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After baseline measurements, norepinephrine (1 60 ng/min) was infused in 7 minute increments (7.5 mL/h) to achieve a stable constriction of 50%to80%0%.
When a stable constriction was established, hypoxia was induced by adjusting the gas concentrations from 5% CO2 in O2 (organ bath PO2>650 mm Hg, ISO2-D, WPI, Fl) to 5% CO2 in N2 resulting in 1% O2 in the organbath (PO2 30 mm Hg).
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We observed that stable mitochondrial constriction sites in gem1Δ cells (mito-TagBFP) were persistently associated with ER tubules (GFP-HDEL).
In addition, similar to ERMES distribution, Yme2-GFP foci were observed associated with stable mitochondrial constrictions in gem1Δ cells, consistent with the idea that the spatial resolution of ER mitochondria contacts following division is fundamentally important for nucleoid distribution.
In this case, the circular muscle reaches a stable steady state of constriction.
These findings are consistent with fluorescence data demonstrating that, in comparison to wild-type cells, ER tubules in gem1Δ cells were observed associated in a relatively stable manner with mitochondrial constriction sites.
Thus, taken together, these observations indicate that the stable ERMES-associated mitochondrial constriction sites in gem1Δ cells is a consequence of defective resolution of mitochondrial tips, perhaps as a consequence of altered ER mitochondria contacts.
Significantly, we also observed stable ERMES-associated mitochondrial constrictions in gem1Δ cells expressing either Gem1S19N or Gem1S462N.
Models retained the predicted lumenal orientation of His17 and Phe25 (Fig. 3A) which caused lumenal constrictions, and were more energetically stable compared to previous de novo models 15, 27 (NMR: −54,472 kcal/mol; de novo: −17,806 kcal/mol).
A vast majority of ERMES-marked mitochondrial constriction sites in gem1Δ cells were stable, typically persisting for the duration of image capture (3 3.5 min).
The photic response of the system can be driven to a stable state (here measured as the photoequilibrium of pupil constriction) that is modulated by and can be predicted from the parameters of prior light exposure.
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