VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates.

α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.

A matter of timing.

A change in the electric charge of autophagosome membranes controls the recruitment of SNARE proteins to ensure that membrane fusion occurs at the right time during autophagy.

Probing the dynamic crosstalk of lysosomes and mitochondria with structured illumination microscopy.

Structured illumination microscopy (SIM) is a super-resolution technology for imaging living cells and has been used for studying the dynamics of lysosomes and mitochondria. Recently, new probes and analyzing methods have been developed for SIM imaging, enabling the quantitative analysis of these subcellular structures and their interactions. This review provides an overview of the working principle and advances of SIM, as well as the organelle-targeting principles and types of fluorescence probes, including small molecules, metal complexes, nanoparticles, and fluorescent proteins. Additionally, quantitative methods based on organelle morphology and distribution are outlined. Finally, the review provides an outlook on the current challenges and future directions for improving the combination of SIM imaging and image analysis to further advance the study of organelles. We hope that this review will be useful for researchers working in the field of organelle research and help to facilitate the development of SIM imaging and analysis techniques.

Mitochondrial nucleoid condensates drive peripheral fission through high membrane curvature.

Mitochondria are dynamic organelles that undergo fusion and fission events, in which the mitochondrial membrane and DNA (mtDNA) play critical roles. The spatiotemporal organization of mtDNA reflects and impacts mitochondrial dynamics. Herein, to study the detailed dynamics of mitochondrial membrane and mtDNA, we rationally develop a dual-color fluorescent probe, mtGLP, that could be used for simultaneously monitoring mitochondrial membrane and mtDNA dynamics via separate color outputs. By combining mtGLP with structured illumination microscopy to monitor mitochondrial dynamics, we discover the formation of nucleoid condensates in damaged mitochondria. We further reveal that nucleoid condensates promoted the peripheral fission of damaged mitochondria via asymmetric segregation. Through simulations, we find that the peripheral fission events occurred when the nucleoid condensates interacted with the highly curved membrane regions at the two ends of the mitochondria. Overall, we show that mitochondrial nucleoid condensates utilize peripheral fission to maintain mitochondrial homeostasis.

Neutral lysophosphatidylcholine mediates α-synuclein-induced synaptic vesicle clustering.

α-synuclein (α-Syn) is a presynaptic protein that is involved in Parkinson's and other neurodegenerative diseases and binds to negatively charged phospholipids. Previously, we reported that α-Syn clusters synthetic proteoliposomes that mimic synaptic vesicles. This vesicle-clustering activity depends on a specific interaction of α-Syn with anionic phospholipids. Here, we report that α-Syn surprisingly also interacts with the neutral phospholipid lysophosphatidylcholine (lysoPC). Even in the absence of anionic lipids, lysoPC facilitates α-Syn-induced vesicle clustering but has no effect on Ca2+-triggered fusion in a single vesicle-vesicle fusion assay. The A30P mutant of α-Syn that causes familial Parkinson disease has a reduced affinity to lysoPC and does not induce vesicle clustering. Taken together, the α-Syn-lysoPC interaction may play a role in α-Syn function.

lpla (lipoprotein lipase a) is a marker of early adipogenesis rather than late adipogenesis in grass carp (Ctenopharyngodon idellus).

Lipoprotein lipase (LPL) functions as a marker of adipocyte differentiation in mammals, but little is known about its role in fish adipogenesis. The aim of this research is to investigate the function of Lpl in adipocyte differentiation in fish. In this paper, we isolated and characterized lipoprotein lipase a (lpla) and lipoprotein lipase b (lplb) from grass carp (Ctenopharyngodon idellus). The complete coding sequence of lpla and lplb was 1524 bp and 1503 bp in length, coding for 507 amino acids and 500 amino acids, respectively. Both lpla and lplb mRNA were expressed in a great number of tissues. During adipogenesis, the level of lpla mRNA reached its maximum at day 2 and then dropped gradually, while the level of lplb mRNA had no significant changes, indicating that lpla and lplb may have different function in the differentiation of grass carp adipocyte. Furthermore, inhibition of lpla by inhibitor of LPL(GSK264220A) at early time points most clearly reduced adipogenesis, whereas these effects were less pronounced at later stages, suggesting that lpla predominantly affects early adipogenesis rather than late adipogenesis. Based on these findings, it can be inferred that lpla and lplb in grass carp may have distinct roles in the differentiation of grass carp adipocyte, and lpla may play an important role in the early adipogenesis rather than late adipogenesis in grass carp.

