mechanism.

Low density lipoproteins bound to Schistosoma mansoni do not alter the rapid lateral diffusion or shedding of lipids in the outer surface membrane.

Schistosomula of Schistosoma mansoni bind human low density lipoproteins (LDL) in a concentration-dependent and saturable manner. The bound LDL could provide phospholipids and sterol to the worm, which cannot synthesize sterol de novo and lacks acyl chain-modifying capability. Here we have used three phospholipid analogues to explore the effect of LDL binding on the parasite's outer tegumental membrane, i.e. the outer of the two membranes that cover its surface syncytium. Fluorescein phosphatidylethanolamine (Fl-PE) and rhodamine phosphatidylethanolamine (Rh-PE) bound to the parasite in a saturable manner and, as shown by fluorescence microscopy, were confined to the surface. Fl-PE fluorescence was completely quenched by Trypan Blue and Fl-PE was lost from the surface following single exponential decay kinetics (t1/2 = 12 h), further suggesting that the probe was confined to the outer membrane. 1,1'-Dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-C18(3); DiI) did not bind saturably and was seen in both the surface and the internal parasite membranes. Fluorescence photobleaching recovery was used to measure the lateral mobility of Fl-PE in the outer membrane. The lateral diffusion coefficient of Fl-PE was approximately 10(-7) cm2s-1. The fractional mobility of Fl-PE was 85% when measured using a laser beam of radius 1.8 microns and 45% using a beam of radius 4.3 microns. These measurements suggest that the outer membrane is composed of micron-scale liquid crystalline-phase lipid domains that lack significant amounts of transmembrane proteins. LDL binding to the parasite surface did not alter the lateral mobility of Fl-PE or the rate of loss of either Fl-PE or Rh-PE.(ABSTRACT TRUNCATED AT 250 WORDS)