Fluorescent detection of fluid phase endocytosis allows for in vivo estimation of endocytic vesicle sizes in plant cells with sub-diffraction accuracy

Traffic
Lars GallTobias Meckel

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.

References

Oct 19, 1979·Biochimica Et Biophysica Acta·F OlsonD Papahadjopoulos
Jul 1, 1977·The Journal of General Virology·F L GrahamR Nairn
Jun 13, 1986·Biochimica Et Biophysica Acta·L D MayerP R Cullis
Jul 11, 1985·Biochimica Et Biophysica Acta·L D MayerA S Janoff
Aug 1, 1965·Journal of Molecular Biology·A D BanghamJ C Watkins
Apr 1, 1993·Chemistry and Physics of Lipids·S KölchensD F O'Brien
Feb 1, 1993·Biophysical Journal·B L MuiT D Madden
Jul 19, 2000·The Plant Cell·J U SutterG Thiel
Nov 5, 2002·Analytical Biochemistry·Takashi KannoTomoji Kawai
Mar 12, 2003·Proceedings of the National Academy of Sciences of the United States of America·David ReigadaCarles Solsona
Jan 21, 2004·The Plant Journal : for Cell and Molecular Biology·Annette C HurstUlrike Homann
Jan 24, 2004·Journal of Experimental Botany·F BaluskaD Volkmann
Apr 28, 2005·European Journal of Pharmaceutical Sciences : Official Journal of the European Federation for Pharmaceutical Sciences·Barbara RuoziMaria Angela Vandelli
Nov 8, 2006·Journal of Experimental Botany·Joseph C Shope, Keith A Mott
Nov 14, 2006·Biophysical Journal·Tobias MeckelGerhard Thiel
Feb 20, 2007·Current Biology : CB·Pankaj DhonuksheJirí Friml
Jul 5, 2007·Nature Reviews. Molecular Cell Biology·Satyajit Mayor, Richard E Pagano
Jul 17, 2007·Current Opinion in Cell Biology·Ernst J Ungewickell, Lars Hinrichsen
Jul 19, 2008·Methods : a Companion to Methods in Enzymology·Stefan Wieser, Gerhard J Schütz
Sep 1, 1988·Planta·O Baron-EpelM Schindler

❮ Previous
Next ❯

Citations

Sep 4, 2010·Protoplasma·Nathalie Leborgne-CastelKarim Bouhidel
Dec 3, 2014·Advanced Drug Delivery Reviews·Florian M LauerTobias Meckel
Apr 22, 2011·Current Opinion in Plant Biology·Christopher GrefenMichael R Blatt
Dec 18, 2012·The FEBS Journal·Li XiangWim Van den Ende
Jan 4, 2012·The Plant Journal : for Cell and Molecular Biology·Vera Bandmann, Ulrike Homann
May 10, 2013·Plant Signaling & Behavior·Ed EtxeberriaJavier Pozueta-Romero
Jul 26, 2017·The Plant Journal : for Cell and Molecular Biology·Hugh C WoolfendenRichard J Morris

❮ Previous
Next ❯

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.