N-way FRET microscopy of multiple protein-protein interactions in live cells.

Fluorescence Resonance Energy Transfer (FRET) microscopy has emerged as a powerful tool to visualize nanoscale protein-protein interactions while capturing their microscale organization and millisecond dynamics. Recently, FRET microscopy was extended to imaging of multiple donor-acceptor pairs, thereby enabling visualization of multiple biochemical events within a single living cell. These methods require numerous equations that must be defined on a case-by-case basis. Here, we present a universal multispectral microscopy method (N-Way FRET) to enable quantitative imaging for any number of interacting and non-interacting FRET pairs. This approach redefines linear unmixing to incorporate the excitation and emission couplings created by FRET, which cannot be accounted for in conventional linear unmixing. Experiments on a three-fluorophore system using blue, yellow and red fluorescent proteins validate the method in living cells. In addition, we propose a simple linear algebra scheme for error propagation from input data to estimate the uncertainty in the computed FRET images. We demonstrate the strength of this approach by monitoring the oligomerization of three FP-tagged HIV Gag proteins whose tight association in the viral capsid is readily observed. Replacement of one FP-Gag molecule with a lipid raft-targeted FP allowed direct observation of Gag oligomerization with no association between FP-Gag and raft-targeted FP. The N-Way FRET method provides a new toolbox for capturing multiple molecular processes with high spatial and temporal resolution in living cells.

A growth factor signaling cascade confined to circular ruffles in macrophages.

The formation of macropinosomes requires large-scale movements of membranes and the actin cytoskeleton. Over several minutes, actin-rich surface ruffles transform into 1-5 µm diameter circular ruffles, which close at their distal margins, creating endocytic vesicles. Previous studies using fluorescent reporters of phosphoinositides and Rho-family GTPases showed that signals generated by macrophages in response to the growth factor Macrophage Colony-Stimulating Factor (M-CSF) appeared transiently in domains of plasma membrane circumscribed by circular ruffles. To address the question of how signaling molecules are coordinated in such large domains of plasma membrane, this study analyzed the relative timing of growth factor-dependent signals as ruffles transformed into macropinosomes. Fluorescent protein chimeras expressed in macrophages were imaged by microscopy and quantified relative to circular ruffle formation and cup closure. The large size of macropinocytic cups allowed temporal resolution of the transitions in phosphoinositides and associated enzyme activities that organize cup closure. Circular ruffles contained transient and sequential spikes of phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)), phosphatidylinositol (3,4,5)-trisphosphate (PIP(3)), diacylglycerol, PI(3,4)P(2), PI(3)P and the activities of protein kinase C-α, Rac1, Ras and Rab5. The confinement of this signal cascade to circular ruffles indicated that diffusion barriers present in these transient structures focus feedback activation and deactivation of essential enzyme activities into restricted domains of plasma membrane.

Ruffles limit diffusion in the plasma membrane during macropinosome formation.

In murine macrophages stimulated with macrophage-colony-stimulating factor (M-CSF), signals essential to macropinosome formation are restricted to the domain of plasma membrane enclosed within cup-shaped, circular ruffles. Consistent with a role for these actin-rich structures in signal amplification, microscopic measures of Rac1 activity determined that disruption of actin polymerization by latrunculin B inhibited ruffling and the localized activation of Rac1 in response to M-CSF. To test the hypothesis that circular ruffles restrict the lateral diffusion of membrane proteins that are essential for signaling, we monitored diffusion of membrane-tethered, photoactivatable green fluorescent protein (PAGFP-MEM) in ruffling and non-ruffling regions of cells. Although diffusion within macropinocytic cups was not inhibited, circular ruffles retained photoactivated PAGFP-MEM inside cup domains. Confinement of membrane molecules by circular ruffles could explain how actin facilitates positive feedback amplification of Rac1 in these relatively large domains of the plasma membrane, thereby organizing the contractile activities that close macropinosomes.

Transient increase in cyclic AMP localized to macrophage phagosomes.

Cyclic AMP (cAMP) regulates many biological processes and cellular functions. The importance of spatially localized intracellular gradients of cAMP is increasingly appreciated. Previous work in macrophages has shown that cAMP is produced during phagocytosis and that elevated cAMP levels suppress host defense functions, including generation of proinflammatory mediators, phagocytosis and killing. However, the spatial and kinetic characteristics of cAMP generation in phagocytosing macrophages have yet to be examined. Using a Förster resonance energy transfer (FRET)-based cAMP biosensor, we measured the generation of cAMP in live macrophages. We detected no difference in bulk intracellular cAMP levels between resting cells and cells actively phagocytosing IgG-opsonized particles. However, analysis with the biosensor revealed a rapid decrease in FRET signal corresponding to a transient burst of cAMP production localized to the forming phagosome. cAMP levels returned to baseline after the particle was internalized. These studies indicate that localized increases in cAMP accompany phagosome formation and provide a framework for a more complete understanding of how cAMP regulates macrophage host defense functions.

