Clathrin mediates membrane fission and budding by constricting membrane pores.

Membrane budding, which underlies fundamental processes like endocytosis, intracellular trafficking, and viral infection, is thought to involve membrane coat-forming proteins, including the most observed clathrin, to form Ω-shape profiles and helix-forming proteins like dynamin to constrict Ω-profiles' pores and thus mediate fission. Challenging this fundamental concept, we report that polymerized clathrin is required for Ω-profiles' pore closure and that clathrin around Ω-profiles' base/pore region mediates pore constriction/closure in neuroendocrine chromaffin cells. Mathematical modeling suggests that clathrin polymerization at Ω-profiles' base/pore region generates forces from its intrinsically curved shape to constrict/close the pore. This new fission function may exert broader impacts than clathrin's well-known coat-forming function during clathrin (coat)-dependent endocytosis, because it underlies not only clathrin (coat)-dependent endocytosis, but also diverse endocytic modes, including ultrafast, fast, slow, bulk, and overshoot endocytosis previously considered clathrin (coat)-independent in chromaffin cells. It mediates kiss-and-run fusion (fusion pore closure) previously considered bona fide clathrin-independent, and limits the vesicular content release rate. Furthermore, analogous to results in chromaffin cells, we found that clathrin is essential for fast and slow endocytosis at hippocampal synapses where clathrin was previously considered dispensable, suggesting clathrin in mediating synaptic vesicle endocytosis and fission. These results suggest that clathrin and likely other intrinsically curved coat proteins are a new class of fission proteins underlying vesicle budding and fusion. The half-a-century concept and studies that attribute vesicle-coat contents' function to Ω-profile formation and classify budding as coat-protein (e.g., clathrin)-dependent or -independent may need to be re-defined and re-examined by considering clathrin's pivotal role in pore constriction/closure.

Ascorbic acid potentiates photodynamic inactivation mediated by octyl gallate and blue light for rapid eradication of planktonic bacteria and biofilms.

This study reported for the first time that Ascorbic acid (AA) could appreciably boost the efficiency of Octyl gallate (OG)-mediated photodynamic inactivation (PDI) on Escherichia coli and Staphylococcus aureus in planktonic and biofilm states. The combination of OG (0.075 mM) and AA (200 mM) with 420 nm blue light (212 mW/cm2) led to a >6 Log killing within only 5 min for E. coli and S. aureus and rapid eradication of biofilms. The mechanism of action appears to be the generation of highly toxic hydroxyl radicals (•OH) via photochemical pathways. OG was exposed to BL irradiation to generate various reactive oxygen radicals (ROS) and the addition of AA could transform singlet oxygen (1O2) into hydrogen peroxide (H2O2), which could further react with AA to generate enormous •OH. These ROS jeopardized bacteria and biofilms by nonspecifically attacking various biomacromolecules. Overall, this PDI strategy provides a powerful microbiological decontamination modality to guarantee safe food products.

Structural insights into the N-terminal APHB domain of HrpA: mediating canonical and i-motif recognition.

RNA helicases function as versatile enzymes primarily responsible for remodeling RNA secondary structures and organizing ribonucleoprotein complexes. In our study, we conducted a systematic analysis of the helicase-related activities of Escherichia coli HrpA and presented the structures of both its apo form and its complex bound with both conventional and non-canonical DNAs. Our findings reveal that HrpA exhibits NTP hydrolysis activity and binds to ssDNA and ssRNA in distinct sequence-dependent manners. While the helicase core plays an essential role in unwinding RNA/RNA and RNA/DNA duplexes, the N-terminal extension in HrpA, consisting of three helices referred to as the APHB domain, is crucial for ssDNA binding and RNA/DNA duplex unwinding. Importantly, the APHB domain is implicated in binding to non-canonical DNA structures such as G-quadruplex and i-motif, and this report presents the first solved i-motif-helicase complex. This research not only provides comprehensive insights into the multifaceted roles of HrpA as an RNA helicase but also establishes a foundation for further investigations into the recognition and functional implications of i-motif DNA structures in various biological processes.

Membrane transformations of fusion and budding.

Membrane fusion and budding mediate fundamental processes like intracellular trafficking, exocytosis, and endocytosis. Fusion is thought to open a nanometer-range pore that may subsequently close or dilate irreversibly, whereas budding transforms flat membranes into vesicles. Reviewing recent breakthroughs in real-time visualization of membrane transformations well exceeding this classical view, we synthesize a new model and describe its underlying mechanistic principles and functions. Fusion involves hemi-to-full fusion, pore expansion, constriction and/or closure while fusing vesicles may shrink, enlarge, or receive another vesicle fusion; endocytosis follows exocytosis primarily by closing Ω-shaped profiles pre-formed through the flat-to-Λ-to-Ω-shape transition or formed via fusion. Calcium/SNARE-dependent fusion machinery, cytoskeleton-dependent membrane tension, osmotic pressure, calcium/dynamin-dependent fission machinery, and actin/dynamin-dependent force machinery work together to generate fusion and budding modes differing in pore status, vesicle size, speed and quantity, controls release probability, synchronization and content release rates/amounts, and underlies exo-endocytosis coupling to maintain membrane homeostasis. These transformations, underlying mechanisms, and functions may be conserved for fusion and budding in general.

How micron-sized exocrine vesicles release content: A comparison with sub-micron endocrine vesicles.

