The use of HaloTag-based technology in flow and laser scanning cytometry analysis of live and fixed cells

<p>Abstract</p> <p>Background</p> <p>Combining the technologies of protein tag labeling and optical microscopy allows sensitive analysis of protein function in cells.</p> <p>Findings</p> <p>Here, we describe development of applications using protein tag technology (HaloTag (HT)-based) for flow and laser scanning cytometry (LSC). Cell lines, expressing recombinant surface β1-integrin-HT and HT-p65 fusion protein, and a CD4 T cell line (Jurkat) infected with human immunodeficiency virus type 1 (HIV-1) reporter virus expressing the unfused HT (HIV-1_Lai-Halo), were stained with different HT ligands and successfully detected by flow cytometers equipped with 488 and 561 nm lasers as well as a laser scanning cytometer (equipped with 488 and 405 nm lasers) alone or combined with cell cycle and viability markers.</p> <p>Conclusions</p> <p>Use of HT technology for cytometric applications has advantages over its use in microscopy as it allows for the statistical measurement of protein expression levels in individual cells within a heterogeneous cell population in combination with cell cycle analysis. Another advantage is the ability of the HaloTag to withstand long fixation and high concentration of fixative, which can be useful in research of infectious agents like HIV and/or mycobacteria.</p

Molecular basis of association of receptor activity-modifying protein 3 with the family B G protein-coupled secretin receptor

The three receptor activity-modifying proteins (RAMPs) have been recognized as being important for the trafficking and function of a subset of family B G protein-coupled receptors, although the structural basis for this has not been well established. In the current work, we use morphological fluorescence techniques, bioluminescence resonance energy transfer, and bimolecular fluorescence complementation to demonstrate that the secretin receptor associates specifically with RAMP3, but not with RAMP1 or RAMP2. We use truncation constructs, peptide competition experiments, and chimeric secretin-GLP1 receptor constructs to establish that this association is structurally specific, dependent on the intramembranous region of the RAMP and TM6 and TM7 of this receptor. There were no observed changes in secretin-stimulated cAMP, intracellular calcium, ERK1/2 phosphorylation, or receptor internalization in receptor-bearing COS or CHO-K1 cells in the presence or absence of exogenous RAMP transfection, although the secretin receptor trafficks normally to the cell surface in these cells in a RAMP-independent manner, resulting in both free and RAMP-associated receptor on the cell surface. RAMP3 association with this receptor was shown to be capable of rescuing a receptor mutant (G241C) that is normally trapped intracellularly in the biosynthetic machinery. Similarly, secretin receptor expression had functional effects on adrenomedullin activity, with increasing secretin receptor expression competing for RAMP3 association with the calcitonin receptor-like receptor to yield a functional adrenomedullin receptor. These data provide important new insights into the structural basis for RAMP3 interaction with a family B G protein-coupled receptor, potentially providing a highly selective target for drug action. This may be representative of similar interactions between other members of this receptor family and RAMP proteins

Differential effects of human and plant N-acetylglucosaminyltransferase I (GnTI) in plants

In plants and animals, the first step in complex type N-glycan formation on glycoproteins is catalyzed by N-acetylglucosaminyltransferase I (GnTI). We show that the cgl1-1 mutant of Arabidopsis, which lacks GnTI activity, is fully complemented by YFP-labeled plant AtGnTI, but only partially complemented by YFP-labeled human HuGnTI and that this is due to post-transcriptional events. In contrast to AtGnTI-YFP, only low levels of HuGnTI-YFP protein was detected in transgenic plants. In protoplast co-transfection experiments all GnTI-YFP fusion proteins co-localized with a Golgi marker protein, but only limited co-localization of AtGnTI and HuGnTI in the same plant protoplast. The partial alternative targeting of HuGnTI in plant protoplasts was alleviated by exchanging the membrane-anchor domain with that of AtGnTI, but in stably transformed cgl1-1 plants this chimeric GnTI still did not lead to full complementation of the cgl1-1 phenotype. Combined, the results indicate that activity of HuGnTI in plants is limited by a combination of reduced protein stability, alternative protein targeting and possibly to some extend to lower enzymatic performance of the catalytic domain in the plant biochemical environment

In situ proximity ligation assays indicate that hemochromatosis proteins Hfe and transferrin receptor 2 (Tfr2) do not interact

The hemochromatosis associated proteins HFE and Transferrin Receptor 2 (TFR2) have been shown to be important for the proper regulation of hepcidin. A number of in vitro studies using transient overexpression systems have suggested that an interaction between HFE and TFR2 is required for the regulation of hepcidin. This model of iron sensing which centers upon the requirement for an interaction between HFE and TFR2 has recently been questioned with in vivo studies in mice from our laboratory and others which suggest that Hfe and Tfr2 can regulate hepcidin independently of each other. To re-examine the postulated interaction between Hfe and Tfr2 we developed a novel expression system in which both proteins are stably co-expressed and used the proximity ligation assay to examine the interactions between Hfe, Tfr1 and Tfr2 at a cellular level. We were able to detect the previously described interaction between Hfe and Tfr1, and heterodimers between Tfr1 and Tfr2; however no interaction between Hfe and Tfr2 was observed in our system. The results from this study indicate that Hfe and Tfr2 do not interact with each other when they are stably expressed at similar levels. Furthermore, these results support in vivo studies which suggest that Hfe and Tfr2 can independently regulate hepcidin