Adam D. Hoppe

Fluorescence Resonance Energy Transfer-Based Stoichiometry in Living Cells

Imaging of fluorescence resonance energy transfer (FRET) between fluorescently labeled molecules can measure the timing and location of intermolecular interactions inside living cells. Present microscopic methods measure FRET in arbitrary units, and cannot discriminate FRET efficiency and the fractions of donor and acceptor in complex. Here we describe a stoichiometric method that uses three microscopic fluorescence images to measure FRET efficiency, the relative concentrations of donor and acceptor, and the fractions of donor and acceptor in complex in living cells. FRET stoichiometry derives from the concept that specific donor-acceptor complexes will give rise to a characteristic FRET efficiency, which, if measured, can allow stoichiometric discrimination of interacting components. A first equation determines FRET efficiency and the fraction of acceptor molecules in complex with donor. A second equation determines the fraction of donor molecules in complex by estimating the donor fluorescence lost due to energy transfer. This eliminates the need for acceptor photobleaching to determine total donor concentrations and allows for repeated measurements from the same cell. A third equation obtains the ratio of total acceptor to total donor molecules. The theory and method were confirmed by microscopic measurements of fluorescence from cyan fluorescent protein (CFP), citrine, and linked CFP-Citrine fusion protein, in solutions and inside cells. Together, the methods derived from these equations allow sensitive, rapid, and repeatable detection of donor-, acceptor-, and donor-acceptor complex stoichiometry at each pixel in an image. By accurately imaging molecular interactions, FRET stoichiometry opens new areas for quantitative study of intracellular molecular networks.

The coordination of signaling during Fc receptor-mediated phagocytosis

Phagocytosis by macrophages can be initiated by Fcγ receptors (FcR) in membranes that bind to Fc regions of immunoglobulin G (IgG). Activated FcR transduce signals to cytoplasm, which regulate the internalization of IgG‐coated particles into plasma membrane‐derived vacuoles, phagosomes. Particles internalized by phagocytosis are much larger than FcR, which prompts questions of if and how the receptors are coordinated with each other. FcR‐mediated signal transduction entails recruitment of proteins from cytoplasm to the receptor, largely via protein phosphorylation. These FcR signaling complexes then activate proteins that regulate actin, myosin, membrane fusion, and the production of reactive oxygen intermediates. Recent fluorescence microscopic studies of phagocytosis in macrophages indicate that signaling by FcR occurs as a sequence of distinct stages, evident in the spatial and temporal patterns of phosphoinositides, protein kinase C, and Rho‐family GTPase activation on forming phagosomes. The coordination of these stages may be regulated by lipids or lipid‐anchored proteins, which diffuse away from FcR complexes. Lateral diffusion of FcR‐derived signals could integrate FcR‐dependent responses over large areas of membrane in the forming phagosome.

The uniformity of phagosome maturation in macrophages.

Many studies of endocytosis and phagocytosis presume that organelles containing a single kind of internalized particle exhibit invariant patterns of protein and phospholipid association as they mature inside cells. To test this presumption, fluorescent protein chimeras were expressed in RAW 264.7 macrophages, and time-lapse ratiometric fluorescence microscopy was used to measure the maturation dynamics of individual phagosomes containing IgG-opsonized erythrocytes. Quantitative analysis revealed consistent patterns of association for YFP chimeras of β-actin, Rab5a, Rab7, and LAMP-1, and no association of YFP chimeras marking endoplasmic reticulum or Golgi. YFP-2xFYVE, recognizing phosphatidylinositol 3-phosphate (PI(3)P), showed two patterns of phagosome labeling. Some phagosomes increased labeling quickly after phagosome closure and then lost the label within 20 min, whereas others labeled more slowly and retained the label for several hours. The two patterns of PI(3)P on otherwise identical phagosomes indicated that organelle maturation does not necessarily follow a single path and that some features of phagosome maturation are integrated over the entire organelle.