Exocytosis releases vesicular contents to mediate physiological functions. In this issue, Biton et al. (https://doi.org/10.1083/jcb.202302112) found four modes of releasing micron-sized exocrine vesicles and the underlying mechanisms involving actomyosin and BAR domain proteins. We highlight their discovery, compare it with much smaller/faster neuroendocrine vesicle fusion, and draw distinct and conserved principles regarding their membrane transformations, pore dynamics, and underlying mechanisms.

Radiomics in Carotid Plaque: A Systematic Review and Radiomics Quality Score Assessment.

Imaging modalities provide information on plaque morphology and vulnerability; however, they are operator dependent and miss a great deal of microscopic information. Recently, many radiomics models for carotid plaque that identify unstable plaques and predict cardiovascular outcomes have been proposed. This systematic review was aimed at assessing whether radiomics is a reliable and reproducible method for the clinical prediction of carotid plaque. A systematic search was conducted to identify studies published in PubMed and Cochrane library from January 1, 2001, to September 30, 2022. Both retrospective and prospective studies that developed and/or validated machine learning models based on radiomics data to classify or predict carotid plaques were included. The general characteristics of each included study were selected, and the methodological quality of radiomics reports and risk of bias were evaluated using the radiomics quality score (RQS) tool and Quality Assessment of Diagnostic Accuracy Studies-2, respectively. Two investigators independently reviewed each study, and the consensus data were used for analysis. A total of 2429 patients from 16 studies were included. The mean area under the curve of radiomics models for diagnostic or predictive performance of the included studies was 0.88 ± 0.02, with a range of 0.741-0.989. The mean RQS was 9.25 (standard deviation: 6.04), representing 25.7% of the possible maximum value of 36, whereas the lowest point was -2, and the highest score was 22. Radiomics models have revealed additional information on patients with carotid plaque, but with respect to methodological quality, radiomics reports are still in their infancy, and many hurdles need to be overcome.

Multiple Modes of Fusion and Retrieval at the Calyx of Held Synapse.

Neurotransmitter in vesicles is released through a fusion pore when vesicles fuse with the plasma membrane. Subsequent retrieval of the fused vesicle membrane is the key step in recycling exocytosed vesicles. Application of advanced electrophysiological techniques to a large nerve terminal, the calyx of Held, has led to recordings of endocytosis, individual vesicle fusion and retrieval, and the kinetics of the fusion pore opening process and the fission pore closure process. These studies have revealed three kinetically different forms of endocytosis-rapid, slow, and bulk-and two forms of fusion-full collapse and kiss-and-run. Calcium influx triggers all kinetically distinguishable forms of endocytosis at calyces by activation of calmodulin/calcineurin signaling pathway and protein kinase C, which may dephosphorylate and phosphorylate endocytic proteins. Polymerized actin may provide mechanical forces to bend the membrane, forming membrane pits, the precursor for generating vesicles. These research advancements are reviewed in this chapter.

Barriers to exocytotic vesicle discharge.

Regulated exocytosis, a universal process of eukaryotic cells, involves the merging between the vesicle membrane and the plasma membrane, plays a key role in cell-to-cell communication, particularly in the release of hormones and neurotransmitters. There are a number of barriers a vesicle needs to pass to discharge vesicle content to the extracellular space. At the pre-fusion site vesicles need to be transported to the sites on the plasma membrane where the merger may begin. Classically cytoskeleton was considered an important barrier for vesicle translocation and was thought to be disintegrated to allow vesicle access to the plasma membrane [1]. However, it was considered later that cytoskeletal elements may also play a role at the post-fusion stage, promoting the vesicle merger with the plasma membrane and fusion pore expansion [4,22,23]. In this Special Issue of Cell Calcium entitled "Regulated Exocytosis", the authors address outstanding issues related to vesicle chemical messenger release by regulated exocytosis, including that related to the question whether vesicle content discharge is complete or only partial upon the merging of the vesicle membrane with the plasma membrane triggered by Ca2+. Among processes that limit vesicle discharge at the post-fusion stage is the accumulation of cholesterol in some vesicles [19], a process that has recently been associated with cell aging [20].

Visualization of Exo- and Endocytosis Membrane Dynamics with Super-Resolution STED Microscopy.

Recent advances in stimulated emission depletion (STED) microscopy offer an unparalleled avenue to study membrane dynamics of exo- and endocytosis, such as fusion pore opening, pore expansion, constriction, and closure, as well as the membrane transformation from flat-shaped to round-shaped vesicles in real time. Here we depict a method of using the state-of-the-art STED microscopy to image these membrane dynamics in bovine chromaffin cells. This method can potentially be applied to study other membrane structure dynamics in other cell model system.

Biochemical and functional characterization of an exonuclease from Chaetomium thermophilum.

Exonucleases are often found associated with polymerase or helicase domains in the same enzyme or can function as autonomous entities to maintain genome stability. Here, we uncovered Chaetomium thermophilum RecQ family proteins that also have exonuclease activity in addition to their main helicase function. The novel exonuclease activity is separate from the helical core domain and coexists with the latter two enzymatic activities on the same polypeptide. The CtRecQ121-366 exonuclease region performs independently as an exonuclease. We describe its catalytic mechanism and biological characteristics. We demonstrate unequivocally that CtRecQ121-366 exclusively displays exonuclease activity and that this activity has a 3'-5' polarity that can both hydrolyze ssDNA and cleave dsDNA substrates. The hydrolytic activity of majority exonuclease is driven by bimetal ions, and this appears to be the case for the CtRecQ121-366 exonuclease as well. Additionally, the maximum activity of CtRecQ121-366 was observed at pH 8.0-9.0, low salt with Mg2+. The two helices in the structure, a6 and a7, play significant roles in the execution by anticipating their shape and changing essential amino acids.