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Mitochondria are highly dynamic organelles whose fragmentation by fission is critical to their functional integrity and cellular homeostasis. Here, we develop a method via optogenetic control of mitochondria–lysosome contacts (MLCs) to induce mitochondrial fission with spatiotemporal accuracy. MLCs can be achieved by blue-light-induced association of mitochondria and lysosomes through various photoactivatable dimerizers. Real-time optogenetic induction of mitochondrial fission is tracked in living cells to measure the fission rate. The optogenetic method partially restores the mitochondrial functions of SLC25A46 −/− cells, which display defects in mitochondrial fission and hyperfused mitochondria. The optogenetic MLCs system thus provides a platform for studying mitochondrial fission and treating mitochondrial diseases. Existing methods can lack spatiotemporal accuracy to manipulate dynamic mitochondrial behaviour in live cells. Here the authors report an optogenetic method to control mitochondria-lysosome contacts and induce mitochondrial fission; they use photoactivatable dimerizers including CRY2/CIB and SspB/iLID.

Synaptic vesicles containing neurotransmitters fuse with the plasma membrane upon the arrival of an action potential at the active zone. Multiple proteins organize trans-SNARE complex assembly and priming, leading to fusion. One target membrane SNARE, syntaxin, forms nanodomains at the active zone, and another, SNAP-25, enters non-fusogenic complexes with it. Here, we reveal mechanistic details of AAA+ protein NSF (N-ethylmaleimide sensitive factor) and SNAP (soluble NSF attachment protein) action before fusion. We show that syntaxin clusters are conserved, that NSF colocalizes with them, and characterize SNARE populations that may exist within or near them using cryo-EM. Supercomplexes of NSF, α-SNAP, and either a syntaxin tetramer or one of two binary complexes of syntaxin—SNAP-25 reveal atomic details of SNARE processing and show how sequential ATP hydrolysis drives disassembly. These results suggest a functional role for syntaxin clusters as reservoirs and a corresponding role for NSF in syntaxin liberation and SNARE protein quality control preceding fusion. NSF hydrolyzes ATP to disassemble SNARE complexes. Here, the authors find NSF colocalizes with syntaxin nanodomains, reveal disassembly of syntaxin oligomers and other pre-fusion cis-SNARE complexes by NSF, and show how sequential hydrolysis drives disassembly.

Background While liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N 6 -methyladenosine (m 6 A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis. Methods Liver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling. Results METTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a ( eIF3a ) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s). Conclusions Our findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

The Golgi apparatus is a central hub for protein trafficking and signaling, yet its rapid imaging and cell-selective disruption remain challenging. Here, we report cycling molecular assemblies (CyMA) for fast Golgi imaging and cell-selective interference. CyMA precursors are acetylated amphiphilic thiopeptides that traverse plasma membrane and are deacetylated by intracellular thioesterases. This exposes thiols that undergo palmitoylation by Golgi-resident palmitoyl acyltransferases utilizing palmitoyl-CoA. The resulting palmitoylated peptides self-assemble into dynamic nanostructures (i.e., CyMA) localized at the Golgi. Their continuous, reversible S -acylation enables near-instantaneous Golgi imaging. Replacing fluorophore with a biphenyl motif promotes CyMA accumulation and disrupts functions such as protein modifications, trafficking, and secretion, leading to cell death. This study establishes dynamic supramolecular assembly as an active and selective strategy for Golgi-targeting, pleiotropically interfering with Golgi functions, which may be applicable to targeting other organelles by utilizing alternative enzyme switches to enable kinetic trapping. Tan and colleagues present “cycling molecular assemblies” that borrow cellular lipidation machinery to build nanostructures inside the Golgi apparatus. These tools enable rapid organelle imaging and selective destruction of cancer cells.

Bio‐waste is a side product of biomedical research containing carbon, which can be utilized for developing carbon dots (CDs). CDs are known to be useful for a variety of applications because of their unique photoluminescence, low toxicity, and straightforward synthesis. In this paper, we employed a one‐step hydrothermal method to prepare CDs from bio‐waste as the only reactant. The as‐synthesized Cell‐CDs were found to be chemically stable and biocompatible. In addition, the spectra of Cell‐CDs’ emissions covered the visible light, which is ideal for super‐resolution imaging. Particularly, dual‐color imaging can be achieved, for example, by staining the plasma membrane with Cell‐CDs emitting one color and staining cytosolic organelles with Cell‐CDs emitting a different color of fluorescence. Here, we demonstrate such applications by studying the subcellular dynamics of live cells. Cell‐CDs were synthesized from bio‐hazard waste via a one‐step hydrothermal method, yielding photoluminescent, chemically stable, and biocompatible probes. These Cell‐CDs enabled super‐resolution live‐cell imaging with dual‐color capability, distinguishing plasma membranes and lysosomes by emitting different fluorescent colors.

Lysosomal hyperacidification is regarded as a hallmark of autophagy. A new pH nanosensor that detects shifts in near-infrared emission bands is used to quantify cellular and intratumoral lysosomal pH.