Membrane perforations inhibit lysosome fusion by altering pH and calcium in Listeria monocytogenes vacuoles

Listeria monocytogenes (Lm) evade microbicidal defences inside macrophages by secreting a pore‐forming cytolysin listeriolysin O (LLO), which allows Lm to escape vacuoles. LLO also inhibits Lm vacuole fusion with lysosomes, which indicates LLO alters vacuole chemistry prior to release of Lm into cytoplasm. Using fluorescent probes to measure membrane permeability, calcium and pH, we identified small membrane perforations in vacuoles containing wild‐type but not LLO‐deficient (hly‐) Lm. The small membrane perforations released small fluorescent molecules and persisted for several minutes before expanding to allow exchange of larger fluorescent molecules. Macropinosomes and hly‐ Lm vacuoles acidified and increased their calcium content ([Ca2+]vac) within minutes of formation; however, the small perforations made by LLO‐expressing bacteria increased vacuolar pH and decreased [Ca2+]vac shortly after infection. Experimental increases in vacuolar pH inhibited Lm vacuole fusion with lysosomes. The timing of perforation indicated that LLO‐dependent delays of Lm vacuole maturation result from disruption of ion gradients across vacuolar membranes.

Kinesin-1 structural organization and conformational changes revealed by FRET stoichiometry in live cells

Kinesin motor proteins drive the transport of cellular cargoes along microtubule tracks. How motor protein activity is controlled in cells is unresolved, but it is likely coupled to changes in protein conformation and cargo association. By applying the quantitative method fluorescence resonance energy transfer (FRET) stoichiometry to fluorescent protein (FP)–labeled kinesin heavy chain (KHC) and kinesin light chain (KLC) subunits in live cells, we studied the overall structural organization and conformation of Kinesin-1 in the active and inactive states. Inactive Kinesin-1 molecules are folded and autoinhibited such that the KHC tail blocks the initial interaction of the KHC motor with the microtubule. In addition, in the inactive state, the KHC motor domains are pushed apart by the KLC subunit. Thus, FRET stoichiometry reveals conformational changes of a protein complex in live cells. For Kinesin-1, activation requires a global conformational change that separates the KHC motor and tail domains and a local conformational change that moves the KHC motor domains closer together.

Arp2 Links Autophagic Machinery with the Actin Cytoskeleton

Macroautophagy involves lysosomal/vacuolar elimination of long-lived proteins and entire organelles from the cytosol. The process begins with formation of a double-membrane vesicle that sequesters bulk cytoplasm, or a specific cargo destined for lysosomal/vacuolar delivery. The completed vesicle fuses with the lysosome/vacuole limiting membrane, releasing its content into the organelle lumen for subsequent degradation and recycling of the resulting macromolecules. A majority of the autophagy-related (Atg) proteins are required at the step of vesicle formation. The integral membrane protein Atg9 cycles between certain intracellular compartments and the vesicle nucleation site, presumably to supply membranes necessary for macroautophagic vesicle formation. In this study we have tracked the movement of Atg9 over time in living cells by using real-time fluorescence microscopy. Our results reveal that an actin-related protein, Arp2, briefly colocalizes with Atg9 and directly regulates the dynamics of Atg9 movement. We propose that proteins of the Arp2/3 complex regulate Atg9 transport for specific types of autophagy.

Sequential signaling in plasma-membrane domains during macropinosome formation in macrophages.

Macropinosomes are large endocytic vesicles that form in ruffling regions of plasma membrane. To analyze signal organization relative to ruffle closure into circular ruffles and cup closure into macropinosomes, this study used quantitative microscopy to measure 3′ phosphoinositides and small-GTPase activities in a representative subset of forming macropinosomes. Macropinocytosis was stimulated by the addition of macrophage colony-stimulating factor (M-CSF) to macrophages expressing fluorescent reporter proteins. Ratiometric and fluorescence resonance energy transfer (FRET) microscopy determined that Rac1 activity and phosphatidylinositol (3,4,5)-trisphosphate [PtdIns(3,4,5)P3] levels increased transiently, peaking 26-30 seconds after ruffle closure. Three-dimensional reconstruction of cells labeled with the fluorescent dye FM4-64 showed that PtdIns(3,4,5)P3 was restricted to open, circular cups in the plasma membrane. Quantitative fluorescence microscopic methods determined the timing of cup closure, which followed 40-100 seconds after Rac1 and PtdIns(3,4,5)P3 deactivation and coincided with accumulation of phosphatidylinositol 3-phosphate and Rab5a. Thus, ruffle closure creates a circular domain of plasma membrane that localizes the activation and deactivation of Rac1 and phosphoinositide 3-kinase (PI3K), followed by recruitment of Rab5a and the contractile activities of cup closure.

