Kevin L. Holmes

Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein.

BACKGROUND Fluorescence resonance energy transfer (FRET) is a powerful technique for measuring molecular interactions at Angstrom distances. We present a new method for FRET that utilizes the unique spectral properties of variants of the green fluorescent protein (GFP) for large-scale analysis by flow cytometry. METHODS The proteins of interest are fused in frame separately to the cyan fluorescent protein (CFP) or the yellow fluorescent protein (YFP). FRET between these differentially tagged fusion proteins is analyzed using a dual-laser FACSVantage cytometer. RESULTS We show that homotypic interactions between individual receptor chains of tumor necrosis factor receptor (TNFR) family members can be detected as FRET from CFP-tagged receptor chains to YFP-tagged receptor chains. Noncovalent molecular complexation can be detected as FRET between fusions of CFP and YFP to either the intracellular or extracellular regions of the receptor chains. The specificity of the assay is demonstrated by the absence of FRET between heterologous receptor pairs that do not biochemically associate with each other. Interaction between a TNFR-like receptor (Fas/CD95/Apo-1) and a downstream cytoplasmic signaling component (FADD) can also be demonstrated by flow cytometric FRET analysis. CONCLUSIONS The utility of spectral variants of GFP in flow cytometric FRET analysis of membrane receptors is demonstrated. This method of analyzing FRET allows probing of noncovalent molecular interactions that involve both the intracellular and extracellular regions of membrane proteins as well as proteins within the cells. Unlike biochemical methods, FRET allows the quantitative determination of noncovalent molecular associations at Angstrom level in living cells. Moreover, flow cytometry allows quantitative analyses to be carried out on a cell-by-cell basis on large number of cells. Published 2001 Wiley-Liss, Inc.

Chromothriptic Cure of WHIM Syndrome

Chromothripsis is a catastrophic cellular event recently described in cancer in which chromosomes undergo massive deletion and rearrangement. Here, we report a case in which chromothripsis spontaneously cured a patient with WHIM syndrome, an autosomal dominant combined immunodeficiency disease caused by gain-of-function mutation of the chemokine receptor CXCR4. In this patient, deletion of the disease allele, CXCR4(R334X), as well as 163 other genes from one copy of chromosome 2 occurred in a hematopoietic stem cell (HSC) that repopulated the myeloid but not the lymphoid lineage. In competitive mouse bone marrow (BM) transplantation experiments, Cxcr4 haploinsufficiency was sufficient to confer a strong long-term engraftment advantage of donor BM over BM from either wild-type or WHIM syndrome model mice, suggesting a potential mechanism for the patients cure. Our findings suggest that partial inactivation of CXCR4 may have general utility as a strategy to promote HSC engraftment in transplantation.

Preparation of Cells and Reagents for Flow Cytometry

Flow cytometry is widely used for analyzing the expression of cell surface and intracellular molecules (on a per cell basis), characterizing and defining different cell types in heterogeneous populations, assessing the purity of isolated subpopulations, and analyzing cell size and volume. This technique is predominantly used to measure fluorescence intensity produced by fluorescent‐labeled antibodies or ligands that bind to specific cell‐associated molecules. A procedure for direct and indirect staining of single‐cell suspensions of lymphoid tissue or peripheral blood lymphocytes to detect cell surface membrane antigens is presented. In addition, support protocols present methods for fluorescence labeling of purified antibodies. A protocol for flow cytometric analysis of intracellular antigens in single‐cell suspensions is also included. Alternate protocols describe intracellular staining of unfixed cells in the presence of a detergent and staining of nonviable cells to facilitate discrimination of dead cells in fixed or permeabilized cell preparations.

Infection and maturation of monocyte-derived human dendritic cells by human respiratory syncytial virus, human metapneumovirus, and human parainfluenza virus type 3

Human respiratory syncytial virus (HRSV), human metapneumovirus (HMPV), and human parainfluenza virus type 3 (HPIV3) are common, important respiratory pathogens, but HRSV has a substantially greater impact with regard to acute disease, long-term effects on airway function, and frequency of re-infection. It has been reported to strongly interfere with the functioning of dendritic cells (DC). We compared HRSV to HMPV and HPIV3 with regard to their effects on human monocyte-derived immature DC (IDC). Side-by-side analysis distinguished between common effects versus those specific to individual viruses. The use of GFP-expressing viruses yielded clear identification of robustly infected cells and provided the means to distinguish between direct effects of robust viral gene expression versus bystander effects. All three viruses infected inefficiently based on GFP expression, with considerable donor-to donor-variability. The GFP-negative cells exhibited low, abortive levels of viral RNA synthesis. The three viruses induced low-to-moderate levels of DC maturation and cytokine/chemokine responses, increasing slightly in the order HRSV, HMPV, and HPIV3. Infection at the individual cell level was relatively benign, such that in general GFP-positive cells were neither more nor less able to mature compared to GFP-negative bystanders, and cells were responsive to a secondary treatment with lipopolysaccharide, indicating that the ability to mature was not impaired. However, there was a single exception, namely that HPIV3 down-regulated CD38 expression at the RNA level. Maturation by these viruses was anti-apoptotic. Inefficient infection of IDC and sub-optimal maturation might result in reduced immune responses, but these effects would be common to all three viruses rather than specific to HRSV.

Chapter 9 Protein labeling with fluorescent probes

Publisher Summary This chapter presents an overview of the field of fluorochrome bioconjugation, including a discussion of the basis of the procedures, optimization of the conjugation, and fluorochrome specific topics. The conjugation of fluorochromes to antibody molecules is emphasized, but the same principles can be applied to other large proteins. The ability to label proteins or antibodies with fluorochrome dyes empowers the researcher with the tools to investigate a myriad of biological questions. Foremost is the ability to couple antibodies with organic dyes and phycobiliproteins for use in flow cytometry and imaging. A reflection of the usefulness of these reagents is the growth of commercially available fluorescent-labeled antibodies in more recent years. However, as new monoclonal antibodies are made or new dyes discovered, it is important to understand the relatively simple chemistries involved in fluorescent conjugation. The most important fact to remember is that optimal conjugation may require slight modification of conditions for each protein labeled.

