Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine

Bomford, Adrian; Young, Stephen P.; Williams, R.J.R.

doi:10.1021/bi00335a013

Cited by 24 publications

(28 citation statements)

References 40 publications

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“…While there are other types of iron chelators/inhibitors that can block cellular iron uptake, methylamine is commonly used to inhibit intercellular iron transport. 34,[54][55][56] Our results are further supported by the well-established transferrin-ferritin intracellular iron transport and storage mechanism. 53 Additional evidences showed that the predominant proteins, upregulated during neural stem cell differentiation, involved in iron storage (ferritin L chain) and cytoskeleton.…”

Section: Discussionsupporting

confidence: 80%

Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)

Chen

Lee

et al. 2015

Tissue Engineering Part C: Methods

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Motor neurons loss plays a pivotal role in the pathoetiology of various debilitating diseases such as, but not limited to, amyotrophic lateral sclerosis, primary lateral sclerosis, progressive muscular atrophy, progressive bulbar palsy, pseudobulbar palsy, and spinal muscular atrophy. However, advancement in motor neuron replacement therapy has been significantly constrained by the difficulties in large-scale production at a costeffective manner. Current methods to derive motor neuron heavily rely on biochemical stimulation, chemical biological screening, and complex physical cues. These existing methods are seriously challenged by extensive time requirements and poor yields. An innovative approach that overcomes prior hurdles and enhances the rate of successful motor neuron transplantation in patients is of critical demand. Iron, a trace element, is indispensable for the normal development and function of the central nervous system. Whether ferric ions promote neuronal differentiation and subsequently promote motor neuron lineage has never been considered. Here, we demonstrate that elevated iron concentration can drastically accelerate the differentiation of human embryonic stem cells (hESCs) toward motor neuron lineage potentially via a transferrin mediated pathway. HB9 expression in 500 nM iron-treated hESCs is approximately twofold higher than the control. Moreover, iron treatment generated more matured and functional motor neuron-like cells that are *1.5 times more sensitive to depolarization when compared to the control. Our methodology renders an expedited approach to harvest motor neuron-like cells for disease, traumatic injury regeneration, and drug screening.

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Section: Discussionsupporting

confidence: 80%

Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)

Chen

Lee

et al. 2015

Tissue Engineering Part C: Methods

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“…Both reticulocytes and K562 cells are known to have a high rate of iron uptake, many transferrin receptors, and a high demand for iron (2,25); thus, exposure of transferrin to a pH below 6 might be expected in these cell types, resulting in the observed release of primarily apotransferrin (25)(26)(27). This hypothesis is currently under investigation using the method described here to measure the acidification kinetics in other cell types.…”