Innate immune signals modulate antiviral and polyreactive antibody responses during severe respiratory syncytial virus infection.

Antiviral antibody production during respiratory syncytial virus (RSV) infection in infants is poorly understood. To characterize local B lymphocyte responses, lung tissue and secretions from infants with RSV bronchiolitis were analyzed for innate B cell-stimulating factors and antiviral antibodies. In lung tissues of infants with fatal RSV bronchiolitis, CD20(+) lymphocytes and IgM-positive, IgG-positive, and IgA-positive plasma cells were prominent but CD4(+) T lymphocytes were not. Type I interferon-induced proteins and B cell tropic factors, including B cell-activating factor (BAFF) and a proliferation-inducing ligand (APRIL), were colocalized in infected epithelium. In nasopharyngeal secretions from infants who survived RSV infection, class-switched antiviral and antinucleosomal antibodies were detected at presentation and correlated with BAFF and APRIL levels. Expression of APRIL and antiviral antibodies of IgA and IgM but not IgG isotype predicted better oxygen saturation. We conclude that B lymphocyte-stimulating factors derived from infected epithelium are primary determinants of the mucosal antibody response in infant RSV bronchiolitis.

Macrophage impairment underlies airway occlusion in primary respiratory syncytial virus bronchiolitis.

Although respiratory syncytial virus (RSV) infection is the most important cause of bronchiolitis in infants, the pathogenesis of RSV disease is poorly described. We studied histopathologic changes in a panel of lung tissue specimens obtained from infants with fatal cases of primary RSV infection. In these tissues, airway occlusion with accumulations of infected, apoptotic cellular debris and serum protein was consistently observed. Similar observations were found after RSV infection in New Zealand black (NZB) mice, which have constitutive deficiencies in macrophage function, but not in BALB/c mice. A deficiency in the number of alveolar macrophages in NZB mice appears to be central to enhanced disease, because depletion of alveolar macrophages in BALB/c mice before RSV exposure resulted in airway occlusion. In mice with insufficient numbers of macrophages, RSV infection yielded an increased viral load and enhanced expression of type I interferon-associated genes at the height of disease. Together, our data suggest that innate, rather than adaptive, immune responses are critical determinants of the severity of RSV bronchiolitis.

Respiratory syncytial virus and influenza virus infections: observations from tissues of fatal infant cases.

Respiratory syncytial virus (RSV) and influenza virus are common causes of infantile lower respiratory tract infection (LRTI). It is widely believed that both viral replication and inappropriately enhanced immune responses contribute to disease severity. In infants, RSV LRTI is known to be more severe than influenza virus LRTI. We compared cytokines and chemokines in secretions of infants surviving various forms of respiratory illness caused by RSV or influenza viruses, to determine which mediators were associated with more severe illness. We analyzed lung tissue from fatal cases of RSV and influenza LRTI to determine the types of inflammatory cells present. Quantities of lymphocyte-derived cytokines were minimal in secretions from infants with RSV infection. Concentrations of most cytokines were greater in influenza, rather than RSV, infection. Lung tissues from fatal RSV and influenza LRTI cases demonstrated extensive presence of viral antigen and a near absence of CD8-positive lymphocytes and natural killer cells, with marked expression of markers of apoptosis. Severe infantile RSV and influenza virus LRTI is characterized by inadequate (rather than excessive) adaptive immune responses, robust viral replication and apoptotic crisis.

Severe human lower respiratory tract illness caused by respiratory syncytial virus and influenza virus is characterized by the absence of pulmonary cytotoxic lymphocyte responses.

BACKGROUND: Respiratory syncytial virus (RSV) and influenza virus are common causes of infantile lower respiratory tract infection (LRTI). It is widely believed that both viral replication and inappropriately enhanced immune responses contribute to disease severity. In infants, RSV LRTI is known to be more severe than influenza virus LRTI. METHODS: We compared cytokines and chemokines in secretions of infants surviving various forms of respiratory illness caused by RSV or influenza viruses, to determine which mediators were associated with more-severe illness. We analyzed lung tissue from infants with fatal cases of RSV and influenza virus LRTI to determine the types of inflammatory cells present. Autopsy tissues were studied for the lymphotoxin granzyme and the apoptosis marker caspase 3. RESULTS: Quantities of lymphocyte-derived cytokines were minimal in secretions from infants with RSV infection. Concentrations of most cytokines were greater in influenza virus, rather than RSV, infection. Lung tissues from infants with fatal RSV and influenza virus LRTI demonstrated an extensive presence of viral antigen and a near absence of CD8-positive lymphocytes and natural killer cells, with marked expression of markers of apoptosis. CONCLUSIONS: Severe infantile RSV and influenza virus LRTI is characterized by inadequate (rather than excessive) adaptive immune responses, robust viral replication, and apoptotic crisis.