Diffusion of Green Fluorescent Protein in the Aqueous-Phase Lumen of Endoplasmic Reticulum

Dayel, Mark J.; Hom, Erik F. Y.; Verkman, A. S.

doi:10.1016/s0006-3495(99)77438-2

Cited by 293 publications

(308 citation statements)

References 42 publications

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“…To further understand the nature of ER dynamics it is important to identify the unknown factors involved, including the factors linking ER tubules to the motor proteins on MTs during sliding dynamics. Unlike other organelles, the entire ER network is completely continuous at all times, even though it constantly rearranges its structure (Lee and Chen 1988;Ellenberg et al 1997;Dayel et al 1999). During ER dynamics, the ER forms threeway junctions by sliding along MTs to fuse with adjacent ER regions it contacts, contributing to the overall "reticular" appearance of the ER.…”

Section: Er Dynamics and Assemblymentioning

confidence: 99%

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

English

Voeltz

2013

Cold Spring Harbor Perspectives in Biology

294

220

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The endoplasmic reticulum (ER) is a large, continuous membrane-bound organelle comprised of functionally and structurally distinct domains including the nuclear envelope, peripheral tubular ER, peripheral cisternae, and numerous membrane contact sites at the plasma membrane, mitochondria, Golgi, endosomes, and peroxisomes. These domains are required for multiple cellular processes, including synthesis of proteins and lipids, calcium level regulation, and exchange of macromolecules with various organelles at ER-membrane contact sites. The ER maintains its unique overall structure regardless of dynamics or transfer at ER-organelle contacts. In this review, we describe the numerous factors that contribute to the structure of the ER.T he endoplasmic reticulum (ER) is a dynamic organelle responsible for many cellular functions, including the synthesis of proteins and lipids, and regulation of intracellular calcium levels. This review focuses on the distinct and complex morphology of the ER. The structure of the ER is complex because of the numerous distinct domains that exist within one continuous membrane bilayer. These domains are shaped by interactions with the cytoskeleton, by proteins that stabilize membrane shape, and by a homotypic fusion machinery that allows the ER membrane to maintain its continuity and identity. The ER also contains domains that contact the plasma membrane (PM) and other organelles including the Golgi, endosomes, mitochondria, lipid droplets, and peroxisomes. ER contact sites with other organelles and the PM are both abundant and dispersed throughout the cytoplasm, suggesting that they too could influence the overall architecture of the ER. As we will discuss here, ER shape and distribution are regulated by many intrinsic and extrinsic forces. ER STRUCTURE AND FORMATION Domains of the ER Are Stabilized by Membrane-Shaping ProteinsThe endoplasmic reticulum (ER) is a large membrane-bound compartment spread throughout the cytoplasm of eukaryotic cells. It is divided into three major morphologies that include the nuclear envelope (NE), peripheral ER cisternae, and an interconnected tubular network

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Section: Er Dynamics and Assemblymentioning

confidence: 99%

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

English

Voeltz

2013

Cold Spring Harbor Perspectives in Biology

294

220

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“…12 Dayel et al studied the diffusion of green fluorescent protein in the endoplasmic reticulum of cells by comparing rotational diffusion times obtained by time-resolved anisotropy measurements with translational diffusion coefficients obtained by fluorescence recovery after photobleaching. 13 Time-resolved fluorescence anisotropy measurements have also been used to study solgel particle growth as an alternative to scattering techniques, 14 and also to elucidate information about pores in sol-gels. 15 Although single-point time-resolved anisotropy measurements of heterogeneous samples, such as cells, can provide very useful information on the torsional dynamics of cell constituents, 16 the extension of this technique to two dimensions is not well established.…”

