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Fluorescent Detection of Fluid Phase Endocytosis Allows for In Vivo Estimation of Endocytic Vesicle Sizes in Plant Cells with Sub‐Diffraction Accuracy

Fluorescent Detection of Fluid Phase Endocytosis Allows for In Vivo Estimation of Endocytic... Using the bright, photostable, charged and hydrophilic fluorescent dye Alexa 488 hydrazide to label the fluid phase around intact guard cells, we show that these cells incorporate the fluid phase during constitutive endocytosis against the high turgor. Mobile, cortical and diffraction‐limited signals were not observed if a concentration <4 mm was used to stain the fluid phase, suggesting that endocytic vesicles had to be loaded with a minimal number of dye molecules to produce a signal above the background. To quantify the number of molecules taken up by the vesicles, we prepared liposomes, filled with various concentrations of Alexa 488 hydrazide, fractionated them according to their size and imaged them under identical conditions as the guard cells. From the size/intensity relations of these liposomes, we extrapolated the molecular brightness of Alexa 488 hydrazide. Using this calibration, the mean fluorescent intensity of single endocytic vesicles translates into a mean number of 573 Alexa 488 molecules. If a vesicle needs to take up 573 molecules from a 4 mm solution, it requires a diameter of at least 87 nm. This number provides the first in vivo estimate for the size of endocytic vesicles in intact, turgid plant cells. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Traffic Wiley

Fluorescent Detection of Fluid Phase Endocytosis Allows for In Vivo Estimation of Endocytic Vesicle Sizes in Plant Cells with Sub‐Diffraction Accuracy

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References (49)

Publisher
Wiley
Copyright
© 2010 John Wiley & Sons A/S
ISSN
1398-9219
eISSN
1600-0854
DOI
10.1111/j.1600-0854.2010.01037.x
pmid
20136778
Publisher site
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Abstract

Using the bright, photostable, charged and hydrophilic fluorescent dye Alexa 488 hydrazide to label the fluid phase around intact guard cells, we show that these cells incorporate the fluid phase during constitutive endocytosis against the high turgor. Mobile, cortical and diffraction‐limited signals were not observed if a concentration <4 mm was used to stain the fluid phase, suggesting that endocytic vesicles had to be loaded with a minimal number of dye molecules to produce a signal above the background. To quantify the number of molecules taken up by the vesicles, we prepared liposomes, filled with various concentrations of Alexa 488 hydrazide, fractionated them according to their size and imaged them under identical conditions as the guard cells. From the size/intensity relations of these liposomes, we extrapolated the molecular brightness of Alexa 488 hydrazide. Using this calibration, the mean fluorescent intensity of single endocytic vesicles translates into a mean number of 573 Alexa 488 molecules. If a vesicle needs to take up 573 molecules from a 4 mm solution, it requires a diameter of at least 87 nm. This number provides the first in vivo estimate for the size of endocytic vesicles in intact, turgid plant cells.

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Traffic – Wiley

Published: Apr 1, 2010

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