Section: Discussionmentioning

confidence: 98%

High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

Sipe

Murphy

1987

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

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The kinetics of acidification of diferric human transferrin in BALB/c mouse 3T3 cells were determined by flow cytometry using a modification of the fluorescein-rhodamine fluorescence ratio technique. For cells labeled at 0°C and warmed to 37C, the minimum pH observed was 6.1 ± 0.2, occurring 5 min after warmup. This step was followed by a slower alkalinization to the pH of the external medium, occurring with a half-time of 5 min. Warmup to 24°C or 17°C resulted in slowing of the time of onset of acidification such that the minimum pH was 6.3 ± 0.2, attained 15 and 25 min after warmup, respectively; the alkalinization step was completely blocked. The limited acidification observed for transferrin corresponds to the initial phase of acidification normally observed for other (nonrecycled) ligands. Since transferrin is not further acidified, the results confirm the existence of two phases of acidification during endocytosis. Measurements of transferrin dissociation at neutral pH after exposure to mildly acidic pH support the conclusion that the transferrin cycle may be completed without exposure of transferrin to a pH below 6. The mildly acidic pH of the endocytic compartments involved in recycling may play a role in regulating enzymatic processing of endocytosed material.Iron delivery to vertebrate cells is primarily mediated by serum transferrin. Uptake of diferric transferrin has been shown to occur through receptor-mediated endocytosis in many different cell types (1-3), and both transferrin and receptor undergo subsequent acidification in the endocytic compartment (4, 5). Unlike most ligands, transferrin does not dissociate from its receptor upon acidification and therefore avoids lysosomal degradation by recycling with the receptor.Several lines of evidence point to the importance of a low endosomal pH in the effective delivery of iron: the increase in the rate of iron release from transfemn upon acidification in vitro (6); the inhibition of iron release caused by lysosomotropic amines known to raise intravesicular pH in vivo (1, 7-9); and the existence of Chinese hamster ovary (CHO) cell mutants defective in acidification, which cannot efficiently extract iron from transferrin (10-12 MATERIALS AND METHODS Preparation of Transferrin Conjugates. Human diferric transfemn was obtained from Miles Scientific (>98% pure). FITC and lissamine rhodamine sulfonyl chloride (LRSC) were obtained from Molecular Probes (Junction City, OR). Transferrin was conjugated with either FITC (FITC-Tf) or LRSC (LRSC-Tf) in 0.1 M sodium carbonate (pH 9.6). Free dye was removed using G-25 columns. The fluorochrome/ protein molar ratio was 4.3 for the FITC conjugate and 5.9 for the LRSC conjugate. These ratios were found to yield optimal specificity (90% or greater), and a signal/noise ratio (ratio of fluorescence from bound labeled transferrin to autofluorescence of unlabeled cells) of -3.0 for each conjugate. The mean number of transferrin binding sites per cell was determined to be 13,000 by using FITC-conjugated calibration b...

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“…In a similar manner, Young and coworkers found the rate of recycling using the loss of cell-associated radiolabeled complexes as a percentage of the initial, or maximum, amount of Tf bound [102]. Therefore, Eq.…”

Section: Obtaining a Quantitative Understanding Of Tf Trafficking mentioning

confidence: 99%

“…In a similar manner, Young and coworkers evaluated the rate constant for the internalization of the holo-Tf/TfR complex, neglecting the dissociation of holo-Tf from TfR [102]. In this case, the authors measured the percentage of cell-surface complexes remaining prior to endocytosis using pronase to distinguish cell-surface bound from internalized complexes.…”

Section: Obtaining a Quantitative Understanding Of Tf Trafficking mentioning

confidence: 99%

The intracellular trafficking pathway of transferrin

Mayle

Kamei

2012

Biochimica et Biophysica Acta (BBA) - General Subjects

370

326

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Background Transferrin (Tf) is an iron-binding protein that facilitates iron-uptake in cells. Iron-loaded Tf first binds to the Tf receptor (TfR) and enters the cell through clathrin-mediated endocytosis. Inside the cell, Tf is trafficked to early endosomes, delivers iron, and then is subsequently directed to recycling endosomes to be taken back to the cell surface. Scope of Review We aim to review the various methods and techniques that researchers have employed for elucidating the Tf trafficking pathway and the cell-machinery components involved. These experimental methods can be categorized as microscopy, radioactivity, and surface plasmon resonance (SPR). Major Conclusions Qualitative experiments, such as total internal reflectance fluorescence (TIRF), electron, laser-scanning confocal, and spinning-disk confocal microscopy, have been utilized to determine the roles of key components in the Tf trafficking pathway. These techniques allow temporal resolution and are useful for imaging Tf endocytosis and recycling, which occur on the order of seconds to minutes. Additionally, radiolabeling and SPR methods, when combined with mathematical modeling, have enabled researchers to estimate quantitative kinetic parameters and equilibrium constants associated with Tf binding and trafficking. General Significance Both qualitative and quantitative data can be used to analyze the Tf trafficking pathway. The valuable information that is obtained about the Tf trafficking pathway can then be combined with mathematical models to identify design criteria to improve the ability of Tf to deliver anticancer drugs.

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Release of iron from the two iron-binding sites of transferrin by cultured human cells: modulation by methylamine

Cited by 24 publications

References 40 publications

Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)

Accelerated Neuronal Differentiation Toward Motor Neuron Lineage from Human Embryonic Stem Cell Line (H9)

High-resolution kinetics of transferrin acidification in BALB/c 3T3 cells: exposure to pH 6 followed by temperature-sensitive alkalinization during recycling.

The intracellular trafficking pathway of transferrin

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