Murine hematopoietic cell surface antigen expression.

Abstract Monoclonal antibodies have identified numerous cell surface antigens expressed at various stages of hematopoietic cell development in mice and humans. Complex systems of nomenclature have evolved as new antigens have been discovered. Here, Kevin Holmes and Herbert Morse III summarize Ly antigen expression during murine hematological cell development and in the accompanying centre-page diagram provide a comprehensive guide to antigen nomenclature.

A Regulatory Role for ADP-ribosylation Factor 6 (ARF6) in Activation of the Phagocyte NADPH Oxidase

In activated neutrophils NADPH oxidase is regulated through various signaling intermediates, including heterotrimeric G proteins, kinases, GTPases, and phospholipases. ADP-ribosylation factor (ARF) describes a family of GTPases associated with phospholipase D (PLD) activation. PLD is implicated in NADPH oxidase activation, although it is unclear whether activation of PLD by ARF is linked to receptor-mediated oxidase activation. We explored whether ARF participates in NADPH oxidase activation by formyl-methionine-leucine-phenylalanine (fMLP) and whether this involves PLD. Using multicolor forward angle light scattering analyses to measure superoxide production in differentiated neutrophil-like PLB-985 cells, we tested enhanced green fluorescent fusion proteins of wild-type ARF1 or ARF6, or their mutant counterparts. The ARF6(Q67L) mutant defective in GTP hydrolysis caused increased superoxide production, whereas the ARF6(T27N) mutant defective in GTP binding caused diminished responses to fMLP. The ARF1 mutants had no effect on fMLP responses, and none of the ARF proteins affected phorbol 12-myristate 13-acetate-elicited oxidase activity. PLD inhibitors 1-butanol and 2,3-diphosphoglycerate, or the ARF6(N48R) mutant assumed to be defective in PLD activation, blocked fMLP-elicited oxidase activity in transfected cells. The data suggest that ARF6 but not ARF1 modulates receptor-mediated NADPH oxidase activation in a PLD-dependent mechanism. Because PMA-elicited NADPH oxidase activation also appears to be PLD-dependent, but ARF-independent, ARF6 and protein kinase C may act through distinct pathways, both involving PLD.

CD23 Trimers Are Preassociated on the Cell Surface Even in the Absence of Its Ligand, IgE

Allergic disease is mediated by high levels of allergen-specific IgE. IgE binding to CD23, the low affinity receptor for IgE, results in a negative feedback signal leading to a decrease in IgE production. Previous studies have shown that CD23 associates as an oligomer and that cooperative binding of at least two lectin domains is required for high affinity IgE binding to CD23. We have previously shown that cooperative binding is required for regulation of IgE production. This study describes the production of several mAbs that bind the stalk region of murine CD23. One of the Abs, 19G5, inhibited the IgE/CD23 interaction at 37°C, but not at 4°C. Analysis of the binding properties of these Abs revealed that CD23 dissociates at high temperatures, such as 37°C; however, the N terminus is constitutively associated, indicating partial, rather than complete, dissociation. A novel finding was that the stalk region, previously thought to mediate trimer association, was not required for oligomerization. These data reveal important information about the structure of CD23 that may be useful in modulating IgE production.

Flow Cytometry Analysis Using the Becton Dickinson FACS Calibur

This unit details the operation of a FACS Calibur flow cytometer for cell analysis. The operation of the Becton Dickinson FACS Calibur and CELLQuest software version 3.0 is described, but the unit is general enough to be helpful for users of all flow cytometers. The FACS Calibur replaces both the FACSCan and FACSort; the information presented here is also applicable to older BD instruments. In this unit, particular emphasis is placed on data acquisition rather than data analysis. Single-color analysis using fluorescein isothiocyanate (FITC)-conjugated antibodies is described along with procedures to check instrument performance and sensitivity, single-color (FITC) analysis with simultaneous live/dead discrimination using propidium iodide (PI), simultaneous two-color analysis using FITC- and phycoerythrin (PE)-conjugated antibodies, two-color FITC/PE analysis with simultaneous live/dead discrimination using PI, simultaneous three-color immunofluorescence with FITC, PE, and the red fluorescent dyes, and finally, simultaneous four-color immunofluorescence with FITC, PE, red fluorescence dyes, and APC.

International Society for the Advancement of Cytometry Cell Sorter Biosafety Standards

Flow cytometric cell sorting of biological specimens has become prevalent in basic and clinical research laboratories. These specimens may contain known or unknown infectious agents, necessitating precautions to protect instrument operators and the environment from biohazards arising from the use of sorters. To this end the International Society of Analytical Cytology (ISAC) was proactive in establishing biosafety guidelines in 1997 (Schmid et al., Cytometry 1997;28:99–117) and subsequently published revised biosafety standards for cell sorting of unfixed samples in 2007 (Schmid et al., Cytometry Part A J Int Soc Anal Cytol 2007;71A:414–437). Since their publication, these documents have become recognized worldwide as the standard of practice and safety precautions for laboratories performing cell sorting experiments. However, the field of cytometry has progressed since 2007, and the document requires an update. The new Standards provides guidance: (1) for laboratory design for cell sorter laboratories; (2) for the creation of laboratory or instrument specific Standard Operating Procedures (SOP); and (3) on procedures for the safe operation of cell sorters, including personal protective equipment (PPE) and validation of aerosol containment. Published