HaloTag as a substrate-based macroautophagy reporter.

Macroautophagy is a conserved cellular degradation pathway that, upon upregulation, confers resilience toward various stress conditions, including protection against proteotoxicity associated with neurodegenerative diseases, leading to cell survival. Monitoring autophagy regulation in living cells is important to understand its role in physiology and pathology, which remains challenging. Here, we report that when HaloTag is expressed within a cell of interest and reacts with tetramethylrhodamine (TMR; its ligand attached to a fluorophore), the rate of fluorescent TMR-HaloTag conjugate accumulation in autophagosomes and lysosomes, observed by fluorescence microscopy, reflects the rate of autophagy. Notably, we found that TMR-HaloTag conjugates were mainly degraded by the proteasome (~95%) under basal conditions, while lysosomal degradation (~10% upon pharmacological autophagy activation) was slow and incomplete, forming a degraded product that remained fluorescent within a SDS-PAGE gel, in agreement with previous reports that HaloTag is resistant to lysosomal degradation when fused to proteins of interest. Autophagy activation is distinguished from autophagy inhibition by the increased production of the degraded TMR-HaloTag band relative to the full-length TMR-HaloTag band as assessed by SDS-PAGE and by a faster rate of TMR-HaloTag conjugate lysosomal puncta accumulation as observed by fluorescence microscopy. Pharmacological proteasome inhibition leads to accumulation of TMR-HaloTag in lysosomes, indicating possible cross talk between autophagy and proteasomal degradation.

Do The Twist: Efficient Heavy-Atom-Free Visible Light Polymerization Facilitated by Spin-Orbit Charge Transfer Inter-system Crossing.

The use of visible light to drive polymerizations with spatiotemporal control offers a mild alternative to contemporary UV-light-based production of soft materials. In this spectral region, photoredox catalysis represents the most efficient polymerization method, yet it relies on the use of heavy-atoms, such as precious metals or toxic halogens. Herein, spin-orbit charge transfer intersystem crossing from boron dipyrromethene (BODIPY) dyads bearing twisted aromatic groups is shown to enable efficient visible light polymerizations in the absence of heavy-atoms. A ≈5-15× increase in polymerization rate and improved photostability was achieved for twisted BODIPYs relative to controls. Furthermore, monomer polarity had a distinct effect on polymerization rate, which was attributed to charge transfer stabilization based on ultrafast transient absorption and phosphorescence spectroscopies. Finally, rapid and high-resolution 3D printing with a green LED was demonstrated using the present photosystem.

Wavelength-Resolved Photon Fluxes of Indoor Light Sources: Implications for HOx Production.

Photochemistry is a largely unconsidered potential source of reactive species such as hydroxyl and peroxy radicals (OH and HO2, "HOx") indoors. We present measured wavelength-resolved photon fluxes and distance dependences of indoor light sources including halogen, incandescent, and compact fluorescent lights (CFL) commonly used in residential buildings; fluorescent tubes common in industrial and commercial settings; and sunlight entering buildings through windows. We use these measurements to predict indoor HOx production rates from the photolysis of nitrous acid (HONO), hydrogen peroxide (H2O2), ozone (O3), formaldehyde (HCHO), and acetaldehyde (CH3CHO). Our results suggest that while most lamps can photolyze these molecules, only sunlight and fluorescent tubes will be important to room-averaged indoor HOx levels due to the strong distance dependence of the fluxes from compact bulbs. Under ambient conditions, we predict that sunlight and fluorescent lights will photolyze HONO to form OH at rates of 106-107 molecules cm-3 s-1, and that fluorescent lights will photolyze HCHO to form HO2 at rates of ∼106 molecules cm-3 s-1; rates could be 2 orders of magnitude higher under high precursor concentrations. Ozone and H2O2 will not be important photochemical OH sources under most conditions, and CH3CHO will generally increase HO2 production rates only slightly. We also calculated photolysis rate constants for nitrogen dioxide (NO2) and nitrate radicals (NO3) in the presence of the different light sources. Photolysis is not likely an important fate for NO3 indoors, but NO2 photolysis could be an important source of indoor O3.