22 research outputs found
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
Membrane Diffusion Barriers Localize Signal Amplification during Macropinocytosis.
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 plasma membrane, thereby organizing the contractile activities that close macropinosomes. Using quantitative fluorescence microscopy, we demonstrate that macropinosome formation is directed by a sequence of chemical changes within the cups of plasma membrane circular ruffles. Stages of receptor-dependent signaling were organized into distinct transient waves of phosphoinositides, diacylglycerol, PKCα, Rac and Ras activities, which preceded cup closure and peak recruitment of Rab5 to macropinosomes. Thus, circular ruffles enclose plasma membrane subdomains that focus receptor signal amplification and the signal transitions that coordinate cell movements.Ph.D.ImmunologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/86450/1/twellive_1.pd
A growth factor signaling cascade confined to circular ruffles in macrophages
Summary
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)P2), phosphatidylinositol (3,4,5)-trisphosphate (PIP3), diacylglycerol, PI(3,4)P2, 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
Innate Immune Signals Modulate Antiviral and Polyreactive Antibody Responses during Severe Respiratory Syncytial Virus Infection
N-Way FRET Microscopy of Multiple Protein-Protein Interactions in Live Cells
<div><p>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.</p></div
Transient burst of cAMP at the developing phagosome.
<p>RAW cells expressing C4 or the Epac-camps biosensor were fed opsonized targets and component images for phase-contrast and FRET were taken every 30 sec to capture the phagocytic process from initiation to closure of the phagocytic cup. A. and B. Left insert: A phase-contrast (top) and corresponding E<sub>A</sub> image (bottom) of intact live macrophages transfected with C4 control (A) or Epac-camps (B), from the designated time intervals. Right inserts: One minute time course of a magnified portion of the cell transfected with the specified plasmid (beginning immediately after the sRBCs were added to the culture). The red circle denotes the location of the opsonized sRBC on the E<sub>A</sub> image. Color bar indicates scale of ratio and scale bar is 10 µm in the left insert and 5 µm.in the right insert C. To normalize for non-specific cAMP-mediated effects during phagocytosis, the data are shown as the phagosome-specific difference in E<sub>A</sub> between C4 control and Epac-camps (ec) biosensor-expressing cells. (ie., (E<sub>A(C4-phago)</sub>/E<sub>A(C4-cell)</sub>) − (E<sub>A(ec-phago)</sub>/E<sub>A(ec-cell)</sub>); n = 10 cells) D. To verify that the differences seen between cells transfected with the C4 control construct and the Epac-camps construct were not due to selective bleaching of one of the fluorescent proteins, the R<sub>I</sub> values are plotted (ie., (R<sub>I(C4-phago)</sub>/R<sub>I(C4-cell)</sub>) − (R<sub>I(ec-phago)</sub>/R<sub>I(ec-cell)</sub>)).</p
Changes in cAMP measured by biochemical and FRET microscopic methods in macrophages.
<p>A. RAW cells were plated overnight at 2×10<sup>6</sup> cells per well and stimulated with 200 µM forskolin for 20 min or with EdTx for 3 h. During the final 20 min incubation, either PBS (blank bars) or opsonized sRBCs (open bars) were added to cells as indicated. Total cAMP was quantified as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013962#s2" target="_blank">material and methods</a>. Data represent the mean ± SEM. B. and C. Cells expressing C4 control (B) or the Epac-camps biosensor (C) were either analyzed directly or following treatment with the indicated compounds. The relative amount of FRET after each condition was determined and the results are graphed as mean ± SEM (n = 50–100 cells per condition). D. A representative phase-contrast (top) and corresponding E<sub>A</sub> image (bottom) of an untreated or EdTx-treated macrophage. Color bar indicates scale of ratio and scale bar is 10 µm.</p
No change in total cAMP during phagocytosis.
