Dynamic variations in mitochondrial shape have been related to function. However, tools to automatically classify and enumerate mitochondrial shapes are lacking, as are systematic studies exploring the relationship of such shapes to mitochondrial stress. Here we show that during increased generation of mitochondrial reactive oxygen species (mtROS), mitochondria change their shape from tubular to donut or blob forms, which can be computationally quantified. Imaging of cells treated with rotenone or antimycin, showed time and dose-dependent conversion of tubular forms to donut-shaped mitochondria followed by appearance of blob forms. Time-lapse images showed reversible transitions from tubular to donut shapes and unidirectional transitions between donut and blob shapes. Blobs were the predominant sources of mtROS and appeared to be related to mitochondrial-calcium influx. Mitochondrial shape change could be prevented by either pretreatment with antioxidants like N-acetyl cysteine or inhibition of the mitochondrial calcium uniporter. This work represents a novel approach towards relating mitochondrial shape to function, through integration of cellular markers and a novel shape classification algorithm.
Two azobenzenesulfonamide
molecules with thermally stable cis configurations
resulting from fluorination of positions ortho to
the azo group are reported that can differentially
regulate the activity of carbonic anhydrase in the trans and cis configurations. These fluorinated probes
each use two distinct visible wavelengths (520 and 410 or 460 nm)
for isomerization with high photoconversion efficiency. Correspondingly,
the cis isomer of these systems is highly stable
and persistent (as evidenced by structural studies in solid and solution
state), permitting regulation of metalloenzyme activity without continuous
irradiation. Herein, we use these probes to demonstrate the visible
light mediated bidirectional control over the activity of zinc-dependent
carbonic anhydrase in solution as an isolated protein, in intact live
cells and in vivo in zebrafish during embryo development.
We report two small molecule azobenzenesulfonamide probes, CAP1 and CAP2, capable of photomodulating the activity of carbonic anhydrase (CA) on demand. In the trans form, CAP azobenzene probes adopt a linear shape, making them suitable for occupying the CA active site and interacting with Zn 2+ , thereby inhibiting enzyme activity. Following irradiation with either 365 or 410 nm light, the CAP probes isomerize to their cis form. Because of the change in steric profile, the probe exits the active site, and the activity of the enzyme is restored. The cis isomer can revert back to the trans isomer through thermal relaxation or via photoirradiation with 460 nm light and thereby inhibit protein activity again. This process can be repeated multiple times without any photodegradation and thus can be used to inhibit or activate the protein reversibly. Importantly, we demonstrate our ability to apply CAP azobenzene probes to regulate CA activity both in an isolated protein solution and in live cells, where the two isomers of CAP1 differentially regulate the intracellular cytosolic pH.
A large-pore version of Mg-CUK-1, a water-stable metal−organic framework (MOF) with 1-D channels, was synthesized in basic water. Mg-CUK-1L has a BET surface area of 2896 m 2 g −1 and shows stark selectivity for CO 2 sorption over N 2 , O 2 , H 2 , and CH 4 . It displays reversible, multistep gated sorption of CO 2 below 0.33 atm. The dehydrated single-crystal structure of Mg-CUK-1L confirms retention of the open-channel structure. The MOF can be loaded with organic molecules by immersion in hot melts, providing single crystals suitable for X-ray diffraction. trans-Azobenzene fills the channels in a 2 × 2 arrangement. Solid-state UV−vis spectroscopy reveals that azobenzene molecules undergo reversible trans−cis isomerization, despite being close-packed; this surprising result is confirmed by DFT-simulated UV−vis spectra.
One‐third of all proteins are estimated to require metals for structural stability and/or catalytic activity. Desthiobiotin probes containing metal binding groups can be used to capture metalloproteins with exposed active‐site metals under mild conditions so as to prevent changes in metallation state. The proof‐of‐concept was demonstrated with carbonic anhydrase (CA), an open active site, Zn2+‐containing protein. CA was targeted by using sulfonamide derivatives. Linkers of various lengths and structures were screened to determine the optimal structure for capture of the native protein. The optimized probes could selectively pull down CA from red blood cell lysate and other protein mixtures. Pull‐down of differently metallated CAs was also investigated.
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