In this study, we report the organization of cytoskeletal and large transmembrane proteins at the inhibitory and activating NK cell immunological or immune synapse (IS). Filamentous actin accumulates at the activating, but not the inhibitory, NK cell IS. However, surprisingly, ezrin and the associated protein CD43 are excluded from the inhibitory, but not the activating, NK cell IS. This distribution of ezrin and CD43 at the inhibitory NK cell IS is similar to that previously seen at the activating T cell IS. CD45 is also excluded from the inhibitory, but not activating, NK cell IS. In addition, electron microscopy reveals wide and narrow domains across the synaptic cleft. Target cell HLA-C, located by immunogold labeling, clusters where the synaptic cleft spans the size of HLA-C bound to the inhibitory killer Ig-like receptor. These data are consistent with assembly of the NK cell IS involving a combination of cytoskeletal-driven mechanisms and thermodynamics favoring the organization of receptor/ligand pairs according to the size of their extracellular domains.

The heat shock response has been extensively studied in a variety of systems and organisms, and generally involves the conserved and coordinated upregulation of heat shock proteins that act to alleviate the cellular stresses imposed by hyperthermic stress. Our current understanding of the cellular responses to subphysiological temperatures (hypothermia) is less extensive. This is somewhat surprising, because of their relevance in medicine for the storage of cells, organs, and tissues, and the treatment of brain damage; as well as in the biopharmaceutical sector, where reduced culture temperature can sometimes improve recombinant protein yields from mammalian cells cultured in vitro [1]. What is clear is that the general response to hypothermia appears to include the global attenuation of transcription and translation, whereas a small group of proteins, termed the cold shock proteins, are selectively induced [2]. However, unlike their heat shock counterparts, these cold shock proteins do not appear to be particularly well conserved between prokaryotic and eukaryotic systems, and their functions, such as have been defined, have to date been described in terms of their RNA rather than their protein biology. Exposure to subphysiological temperature is also known to generally lead to changes in the lipid make-up of membranes, resulting in increased membrane rigidity, Mammalian cells cultured in vitro are able to recover from cold stress. However, the mechanisms activated during cold stress and recovery are still being determined. We here report the effects of hypothermia on cellular architecture, cell cycle progression, mRNA stability, protein synthesis and degradation in three mammalian cell lines. The cellular structures examined were, in general, well maintained during mild hypothermia (27-32°C) but became increasingly disrupted at low temperatures (4-10°C). The degradation rates of all mRNAs and proteins examined were much reduced at 27°C, and overall protein synthesis rates were gradually reduced with temperature down to 20°C. Proteins involved in a range of cellular activities were either upregulated or downregulated at 32 and 27°C during cold stress and recovery. Many of these proteins were molecular chaperones, but they did not include the inducible heat shock protein Hsp72. Further detailed investigation of specific proteins revealed that the responses to cold stress and recovery are at least partially controlled by modulation of p53, Grp75 and eIF3i levels. Furthermore, under conditions of severe cold stress (4°C), lipid-containing structures were observed that appeared to be in the process of being secreted from the cell that were not observed at less severe cold stress temperatures. Our findings shed light on the mechanisms involved and activated in mammalian cells upon cold stress and recovery.Abbreviations CCT, chaperonin containing T-complex polypeptide1; Cirp, cold-inducible RNA-binding protein; ER, endoplasmic reticulum; HSF, heat shock factor; NEPHGE, non-equilibrium pH gradient gel electrophoresis;...

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99mTc-SnF2 colloid (Radpharm LLK) leucocyte labelling agent is used in whole blood, exploiting phagocytosis. The objectives of this work were to optimize leucocyte labelling in leucocyte-enriched plasma, and to investigate: (i) the effect of temperature and other factors on labelling efficiency; (ii) the selectivity for different leucocyte types; (iii) the viability of the labelled cells and efflux of the radiolabel; and (iv) the physical characteristics of the colloid. Density gradient centrifugation was used to investigate the labelling efficiency, cell selectivity and efflux, Trypan blue to study the viability, and laser scattering, electron microscopy and membrane filtration to investigate particle size and morphology. Particles appeared as loose, coiled, chain-like aggregates of much smaller particles (<0.05 microm). The aggregate diameter ranged from <0.1 to >5 microm and increased with time. The distribution of radioactivity amongst the particle sizes varied widely. The labelling efficiency in leucocyte-rich plasma was enhanced at 37 degrees C compared to room temperature, and by centrifuging during labelling. The selectivity for different leucocyte types varied markedly between batches and blood samples, in some cases showing preference for mononuclear cells and in others for granulocytes. Viability was excellent and comparable with 99mTc-hexamethylpropyleneamine oxime (99mTc-HMPAO)-labelled cells. A significant fraction of radiolabel, comparable to that observed with 99mTc-HMPAO, was lost from leucocytes during incubation in vitro over 4 h. Thus, 99mTc-SnF2 is a convenient, efficient labelling agent for leucocytes, but shows variable cell selectivity which may be linked to particle size variability, and there is significant efflux of radioactivity from labelled cells.