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Paola Bargagna-Mohan, Santosh Keshipeddy, Dennis Wright, Royce Mohan; A NOVEL VIMENTIN PROBE ILLUMINATES MITOCHONDRIAL DYNAMICS. Invest. Ophthalmol. Vis. Sci. 2017;58(8):2001. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
WFA-Verde is the first-in-class vimentin-binding imaging probe. As vimentin is known to bind to mitochondria, we have employed WFA-Verde as a probe to investigate vimentin-dependent mitochondrial activity.
We employed baby hamster kidney (BHK-21) cells and primary rabbit corneal fibroblasts (RbCFibros). Cells were cultured in glass bottom dishes for 18 h in complete DMEM, prior to experiments. BHK-21 cells were treated for 30 min with MitoTracker Red CMXRos (50 nM) in the presence or absence of nocodazole (10 μM), and then pulsed with WFA-Verde (250 nM) for 5 min. WFA-Verde labeled filaments and mitochondria were recorded live using epifluorescence. In other experiments, BHK-21 cells were treated only with nocodazole, and WFA-Verde labeled cells were fixed/permeabilized, and stained with β-tubulin antibody. To follow mitochondrial dynamics, BHK-21 cells were starved for 2 h with glucose-free DMEM medium, treated with MitoTracker, washed, pulsed with WFA-Verde and then treated with sodium azide (NaN3, 0.05%) and 2-deoxy-glucose (2-DG, 50 mM). Mitochondrial fission was followed live as described above. Mitochondria-vimentin interactions were also tested in RbCFibros. Cells were treated with MitoTracker Red (50 nM) for 30 min, pulsed with WFA-Verde (250 nM) for 5 min, and followed live as described above.
WFA-labeled vimentin co-localizes with mitochondria in both BHK-21 cells and RbCFibros. Disruption of the microtubules (MTs) with nocodazole causes collapse of both WFA-Verde structures and mitochondria in BHK-21 cells. Interestingly, WFA-Verde-labeled vimentin did not overlap with β-tubulin staining, revealing a specific correlation with mitocochondria rather than with MTs. Glucose deprivation forces mitochondria to elongate, and MitoTracker labeled the tubular structures. WFA-Verde co-stained these long mitochondria showing overlap with MitoTracker labeling. The induction of reactive oxygen species by treatment with 2-DG and NaN3 caused WFA-Verde-labelled vimentin filaments to became fragmented, following mitochondria fission.
WFA-Verde labels vimentin structures that co-localize with mitochondria and faithfully reflects mitochondrial dynamics after perturbation of MTs. This probe also affords the monitoring of mitochondrial reorganization, and fragmentation after induction of fission. WFA-Verde can be a useful diagnostic tool to study vimentin-mitochondria interactions in ocular injury and disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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