Advanced imaging techniques for tracking drug dynamics at the subcellular level.

Optical microscopes are an important imaging tool that have effectively advanced the development of modern biomedicine. In recent years, super-resolution microscopy (SRM) has become one of the most popular techniques in the life sciences, especially in the field of living cell imaging. SRM has been used to solve many problems in basic biological research and has great potential in clinical application. In particular, the use of SRM to study drug delivery and kinetics at the subcellular level enables researchers to better study drugs' mechanisms of action and to assess the efficacy of their targets in vivo. The purpose of this paper is to review the recent advances in SRM and to highlight some of its applications in assessing subcellular drug dynamics.

Double-Transmembrane Domain of SNAREs Decelerates the Fusion by Increasing the Protein-Lipid Mismatch.

SNARE is the essential mediator of membrane fusion that highly relies on the molecular structure of SNAREs. For instance, the protein syntaxin-1 involved in neuronal SNAREs, has a single transmembrane domain (sTMD) leading to fast fusion, while the syntaxin 17 has a V-shape double TMDs (dTMDs), taking part in the autophagosome maturation. However, it is not clear how the TMD structure influences the fusion process. Here, we demonstrate that the dTMDs significantly reduce fusion rate compared with the sTMD by using an in vitro reconstitution system. Through theoretical analysis, we reveal that the V-shape dTMDs can significantly increase protein-lipid mismatch, thereby raising the energy barrier of the fusion, and that increasing the number of SNAREs can reduce the energy barrier or protein-lipid mismatch. This study provides a physicochemical mechanistic understanding of SNARE-regulated membrane fusion.

2D MoS2 and BN Nanosheets Damage Mitochondria through Membrane Penetration.

With the progression of nanotechnology, a growing number of nanomaterials have been created and incorporated into organisms and ecosystems, which raises significant concern about potential hazards of these materials on human health, wildlife, and the environment. Two-dimensional (2D) nanomaterials are one type of nanomaterials with thicknesses ranging from that of a single atom or of several atoms and have been proposed for a variety of biomedical applications such as drug delivery and gene therapy, but the toxicity thereof on subcellular organelles remains to be studied. In this work, we studied the impact of two typical 2D nanomaterials, MoS2 and BN nanosheets, on mitochondria, which are a type of membranous subcellular organelle that provides energy to cells. While 2D nanomaterials at a low dose exhibited a negligible cell mortality rate, significant mitochondrial fragmentation and partially reduced mitochondrial functions occurred; cells initiate mitophagy in response to mitochondrial damages, which cleans damaged mitochondria to avoid damage accumulation. Moreover, the molecular dynamics simulation results revealed that both MoS2 and BN nanosheets can spontaneously penetrate the mitochondrial lipid membrane through the hydrophobic interaction. The membrane penetration induced heterogeneous lipid packing resulting in damages. Our results demonstrate that even at a low dose 2D nanomaterials can physically damage mitochondria by penetrating the membrane, which draws attention to carefully evaluating the cytotoxicity of 2D nanomaterials for the potential biomedical application.

An ER-targeted "reserve-release" fluorogen for topological quantification of reticulophagy.

The endoplasmic reticulum's (ER) dynamic nature, essential for maintaining cellular homeostasis, can be influenced by stress-induced damage, which can be assessed by examining the morphology of ER dynamics and, more locally, ER properties such as hydrophobicity, viscosity, and polarity. Although numerous ER-specific chemical probes have been developed to monitor the ER's physical and chemical parameters, the quantitative detection and super-resolution imaging of its local hydrophobicity have yet to be explored. Here, we describe a photostable ER-targeted probe with high signal-to-noise ratio for super-resolution imaging that can specifically respond to changes in ER hydrophobicity under stress based on a "reserve-release" mechanism. The probe shows an excellent ability to target ER over commercial ER dyes and can be used to track local changes of hydrophobicity by fluorescence intensity and morphology during the selective autophagy of ER (i.e., reticulophagy). By correlating the level and location of ER damage with the distribution of fluorescence intensity, we were able to assess reticulophagy at the subcellular level. Beyond that, we developed a topological analytical tool adaptable to any ER probe for detecting structural changes in ER and thus quantitatively identifying reticulophagy. The algorithm-assisted tool can also be adapted to a wide range of molecular probes and organelles. Altogether, the new probe and analytical strategy described here show promise for the quantitative detection and analysis of subtle ER damage and stress.