Hypoxic tumours are a major problem for cancer photodynamic therapy. Here, we show that photoredox catalysis can provide an oxygen-independent mechanism of action to combat this problem. We have designed a highly oxidative Ir( iii ) photocatalyst, [Ir(ttpy)(pq)Cl]PF 6 ([ 1 ]PF 6 , where ‘ttpy’ represents 4′-( p -tolyl)-2,2′:6′,2′′-terpyridine and ‘pq’ represents 3-phenylisoquinoline), which is phototoxic towards both normoxic and hypoxic cancer cells. Complex 1 photocatalytically oxidizes 1,4-dihydronicotinamide adenine dinucleotide (NADH)—an important coenzyme in living cells—generating NAD • radicals with a high turnover frequency in biological media. Moreover, complex 1 and NADH synergistically photoreduce cytochrome c under hypoxia. Density functional theory calculations reveal π stacking in adducts of complex 1 and NADH, facilitating photoinduced single-electron transfer. In cancer cells, complex 1 localizes in mitochondria and disrupts electron transport via NADH photocatalysis. On light irradiation, complex 1 induces NADH depletion, intracellular redox imbalance and immunogenic apoptotic cancer cell death. This photocatalytic redox imbalance strategy offers a new approach for efficient cancer phototherapy. Current photodynamic therapy photosensitizers require oxygen; however, tumours are often hypoxic. Now, an organoiridium complex with an unusually high redox potential, which is effective in normoxia and hypoxia, has been developed. The organoiridium complex kills cancer cells by an immunogenic apoptotic mechanism involving efficient photocatalytic oxidation of NADH to NAD radicals, and reduction of cytochrome c .

Because lysosomes play critical roles in multiple cellular functions and are associated with many diseases, studying them at the subcellular level could elucidate their functionality and support the discovery of therapeutic drugs for treating those diseases. The commonly used dyes for super-resolution imaging of lysosomes are the commercial molecular LysoTrackers. But the tolerance to changes in the lysosomal microenvironment and to lysosomal membrane permeabilization (LMP) and the photostability of the LysoTrackers are worrisome. The purpose of our study was to evaluate the feasibility of performing a fluorescent gold nanoprobe for super-resolution observation of lysosomal dynamics in living cells and compare it to the commercial LysoTrackers. Methods: The nanoprobe Cy5@Au NP contained three parts: a bio-inert gold core, a biocompatible polyethylene glycol spacer, and a fluorophore cyanine 5. Structured illumination microscopy (SIM) was employed to capture the fluorescence of Cy5@Au NPs in cells. The tolerance assays to changes in the lysosomal microenvironment and to LMP, the photobleaching assay, and the long-term lysosomes labelling assay of Cy5@Au NPs were compared with commercial LysoTrackers. The super-resolution observation of lysosomal dynamics with Cy5@Au NPs was performed. Results: Cy5@Au NPs can light up lysosomes specifically under SIM. Compared with commercial lysosomal molecular probes, Cy5@Au NPs exhibited stronger tolerance in lysosomes during various treatments, and changes in the lysosomal microenvironment and LMP did not cause Cy5@Au NPs to lose track of their targets. Cy5@Au NPs demonstrated an excellent anti-photobleaching ability, and a long-term labelling assay revealed that they could label lysosomes more than 3 d. Biological events of lysosomes such as the kiss-and-run process, fusion, fission, and mitophagy were recorded with the fluorescent Cy5@Au NPs under SIM. Conclusions: The nanoprobe Cy5@Au NP was successfully used as a lysosomal probe for the super-resolution observation in living cells and found to overcome the limitations of commercial LysoTrackers. Our results thus confirm that nanoparticles can be useful tools for subcellular super-resolution imaging and highlight new avenues for using nanoparticles in biology.

Mitochondrial dynamics, including fission and fusion, control the morphology and function of mitochondria, and disruption of mitochondrial dynamics leads to Parkinson’s disease, Alzheimer’s disease, metabolic diseases, and cancers. Currently, many types of commercial mitochondria probes are available, but high excitation energy and low photo-stability render them unsuitable for tracking mitochondrial dynamics in living cells. Therefore, mitochondrial targeting agents that exhibit superior anti-photo-bleaching ability, deep tissue penetration and intrinsically high three-dimensional resolutions are urgently needed. Two-photon-excited compounds that use low-energy near-infrared excitation lasers have emerged as non-invasive tools for cell imaging. In this work, terpyridyl cyclometalated Ir(III) complexes ( Ir1-Ir3 ) are demonstrated as one- and two-photon phosphorescent probes for real-time imaging and tracking of mitochondrial morphology changes in living cells.

Fluorescent tracking gene delivery could provide us with a better understanding of the critical steps in the transfection process. However, for in vivo tracking applications, a small diameter (<10 nm) is one of the rigorous requirements for tracking vectors. Herein, we have demonstrated a new paradigm for two-photon tracking gene delivery based on a dendritic nano-sized hexanuclear ruthenium(II) polypyridyl complex. Because this metallodendrimer has a multivalent periphery, the complex, which is 6.1 nm, showed high stability and excellent dispersibility and could stepwise condense DNA in vitro . With the outstanding photochemical properties of Ru(II) polypyridyl, this complex could track gene delivery in vivo using one- and two-photon imaging.