Section: Introductionmentioning

confidence: 99%

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

Siegel

Suhling

Lévêque‐Fort

et al. 2003

Review of Scientific Instruments

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We report a picosecond time-gated fluorescence lifetime imaging ͑FLIM͒ system extended to perform time-resolved fluorescence anisotropy imaging ͑TR-FAIM͒. Upon excitation with linearly polarized laser pulses, the parallel and perpendicular components of the fluorescence emission from a sample are imaged simultaneously using a polarization-resolved imager. The imaging technique presented here quantitatively reports the rotational mobility of a fluorophore as it varies according to the local environment. In a single acquisition run it yields maps of both rotational correlation time and fluorescence lifetime as they vary across a sample. TR-FAIM has been applied to imaging standard multiwell plate samples of rhodamine 6G dissolved in methanol, ethylene glycol, trimethylene glycol, and glycerol. The observed rotational correlation times and fluorescence lifetimes, which report the local viscosity and refractive index of the local rhodamine 6G environment, respectively, are in good agreement with previously published single point measurements. By considering the linear dependence of the rotational correlation time on viscosity up to 20 cP, we are able to obtain a two-dimensional viscosity map. Wide-field maps of rotational correlation time, and therefore viscosity, have been obtained. This illustrates the potential to image the local viscosity and fluorescence lifetime distributions of fluorophore tagged proteins in cells.

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“…This technique has been used successfully to measure the movement of a number of macromolecules in bacteria (21)(22)(23)(24)(25). The fluorescent molecule we chose for use was the GFP from Aequoria victoria, because this protein and its many variants have been used to measure the mobility of soluble proteins in a number of systems (26)(27)(28). GFP was expressed to high levels in the cytoplasm or core of spores of Bacillus subtilis (29), and FRAP analysis was used to determine the mobility of this GFP in dormant spores as well as in spores at both stages I and II of spore germination.…”

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confidence: 99%

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

Cowan

Koppel²,

Setlow³

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

146

116

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Fluorescence redistribution after photobleaching has been used to show that a cytoplasmic GFP fusion is immobile in dormant spores of Bacillus subtilis but becomes freely mobile in germinated spores in which cytoplasmic water content has increased Ϸ2-fold. The GFP immobility in dormant spores is not due to the high levels of dipicolinic acid in the spore cytoplasm, because GFP was also immobile in germinated cwlD spores that had excreted their dipicolinic acid but where cytoplasmic water content had only increased to a level similar to that in dormant spores of several other Bacillus species. The immobility of a normally mobile protein in dormant wild-type spores and germinated cwlD spores is consistent with the lack of metabolism and enzymatic activity in these spores and suggests that protein immobility, presumably due to low water content, is a major reason for the metabolic dormancy of spores of Bacillus species.S pores of various Bacillus species are formed in the process of sporulation, and these spores are adapted for long-term survival because they are resistant to many environmental stresses and are metabolically dormant (1-4). However, given the proper stimulus, generally the appearance of specific nutrients in the environment, the dormant spores can return to life through the process of spore germination and then outgrowth (2). A major factor in spore dormancy and resistance is the low level of water in the spore cytoplasm or core [25-55% of the mass of the hydrated dormant spore core depending on the species (1, 3-5)]; another factor may be the high level of pyridine-2,6-dicarboxylic acid [dipicolinic acid (DPA)] (Ϸ10% of the mass of the hydrated stage II-germinated spore core) in the spore core, likely present as a chelate with divalent cations, predominantly Ca 2ϩ (1,6). DPA is released in the first minutes of spore germination and is replaced with water as the spore-core water content rises slightly; these spores are said to have completed stage I of germination (2, 7). Subsequently, the large peptidoglycan cortex around the spore core is degraded, and removal of this restraint allows the spore core to expand rapidly by uptake of water and thus complete stage II of germination (2, 7). This latter event results in a level of core water (75-80% of wet weight) that is similar to that in growing cells (8). As noted above, there is neither metabolism nor enzyme action in the cytoplasm of dormant spores, although there are several enzyme-substrate pairs located in this region of the spore (3). There is also no detectable metabolism or enzyme action in the core of stage I-germinated spores that have excreted their DPA but have not degraded their cortex (7). However, in fully germinated (stage II) spores in which the cortex has been degraded and the cytoplasm is fully hydrated, enzyme action and metabolism begin rapidly (2).One explanation that has been put forward for the lack of enzyme action in dormant and stage I-germinated spores is that the level of water in these spores is too low to allow enzyme a...

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Diffusion of Green Fluorescent Protein in the Aqueous-Phase Lumen of Endoplasmic Reticulum

Cited by 293 publications

References 42 publications

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

Endoplasmic Reticulum Structure and Interconnections with Other Organelles

Wide-field time-resolved fluorescence anisotropy imaging (TR-FAIM): Imaging the rotational mobility of a fluorophore

A soluble protein is immobile in dormant spores of Bacillus subtilis but is mobile in germinated spores: Implications for spore dormancy

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