Light-activated mitochondrial fission through optogenetic control of mitochondria-lysosome contacts.

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.

De Novo Design of A Membrane-Anchored Probe for Multidimensional Quantification of Endocytic Dynamics.

As a process of cellular uptake, endocytosis, with gradient acidity in different endocytic vesicles, is vital for the homeostasis of intracellular nutrients and other functions. To study the dynamics of endocytic pathway, a membrane-anchored pH probe, ECGreen, is synthesized to visualize endocytic vesicles under structured illumination microscopy (SIM), a super-resolution technology. Being sensitive to acidity with increasing fluorescence at low pH, ECGreen can differentiate early and late endosomes as well as endolysosomes. Meanwhile, membrane anchoring not only improves the durability of ECGreen, but also provides an excellent anti-photobleaching property for long-time imaging with SIM. Moreover, by taking these advantages of ECGreen, a multidimensional analysis model containing spatial, temporal, and pH information is successfully developed for elucidating the dynamics of endocytic vesicles and their interactions with mitochondria during autophagy, and reveals a fast conversion of endosomes near the plasma membrane.

Prefused lysosomes cluster on autophagosomes regulated by VAMP8.

Lysosome-autophagosome fusion is critical to autophagosome maturation. Although several proteins that regulate this fusion process have been identified, the prefusion architecture and its regulation remain unclear. Herein, we show that upon stimulation, multiple lysosomes form clusters around individual autophagosomes, setting the stage for membrane fusion. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein on lysosomes-vesicle-associated membrane protein 8 (VAMP8)-plays an important role in forming this prefusion state of lysosomal clusters. To study the potential role of phosphorylation on spontaneous fusion, we investigated the effect of phosphorylation of C-terminal residues of VAMP8. Using a phosphorylation mimic, we observed a decrease of fusion in an ensemble lipid mixing assay and an increase of unfused lysosomes associated with autophagosomes. These results suggest that phosphorylation not only reduces spontaneous fusion for minimizing autophagic flux under normal conditions, but also preassembles multiple lysosomes to increase the fusion probability for resuming autophagy upon stimulation. VAMP8 phosphorylation may thus play an important role in chemotherapy drug resistance by influencing autophagosome maturation.

A Continuous Add-On Probe Reveals the Nonlinear Enlargement of Mitochondria in Light-Activated Oncosis.

Oncosis, depending on DNA damage and mitochondrial swelling, is an important approach for treating cancer and other diseases. However, little is known about the behavior of mitochondria during oncosis, due to the lack of probes for in situ visual illumination of the mitochondrial membrane and mtDNA. Herein, a mitochondrial lipid and mtDNA dual-labeled probe, MitoMN, and a continuous add-on assay, are designed to image the dynamic process of mitochondria in conditions that are unobservable with current mitochondrial probes. Meanwhile, the MitoMN can induce oncosis in a light-activated manner, which results in the enlargement of mitochondria and the death of cancer cells. Using structured illumination microscopy (SIM), MitoMN-stained mitochondria with a dual-color response reveals, for the first time, how swelled mitochondria interacts and fuses with each other for a nonlinear enlargement to accelerate oncosis into an irreversible stage. With this sign of irreversible oncosis revealed by MitoMN, oncosis can be segregated into three stages, including before oncosis, initial oncosis, and accelerated oncosis.

A nano-predator of pathological MDMX construct by clearable supramolecular gold(I)-thiol-peptide complexes achieves safe and potent anti-tumor activity.