<p>RAW cells were transfected with plasmids for C4 control or the Epac-camps biosensor. Total cellular E<sub>A</sub> was measured over time and plotted relative to the first measured value. A. Measurements of unfed macrophages showed small decreases in FRET, indicating selective photobleaching of mCit. B. Transfected cells were fed opsonized targets and phagocytosis was synchronized as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013962#s2" target="_blank">material and methods</a>. No significant changes in cAMP were detectable during phagocytosis. Results are shown as mean ± SEM of 4–7 cells.</p
Linear unmixing of FRET amplitudes and N-Way FRET analysis in living cells.
<p><b>A</b>) Images of live COS7 cells expressing a CFP-RFP linked construct were captured using paired excitation and emission filter combinations (e.g. cc = CFP excitation, CFP emission) to sample the N-Way FRET landscape. <b>D</b>) ROIs to provide the raw intensities from these cells. Unmixing of these images using matrix <b>A</b> recovers the fluorescence abundances (e.g. xC) and showed that FRET could be observed only between CFP and RFP (e.g. xCR) (<b>B,</b> images are scaled independently) and was reproducible over multiple cells (<b>E</b>). <b>C</b>) Quantitative unmixing with N-Way FRET showed that equal abundances of CFP and RFP were present and FRET was observed as E[CR], but no was observed for E[CY] or E[YR] as expected (<b>F</b>). 20 cells per condition; data from Scope 2; bars not connected by the same letter are significantly different (p<0.05) by Tukey HSD post hoc comparison of means; error bars are standard deviation.</p
N-Way FRET on the excitation-emission landscape.
<p>Spectroscopically, FRET is a coupling between donor excitation and acceptor emission. This excitation-emission coupling (Φ) can be described by the outer product of the excitation vector <b>ε</b> and an emission vector <b>s</b>. The Φ signatures define the spectral library <b>A</b> for the N-Way FRET linear unmixing problem (<b>d</b> = <b>Ax</b> = <b>Bc</b>) that can be viewed on the 2D excitation-emission landscape in addition to viewing the data (<b>d</b>). Specifically, these appear as topographical features with light green = 0, warmer colors are increasing height and dark blue colors are negative. <b>A</b>) The 2D spectrum for CFP-YFP FRET can be decomposed into the superposition of CFP (Φ<sub>C,C</sub>), YFP (Φ<sub>Y,Y</sub>) and CFP-YFP FRET (Φ<sub>C,Y</sub>). Recovering <b>ε</b> and <b>s</b> for each fluorophore in the system allows calculation of the unmixing matrix, <b>A</b> which can be linearly unmixed to estimate the fluorescence from CFP (x<sub>CFP</sub>), YFP (x<sub>YFP</sub>) and the FRET sensitized emission (x<sub>CY</sub>). <b>B</b> can be obtained by calibration with known FRET efficiency standards. Linear unmixing with <b>B</b> to allows estimation of concentrations of total fluorophores ([CFP] and [YFP]) and apparent FRET (E<sub>CY</sub>[CFP-YFP]) which are contained in vector <b>c</b>. During this step, a negative component (blue color) couples the FRET-associated decrease in donor fluorescence to an increase in acceptor fluorescence. <b>B</b>) For most instruments, the complete landscape is not measured, rather, excitation and emission bandpass filters (boxes) define portions of the excitation-emission landscape. For 2-Way FRET the three images needed are d<sub>c,c</sub>, d<sub>y,y</sub> and d<sub>c,y</sub>. <b>C</b>) As more fluorophores are added to the system (e.g. the addition of RFP), the spectral landscape grows by the addition of direct fluorescence components along the diagonal (d<sub>1,1,</sub> d<sub>2,2</sub>, and d<sub>3,3</sub>) and their possible FRET interactions which appear as off-diagonal peaks (e.g. d<sub>1,2</sub>, d<sub>1,3</sub> and d<sub>2,3</sub>). <b>D</b>) The mathematical form of this problem generalizes to account for multiple fluorophores engaged in FRET.</p