Live-cell imaging reveals kinetic determinants of quality control triggered by ribosome stalling.

Translation of problematic mRNA sequences induces ribosome stalling, triggering quality-control events, including ribosome rescue and nascent polypeptide degradation. To define the timing and regulation of these processes, we developed a SunTag-based reporter to monitor translation of a problematic sequence (poly[A]) in real time on single mRNAs. Although poly(A)-containing mRNAs undergo continuous translation over the timescale of minutes to hours, ribosome load is increased by ∼3-fold compared to a control, reflecting long queues of ribosomes extending far upstream of the stall.

Puromycin reactivity does not accurately localize translation at the subcellular level.

Puromycin is a tyrosyl-tRNA mimic that blocks translation by labeling and releasing elongating polypeptide chains from translating ribosomes. Puromycin has been used in molecular biology research for decades as a translation inhibitor. The development of puromycin antibodies and derivatized puromycin analogs has enabled the quantification of active translation in bulk and single-cell assays.

Non-equilibrium dynamics of a nascent polypeptide during translation suppress its misfolding.

Protein folding can begin co-translationally. Due to the difference in timescale between folding and synthesis, co-translational folding is thought to occur at equilibrium for fast-folding domains. In this scenario, the folding kinetics of stalled ribosome-bound nascent chains should match the folding of nascent chains in real time.

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells.

ATP-dependent force generation and membrane scission by ESCRT-III and Vps4.

The endosomal sorting complexes required for transport (ESCRTs) catalyze reverse-topology scission from the inner face of membrane necks in HIV budding, multivesicular endosome biogenesis, cytokinesis, and other pathways. We encapsulated ESCRT-III subunits Snf7, Vps24, and Vps2 and the AAA+ ATPase (adenosine triphosphatase) Vps4 in giant vesicles from which membrane nanotubes reflecting the correct topology of scission could be pulled. Upon ATP release by photo-uncaging, this system generated forces within the nanotubes that led to membrane scission in a manner dependent upon Vps4 catalytic activity and Vps4 coupling to the ESCRT-III proteins.

Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks.

The RNA-guided DNA endonuclease Cas9 has emerged as a powerful tool for genome engineering. Cas9 creates targeted double-stranded breaks (DSBs) in the genome. Knockin of specific mutations (precision genome editing) requires homology-directed repair (HDR) of the DSB by synthetic donor DNAs containing the desired edits, but HDR has been reported to be variably efficient.

Ribosome. Mechanical force releases nascent chain-mediated ribosome arrest in vitro and in vivo.

Protein synthesis rates can affect gene expression and the folding and activity of the translation product. Interactions between the nascent polypeptide and the ribosome exit tunnel represent one mode of regulating synthesis rates. The SecM protein arrests its own translation, and release of arrest at the translocon has been proposed to occur by mechanical force.

Mechanisms of cellular proteostasis: insights from single-molecule approaches.

Cells employ a variety of strategies to maintain proteome homeostasis. Beginning during protein biogenesis, the translation machinery and a number of molecular chaperones promote correct de novo folding of nascent proteins even before synthesis is complete. Another set of molecular chaperones helps to maintain proteins in their functional, native state.

Role of arachidonic acid lipoxygenase metabolites in acetylcholine-induced relaxations of mouse arteries.

Arachidonic acid (AA) metabolites function as EDHFs in arteries of many species. They mediate cyclooxygenase (COX)- and nitric oxide (NO)-independent relaxations to acetylcholine (ACh). However, the role of AA metabolites as relaxing factors in mouse arteries remains incompletely defined.

Semiautomatic vessel wall detection and quantification of wall thickness in computed tomography images of human abdominal aortic aneurysms.

Purpose: Quantitative measurements of wall thickness in human abdominal aortic aneurysms (AAAs) may lead to more accurate methods for the evaluation of their biomechanical environment.

Methods: The authors describe an algorithm for estimating wall thickness in AAAs based on intensity histograms and neural networks involving segmentation of contrast enhanced abdominal computed tomography images. The algorithm was applied to ten ruptured and ten unruptured AAA image data sets.

14,15-Epoxyeicosa-5,8,11-trienoic acid (14,15-EET) surrogates containing epoxide bioisosteres: influence upon vascular relaxation and soluble epoxide hydrolase inhibition.

All-cis-14,15-epoxyeicosa-5,8,11-trienoic acid (14,15-EET) is a labile, vasodilatory eicosanoid generated from arachidonic acid by cytochrome P450 epoxygenases. A series of robust, partially saturated analogues containing epoxide bioisosteres were synthesized and evaluated for relaxation of precontracted bovine coronary artery rings and for in vitro inhibition of soluble epoxide hydrolase (sEH). Depending upon the bioisostere and its position along the carbon chain, varying levels of vascular relaxation and/or sEH inhibition were observed.

11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid: an endothelium-derived 15-lipoxygenase metabolite that relaxes rabbit aorta.

Previous studies indicate that 11,12,15-trihydroxyeicosatrienoic acid (11,12,15-THETA), an endothelium-derived hyperpolarizing factor in the rabbit aorta, mediates a portion of the relaxation response to acetylcholine by sequential metabolism of arachidonic acid by 15-lipoxygenase, hydroperoxide isomerase, and epoxide hydrolase. To determine the stereochemical configuration of the endothelial 11,12,15-THETA, its activity and chromatographic migration were compared with activity and migration of eight chemically synthesized stereoisomers of 11,12,15(S)-THETA. Of the eight isomers, only 11(R),12(S),15(S)-trihydroxyeicosa-5(Z),8(Z),13(E)-trienoic acid comigrated with the biological 11,12,15-THETA on reverse- and normal-phase HPLC and gas chromatography.