DMT1 bridges endosomes and mitochondria to modulate mitochondrial iron translocation

Abstract

Transient &quotkiss-and-run&quot endosome-mitochondria interactions can mediate mitochondrial iron translocation (MIT) but the associated mechanisms are still elusive. We show that Divalent Metal Transporter 1 (DMT1) modulates MIT via endosome-mitochondria interactions in invasive MDA-MB-231, but not in non-invasive T47D breast cancer cells. CRISPR/Cas9-based DMT1 knockout (KO) stable cells were used to demonstrate that DMT1 regulates MIT, endosomal speed, and labile iron pool (LIP) levels only in MDA-MB-231. DMT1 silencing increases PINK1/Parkin mitophagy markers, the autophagy marker LC3B, as well as mitochondrial ferritin in MDA-MB-231, but not in T47D. Strikingly, re-expression of DMT1 in MDA-MB-231 DMT KO cells rescues all protein levels evaluated. DMT1 silencing decreases Tom20 colocalization with PMPCB, a DMT1 interactor that regulates mitophagy hyperactivation. In MDA-MB-231 both mitochondrial metabolism and invasion were impaired by DMT1 silencing and rescued by DMT1 re-expression. DMT1 acts as a bridge between endosomes and mitochondria to support higher MIT/lower LIP levels, which are necessary for sustaining mitochondrial bioenergetics and invasive cancer cell migration.

Assessment of Raman hyperspectral spectra upon depletion of proteins involved in iron transport

“…Strikingly, upon DMT1 silencing, we can detect a reduction in the 1300 cm -1 Raman peak. In agreement, TfR expression is reduced in DMT1 KO cells 9 .…”

“…Importantly, both Rab4A and DMT1 play a role in intracellular iron transport and metabolism, although in distinct pathways. Rab4A affects iron transport indirectly, most probably by regulating TfR-Tf endosomal trafficking 8 , whereas DMT1 is responsible for iron translocation from endosomes into mitochondria 9 . Previously, we have shown that ironbound Tf can be monitored by the 1300 cm -1 Raman peak in breast cancer cells 16,17 .…”

“…Importantly, recycling of TfR is regulated by Rab4A 7 , and Rab4A modulates early endosomal volume and epidermal growth factor receptor (EGFR) activation in breast cancer cells 8 . Recently, we have shown that the iron transporter DMT1 acts as a bridge between endosome and mitochondria to regulate endosome-mitochondria interaction, as well as mitochondrial iron translocation in triple negative MDA-MB-231 cells 9 . However, the role of DMT1 and Rab4A in the regulation of iron transport and release of iron from Tf-TfR complexes, as well as in the overall cellular metabolism of breast cancer cells, is an unexplored area.…”

Live microscopy of multicellular spheroids with the multi-modal near-infrared nanoparticles reveals differences in oxygenation gradients

“…This is in agreement with previously reported metabolic features of spheroids, which require at least 10 minutes to display a clear uncoupling effect. [61][62][63] Rotenone strongly inhibited mitochondrial respiration causing spheroids reoxygenation, leading to an increased ratio % at both spheroid's periphery and core within ~30 seconds after stimulation (Fig. S9, B).…”

“…61 Surprisingly, there has been little attention to the use of far-red and near-infrared (NIR) dyes in O 2 imaging so far. [62][63][64][65] Using such structures with long-wave excitation and emission would provide decreased phototoxicity, ensure better filtering of the autofluorescence 66 and provide deeper light penetration across the volume of multicellular spheroids. While some progress has been achieved with the dye-and fluorescent protein-based structures, their use in ratiometric measurements in imaging 3D microtissues is still rare.…”