Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

Brock, Roland; Jovin, Thomas M.

doi:10.1242/jcs.114.13.2437

Cited by 23 publications

(1 citation statement)

References 43 publications

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“…Another important question is whether focal receptor stimulation gets generalized through the spread of receptor activation. To this end we used magnetic beads covalently coupled to EGF or Herceptin and showed that receptor activation remained localized to the site where the ligand-or antibody-coated bead bound (Brock and Jovin 2001;Brock and Jovin 2003;Fig. 2.4).…”

Section: Focal Stimulation Of Cells With Magnetic Beadsmentioning

confidence: 99%

Novel Single Cell Fluorescence Approaches in the Investigation of Signaling at the Cellular Level

Nagy¹,

Vereb²,

Post³

et al.

Springer Series in Biophysics

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Section: Focal Stimulation Of Cells With Magnetic Beadsmentioning

confidence: 99%

Novel Single Cell Fluorescence Approaches in the Investigation of Signaling at the Cellular Level

Nagy¹,

Vereb²,

Post³

et al.

Springer Series in Biophysics

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Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking

Derfus

Chan

Bhatia³

2004

Advanced Materials

526

488

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time. This might be a promising antimicrobial coating for a wide range of materials for biomedical or daily-life applications. ExperimentalPEI (MW 5000 g/mol) was purchased from Hyperpolymers (Freiburg, Germany). All other chemicals, if not stated otherwise, were from Fluka and used without further purification. One gram of PEI was suspended in 50 mL acetone and the mixture was cooled to 0 C . Methacryloyl chloride (0.83 mL dissolved in 35 mL acetone) was added dropwise to the stirred suspension within 20 min. After 30 min at 0 C, 150 mL of methanol and 20.4 mL of HEA were added to give a clear solution. The solvents were removed under reduced pressure, the PEI-MA/HEA stock solution was further diluted with HEA to control the PEI content, and 1 mg of the photoinitiator Irgacure 651 (Ciba) was dissolved in 1 mL of the solution. 20 lL of the this mixture was spread on a commercial glass slide previously modified with methacryloyloxy-propyltrimethoxysilane using a standard procedure [20]. The slide was then covered with another glass slide, previously coated with a polypropylene film. The liquid layer between the two slides was UV cured in the UV reactor Heraflash from Heraeus-Kulzer (Hanau, Germany) for 180 s to give a transparent film. The films were then washed with water/methanol/triethylamine (TEA) (1:1:1 v/v/v), water/methanol (1:1 v/v), and water, than immersed in a solution of 250 mg AgNO 3 in 8 mL water for 30 s, washed with water, and finally added to 50 mL of an aqueous solution of ascorbic acid (10 mg mL ±1 ). The modification of the films with PEG was performed as follows: The samples were washed with the water/methanol/TEA mixture, methanol, and acetone, then immersed into a solution of 2 g cyanuric chloride in 10 mL acetone at room temperature overnight, rinsed with acetone and chloroform, and finally left in a solution of 2 g O-2-aminoethyl-O¢-methoxy polyethylene glycol (MW 5000 g/ mol) in 10 mL chloroform for 24 h at room temperature. Prior to use the films were thoroughly rinsed with chloroform and immersed in a large amount of water for at least 24 h.UV-vis measurements were carried out with the photospectrometer Lambda 11 from Perkin Elmer. AFM images were recorded with a Nanoscope III scanning probe microscope using Si cantilevers with a fundamental resonance frequency of around 200 kHz. TEM measurements were carried out using a LEO 912 transmission electron microscope applying an acceleration voltage of 120 kV. The silver content was determined using the flame-atom absorption spectrometer Vario 6 from Analytik-Jena-AG (Jena, Germany).The bacterial susceptibility measurement was carried out according to a modified procedure described earlier [21]. S. aureus (ATCC 25123) cells were cultivated by adding 100 lL of a suspension of the bacterial cells in PBS (10 11 cells/mL) to 50 mL of a standard growth medium from Merck and incubating it under shaking at 37 C for 6 h. The bacterial suspension was then centrifuged at 2750 rpm for 10 min, the cells were washed twice with PBS, pH 7.0, re-suspended in...

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Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: Application to the EGF Receptor

Brock

Jovin

2003

Cytometry Pt A

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Background:The molecular analysis of intracellular signal transduction requires technologies that address quantitatively the activation of signaling proteins and formation of molecular complexes without disrupting cellular integrity. Methods: Cells expressing the epidermal growth factor receptor (EGFR) in its endogenous form or fused to green fluorescent protein were incubated with 1-m microspheres covalently functionalized with EGF. The disposition of the plasma membrane about the microspheres was analyzed by high-resolution confocal microscopy in combination with computational resolution enhancement and optimized fixation procedures. Receptor activation and translocation of signaling proteins to the microspheres was quantitated by image processing protocols for recovering the microsphere-associated fluorescence and the fluorescence in the local environment. Results: EGF-functionalized microspheres were internalized in an activation-dependent manner similar to that of

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Heterogeneity of signal transduction at the subcellular level: microsphere-based focal EGF receptor activation and stimulation of Shc translocation

Cited by 23 publications

References 43 publications

Novel Single Cell Fluorescence Approaches in the Investigation of Signaling at the Cellular Level

Novel Single Cell Fluorescence Approaches in the Investigation of Signaling at the Cellular Level

Intracellular Delivery of Quantum Dots for Live Cell Labeling and Organelle Tracking

Quantitative image analysis of cellular protein translocation induced by magnetic microspheres: Application to the EGF Receptor

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