As alternatives to small-molecular proteolysis-targeting chimeras (PROTAC), peptide-based molecular glues (MG) are a broad range of dual-functional ligands that simultaneously bind with targetable proteins and E3 ligases by mimicking proteinprotein interaction (PPI) partners. Methods: Herein, we design a peptide-derived MG to target a tumor-driving protein, MDMX, for degradation, and nanoengineered it into a supramolecular gold(I)-thiol-peptide complex (Nano-MP) to implement the proteolysis recalcitrance, cellular internalization, and glutathione-triggered release. To optimize the tumor targeting, a pH-responsive macromolecule termed polyacryl sulfydryl imidazole (PSI) was synthesized to coat Nano-MP. Results: As expected, Nano-MP@PSI induced the MDMX degradation by ubiquitination and subsequently restored the anti-cancer function of p53 and p73. Nano-MP@PSI revealed potent anti-cancer activities in an orthotopic xenograft mouse model of retinoblastoma by intraocular injection and a patient-derived xenograft model of malignant pancreatic cancer by systemic injection, while maintaining a favorable safety profile and showing a highly favorable clearable profile of excretion from the living body. Conclusion: Collectively, this work not only provided a clinically viable paradigm for the treatment of a wide variety of tumors by multiple administration types, but, more importantly, it bridged the chasm between peptides and PROTACs, and likely reinvigorated the development of peptide-derived proteolysis-targeting chimeras for a great variety of diseases.

Simultaneous Zn2+ tracking in multiple organelles using super-resolution morphology-correlated organelle identification in living cells.

Zn2+ plays important roles in metabolism and signaling regulation. Subcellular Zn2+ compartmentalization is essential for organelle functions and cell biology, but there is currently no method to determine Zn2+ signaling relationships among more than two different organelles with one probe. Here, we report simultaneous Zn2+ tracking in multiple organelles (Zn-STIMO), a method that uses structured illumination microscopy (SIM) and a single Zn2+ fluorescent probe, allowing super-resolution morphology-correlated organelle identification in living cells. To guarantee SIM imaging quality for organelle identification, we develop a new turn-on Zn2+ fluorescent probe, NapBu-BPEA, by regulating the lipophilicity of naphthalimide-derived Zn2+ probes to make it accumulate in multiple organelles except the nucleus. Zn-STIMO with this probe shows that CCCP-induced mitophagy in HeLa cells is associated with labile Zn2+ enhancement. Therefore, direct organelle identification supported by SIM imaging makes Zn-STIMO a reliable method to determine labile Zn2+ dynamics in various organelles with one probe. Finally, SIM imaging of pluripotent stem cell-derived organoids with NapBu-BPEA demonstrates the potential of super-resolution morphology-correlated organelle identification to track biospecies and events in specific organelles within organoids.

A dual-labeling probe to track functional mitochondria-lysosome interactions in live cells.

Mitochondria-lysosome interactions are essential for maintaining intracellular homeostasis. Although various fluorescent probes have been developed to visualize such interactions, they remain unable to label mitochondria and lysosomes simultaneously and dynamically track their interaction. Here, we introduce a cell-permeable, biocompatible, viscosity-responsive, small organic molecular probe, Coupa, to monitor the interaction of mitochondria and lysosomes in living cells. Through a functional fluorescence conversion, Coupa can simultaneously label mitochondria with blue fluorescence and lysosomes with red fluorescence, and the correlation between the red-blue fluorescence intensity indicates the progress of mitochondria-lysosome interplay during mitophagy. Moreover, because its fluorescence is sensitive to viscosity, Coupa allowed us to precisely localize sites of mitochondria-lysosome contact and reveal increases in local viscosity on mitochondria associated with mitochondria-lysosome contact. Thus, our probe represents an attractive tool for the localization and dynamic tracking of functional mitochondria-lysosome interactions in living cells.

Multiple-Color Platinum Complex with Super-Large Stokes Shift for Super-Resolution Imaging of Autolysosome Escape.

It is of great significance to track the platinum drugs in real time with super-resolution to elucidate their mechanism of action, such as their behavior and distribution in live cells. Such information is required for further drug development. However, it is always challenging to design platinum complexes suitable for such research. Herein, we design a luminescent building block (L) for metal complexes and a dinuclear platinum complex (Pt2 L) for super-resolution imaging. Because of its super-large Stokes shift and excellent photophysical properties, Pt2 L is capable of serving as an ideal candidate for super-resolution imaging with extremely low luminescence background and high photobleaching resistance. Moreover, upon light stimulation, a matter flux of Pt2 L escaping from autolysosomes to nucleus was observed, which represents a new transportation path. Utilizing the photoactivated escape properties, we can regulate the nuclear accessibility of Pt2 L form autolysosomes with photo-selectivity, which provides a new way to improve the targeting of platinum drugs.