Isolation of a Novel Swine Influenza Virus from Oklahoma in 2011 Which Is Distantly Related to Human Influenza C Viruses

Of the Orthomyxoviridae family of viruses, only influenza A viruses are thought to exist as multiple subtypes and has non-human maintenance hosts. In April 2011, nasal swabs were collected for virus isolation from pigs exhibiting influenza-like illness. Subsequent electron microscopic, biochemical, and genetic studies identified an orthomyxovirus with seven RNA segments exhibiting approximately 50% overall amino acid identity to human influenza C virus. Based on its genetic organizational similarities to influenza C viruses this virus has been provisionally designated C/Oklahoma/1334/2011 (C/OK). Phylogenetic analysis of the predicted viral proteins found that the divergence between C/OK and human influenza C viruses was similar to that observed between influenza A and B viruses. No cross reactivity was observed between C/OK and human influenza C viruses using hemagglutination inhibition (HI) assays. Additionally, screening of pig and human serum samples found that 9.5% and 1.3%, respectively, of individuals had measurable HI antibody titers to C/OK virus. C/OK virus was able to infect both ferrets and pigs and transmit to naive animals by direct contact. Cell culture studies showed that C/OK virus displayed a broader cellular tropism than a human influenza C virus. The observed difference in cellular tropism was further supported by structural analysis showing that hemagglutinin esterase (HE) proteins between two viruses have conserved enzymatic but divergent receptor-binding sites. These results suggest that C/OK virus represents a new subtype of influenza C viruses that currently circulates in pigs that has not been recognized previously. The presence of multiple subtypes of co-circulating influenza C viruses raises the possibility of reassortment and antigenic shift as mechanisms of influenza C virus evolution.

A Cdc42 activation cycle coordinated by PI 3-kinase during Fc receptor-mediated phagocytosis

During Fc receptor-mediated phagocytosis in macrophages, PI 3-kinase mediates transitions in the signaling by Rho-family GTPases. Receptor-activated Cdc42 increases PI 3-kinase activity. Increased 3′ phosphoinositide concentrations in phagocytic cups then deactivate Cdc42.

Abnormal small heat shock protein interactions involving neuropathy-associated HSP22 (HSPB8) mutants

Two mutations (K141E, K141N) in the small heat shock protein (sHSP) HSP22 (HSPB8) are associated with the inherited peripheral motor neuron disorders distal hereditary motor neuropathy type II and axonal Charcot‐Marie‐Tooth disease type 2L. HSP22 is known to form homodimers, heterodimers with other sHSPs, and larger oligomers. In an effort to elucidate the cellular basis for these diseases, we have determined the ability of mutant HSP22 to interact with itself, with wild‐type HSP22, and with other sHSPs that are abundant in neurons. Using the yeast two‐hybrid method, quantitative fluorescence resonance energy transfer in live cells, and cross‐linking, we found aberrantly increased interactions of mutant HSP22 forms with themselves, with wild‐type HSP22, and with the other sHSPs, αB‐crystallin, and HSP27. Interaction with HSP20 was not affected by the mutations. The data suggest that each mutant form of HSP22 has a characteristic pattern of abnormal interaction properties. A mutation (S135F) in HSP27 that is also associated with these disorders showed increased interaction with wild‐type HSP22 also, suggesting linkage of these two etiologic factors, HSP22 and HSP27, into one common pathway. Increased interactions involving mutant sHSPs may be the molecular basis for their increased tendency to form cytoplasmic protein aggregates, and for the occurrence of the associated neuropathies.—Jean‐Marc Fontaine, Xiankui Sun, Adam D. Hoppe, Stephanie Simon, Patrick Vicart, Michael J. Welsh, and Rainer Benndorf. Abnormal small heat shock protein interactions involving neuropathy‐associated HSP22 (HSPB8) mutants. FASEB J. 20,E1579–E1588 (2006)