Technical Reference #1641

1641.

A Phosphoinositide Switch Controls the Maturation and Signaling Properties of APPL Endosomes Roberto Zoncu; Rushika Perera; Daniel Balkin; Michelle Pirruccello; Derek Toomre; Pietro De Camilli, Howard Hughes Medical Insitute, Cell, 136(1641), (2009)
Link To Paper

Abstract:
The recent identification of several novel endocytic compartments has challenged our current understandingof the topological and functional organizationof the endocytic pathway. Using quantitativesingle vesicle imaging and acute manipulation ofpho

Materials & Methods:
Materials Plasmids antibodies and other reagents are listed in the Supplemental Data. Cell culture Cell culture transfection fixation and immunofluorescence were performed as described (Perera et al. 2006). Cells were seeded in glass-bottomed 35 mm dishes (Mattek Corporation Ashland MA) and imaged by epifluorescence TIRF (Olympus Center Valley PA) or spinning disk confocal (Perkin Elmer Waltham MA) microscopy. See Supplemental Data. Total Internal Reflection Fluorescence Imaging Cells were imaged at 37 C using an Olympus objective-type IX-70 inverted microscopy fitted with a 60 3 1.45 N.A. TIRFM lens (Olympus Center Valley PA) controlled by Andor iQ software (Andor Technologies Belfast Ireland). Cells were typically imaged in two channels by sequential excitation at 0.25 Hz or 0.5 Hz without binning with 0.2 to 0.5 s exposures and detected with a back-illuminated Andor iXon 897 EMCCD camera (512 3 512 14 bit; Andor Technologies). The depth of the evanescent field was 100 nm. Image Analysis Analysis of fluorescence spots in TIRF images was conducted as described (Perera et al. 2006; Zoncu et al. 2007). Briefly average fluorescence/time plots were generated from 10–15 fluorescent spots which were subsequently time-aligned by the conversion of one fluorescent marker to another using Andor software (Andor Technologies Belfast Ireland). Manual tracking of the conversion of fluorescent spots (macropinosomes) was conducted using the ‘‘manual tracking’’ Image J plug-in (National Institutes of Health). Particle count was conducted using Image J as previously described (Zoncu et al. 2007). All data were analyzed for significance using student’s t test. Rapalogue-Mediated Depletion of Phosphoinositides Experiments involving rapalogue -mediated recruitment of an inositol 5-phosphatase to the plasma membrane were performed as described (Zoncu et al. 2007). For PI3P depletion from Rab5-positive endosomes cells were transiently transfected with CFP-FKBP32-Rab5 mRFP-FRB-MTM1 (kind gift of B. Larijani) and a GFP-tagged endosomal marker (APPL1 WDFY2 or EEA1) and imaged by TIRF sequentially at 488 and 568 nm. After 10 min 500 nM rapalogue (AP 021967 Ariad Pharmaceuticals Cambridge MA) was added and changes in fluorescent protein distribution were monitored for a further 20 min. Cell-Signaling Assays Cells were transiently transfected with GFP-FKBP32-Rab5 and mRFP-FRBMTM1 or mRFP-FRB-MTM1C375S and treated with 500 nM rapalogue at 37 C for 15 min in serum free medium. Subsequently an equal volume of serum free medium containing 3 ng/ml of EGF was added to the dishes (yielding a final concentration of 1.5 ng/ml of EGF) and incubated at 37 C. At different time points cells were washed in cold PBS and lysed in lysis buffer (1% SDS 25mMTris [pH 7.4] 150mMNaCl 1mMEDTA containing Protease Inhibitor cocktail) prior to western blot analysis.

Microscopic Technique
Fluorescence Microscopy, Epifluorescence

Cell Type(s)
COS-7