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Blood, Vol. 110, Issue 1, 125-132, July 1, 2007
Direct interorganellar transfer of iron from endosome to mitochondrion
Blood Sheftel et al.
110: 125
Supplemental materials for: Sheftel et al, Volume 110, Issue 1, 125-132
Files in this Data Supplement:
- Video 1. W-7 inhibits Tf endocytosis (MOV, 1.6 MB)
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Reticulocytes, with MitoTracker Red–labeled mitochondria (red) were incubated for 60 minutes at 4°C in the presence of 60 µM W-7 and then at 37°C in the presence of 500 nM Alexa Green 488 transferrin (green) on a confocal microscope stage. Four hundred images of a single, representative cell were acquired at a rate of 20 frames per second (fps). In order to examine the entire cell, various focal planes were examined during acquisition.
- Video 2. W-7 inhibits the movement of Tf-containing vesicles (MOV, 892 KB)
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Reticulocytes with MitoTracker Red–labeled mitochondria (red) were warmed for 2 minutes in the presence of 500 nM Alexa Green 488 transferrin (green), placed on ice, and treated for 60 minutes with 60 µM W-7. Cells were subsequently incubated at 37°C, and a representative cell was imaged as in Video 1.
- Video 3. Tf-containing vesicles move to close proximity of mitochondria (MOV, 2.58 MB)
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Reticulocytes with MitoTracker Red–labeled mitochondria (red) were incubated (37°C) with 500 nM Alexa Green 488 transferrin (green) on a confocal microscope stage. A representative cell was then imaged as in Video 1 (except that the focal plane remained unchanged).
- Video 4. Proximity of Tf-containing vesicles to mitochondria increases mitochondrial, chelatable iron (MOV, 100 KB)
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Wide-field videomicrograph of the representative reticulocyte/mitochondrion displayed in Figure 6. Cells with mitochondria labeled with the iron-sensitive dye, RDA (red), were treated with AG-Tf (green) and imaged in real-time on the Olympus microscope described in “Experimental procedures.”
- Figure S1. W-7 does not appreciably block heme biosynthesis (JPG, 55.6 KB)
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(A) In order to determine whether protoporphyrin synthesis was affected by the inhibitor, we compared the effects of W-7 on the utilization of an intramitochondrial source of 59Fe with that of 59Fe-Tf. (i) W-7 blocks the incorporation of 59Fe-Tf into heme. Reticulocytes were preincubated on ice with 1 µM 59Fe-Tf for 30 minutes. Where indicated, 60 µM W-7 was included in this step. Cells were then warmed to 37°C for 60 minutes and treated with pronase (30 min / 4°C) to remove surface-bound 59Fe-Tf; the heme and nonheme fractions evaluated. (ii) W-7 does not block heme synthesis. Cells were treated for 60 minutes on ice in the presence of 1 µM 59Fe-Tf, and 2 mM succinylacetone (SA; heme biosynthesis inhibitor) was added for the final 30 minutes of this incubation. The cells were then warmed to 37°C for 30 minutes to allow the uptake of 59Fe. The first lane of panel ii represents the distribution of 59Fe in the samples after this 37°C step; it is expected that the majority of the 59Fe in this sample was mitochondrial.1 Next, the SA was removed by extensive washing (denoted by “+/-”) and the surface-bound 59FeTf removed by pronase digestion. The cells were then reincubated for 60 minutes at 37°C in the presence or absence of 60 µM W-7, washed, and separated into heme and nonheme fractions. Error bars represent standard deviations (n=3). B. In the presence of W-7, the incorporation of iron from 59Fe-Tf into heme is dramatically reduced, whereas, when 59Fe-SIH was used, only a small decrease in iron incorporation into heme was observed. Reticulocytes were incubated at 4°C for 30 minutes in the presence of 30 µM W-7, where indicated, and then treated with the indicated source of Fe for 60 minutes at 37°C. Excess 59Fe was washed from the cells, and the heme and nonheme fractions evaluated. Error bars represent standard deviations (n=3).
REFERENCES
1. Richardson D.R., Ponka P., Vyoral D. Distribution of iron in reticulocytes after inhibition of heme synthesis with succinylacetone: examination of the intermediates involved in iron metabolism. Blood. 1996;87:3477-3488.
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