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First published online 14 August 2007
doi: 10.1242/jcs.007260

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Differential targeting of secretory lysosomes and recycling endosomes in mast cells revealed by patterned antigen arrays

¹ Department of Chemistry and Chemical Biology, Cornell University, Ithaca, NY 14853, USA
² School of Applied and Engineering Physics, Cornell University, Ithaca, NY 14853, USA

View larger version (78K): [in this window] [in a new window]   Fig. 1. Stimulated exocytosis detected by annexin V binding. (A,B) Substantial differences are observed for exocytosis stimulated by patterned bilayers (A) with and (B) without lipid ligand DNP-cap-DPPE (`Ag'). (C,D) The Alexa Fluor-488-annexin V label (green) is localized to cells on patterned regions (red) compared with cells (reflected light overlay) off patterns when only half of the chip is covered by haptenated lipids (C), but it is not localized to the defined regions of stimulus at the subcellular level (D). Cells were incubated with the patterns for 30 minutes at 37°C. Bars, 100 µm (A-C), 20 µm (D).

View larger version (35K): [in this window] [in a new window]   Fig. 2. Secretory lysosome labeled by transmembrane protein CD63-EGFP undergoes antigen-stimulated exocytosis as observed by confocal fluorescence microscopy. Vesicular secretion is characterized by the instantaneous loss of LysoTracker Red (LS) fluorescence (red) and flattening of the CD63-EGFP-labeled granular membrane (green) with the plasma membrane. The profile analysis shows a more gradual spreading of membrane bound fluorescence compared with release of LysoTracker Red. Blue, green, orange lines correspond to 0 seconds, 6 seconds and 9 seconds, respectively. Bars, 10 µm.

View larger version (68K): [in this window] [in a new window]   Fig. 3. TIRFM reveals that exocytosis occurs away from the initial stimulus. (A,B) Representative single exocytosis event (A) in which the fluorescence signal of a single secretory granule labeled by CD63-EGFP (arrows) undergoes a two-stage change, quantified in B by granule peak intensity (green) and width of the bright spot (red) that is fitted by a 2D Gaussian distribution. (C) Summary of visualized exocytosis sites (red circles) from a single representative cell. Boundaries of patterned bilayers in A and C are shown in green. (D) Frequency of exocytosis events on pattern, near pattern and off pattern; the distance from fusion site to the nearest pattern bilayer is less than 0.6 µm, between 0.6 µm and 1.2 µm, and larger than 1.2 µm, respectively. The radius of the pattern feature size is 0.6 µm, and the distance between closest features is 4.8 µm. (E) Three exocytotic events that happened within 40 seconds and took place at exactly the same location; all three events are full fusion. (F) Example fusion events representing three different scenarios before the final full fusion: (I) fusion event shortly after the appearance of the granule, (II) granule is close to membrane but moves in x-y direction before fusion, (III) granule is docked at fusion site for some time before fusion. (G) Quantification of the events I, II and III in terms of peak intensity (green) and width (red). The fourth panel shows normalized average intensity of a 7x7 pixel area centered at peak for several representative cases of transient (I, blue), intermediate (II, purple) or long-docking step (III, green) prior to fusion. Time is indicated in (A,B). (F,G) is referenced by setting the initial fusion frame as 10 seconds. Bars, 2 µm (A), 5 µm (C). Image size of E and G is 2.5 µm.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Recycling endosomes labeled by Alexa Fluor-488 CTB-GM1 are targeted locally to the site of initial stimulus. (A) Confocal fluorescence images illustrate the intracellular pool of CTB-GM1 (equatorial view) and the enrichment of CTB-GM1 over the patterned bilayer (interfacial view). (B,C) Differential mobility of (B) Lyn-EGFP and (C) CTB-GM1 in patterns measured by FPR. (D,E) Slow mobility of surface CTB-GM1 measured by FPR in unstimulated cell (D) with or (E) without the internal perinuclear pool fluorescently labeled to assess contribution of outward trafficking to the fluorescence recovery on the plasma membrane. Bars, 25 µm (A), 10 µm (B-E).