Lysosomes are the degradation centers and signaling hubs in the cell. Lysosomes undergo adaptation to maintain cell homeostasis in response to a wide variety of cues. Dysfunction of lysosomes leads to aging and severe diseases including lysosomal storage diseases (LSDs), neurodegenerative disorders, and cancer. To understand the complexity of lysosome biology, many research approaches and tools have been developed to investigate lysosomal functions and regulatory mechanisms in diverse experimental systems. This review summarizes the current approaches and tools adopted for studying lysosomes, and aims to provide a methodological overview of lysosomal research and related fields.

In animal cells, the Golgi apparatus serves as the central hub of the endomembrane secretory pathway. It is responsible for the processing, modification, and sorting of proteins and lipids. The unique stacking and ribbon-like architecture of the Golgi apparatus forms the foundation for its precise functionality. Under cellular stress or pathological conditions, the structure of the Golgi and its important glycosylation modification function may change. It is crucial to employ suitable methodologies to study the structure and function of the Golgi apparatus, particularly when assessing the involvement of a target protein in Golgi regulation. This article provides a comprehensive overview of the diverse microscopy techniques used to determine the specific location of the target protein within the Golgi apparatus. Additionally, it outlines methods for assessing changes in the Golgi structure and its glycosylation modification function following the knockout of the target gene.

The lipid droplet (LD) is a conserved organelle that exists in almost all organisms, ranging from bacteria to mammals. Dysfunctions in LDs are linked to a range of human metabolic syndromes. The formation of protein complexes on LDs is crucial for maintaining their function. Investigating how proteins interact on LDs is essential for understanding the role of LDs. We have developed an effective method to uncover protein–protein interactions and protein complexes specifically on LDs. In this method, we conduct co-immunoprecipitation (co-IP) experiments using LD proteins extracted directly from isolated LDs, rather than utilizing proteins from cell lysates. To elaborate, we begin by purifying LDs with high-quality and extracting LD-associated proteins. Subsequently, the co-IP experiment is performed on these LD-associated proteins directly, which would enhance the co-IP experiment specificity of LD-associated proteins. This method enables researchers to directly unveil protein complexes on LDs and gain deeper insights into the functional roles of proteins associated with LDs.

Migrasomes are a novel type of cell organelle that form on the retraction fibers at the rear of migrating cells. In recent years, numerous studies have unveiled the mechanisms of migrasome formation and have highlighted significant roles of migrasomes in both physiological and pathological processes. Building upon the strategies outlined in published works and our own research experiences, we have compiled a comprehensive set of protocols for observing migrasomes. These step-by-step instructions encompass various aspects such as cell culture, labeling, imaging, in vitro reconstitution, and statistical analysis. We believe that these protocols serve as a valuable resource for researchers exploring migrasome biology.

The endoplasmic reticulum (ER) is an essential component of the endomembrane system in eukaryotes and plays a crucial role in protein and lipid synthesis, as well as the maintenance of calcium homeostasis. Morphologically, the ER is composed primarily of sheets and tubules. The tubular ER is composed of a network of tubular membrane structures, each with diameters ranging from 30 to 50 nanometers. In recent years, there has been in-depth research on the molecular mechanisms of membrane shaping and membrane fusion of the tubular ER. However, there is still limited understanding of the specific physiological functions of the tubular ER. Here, we report a protocol that combines differential centrifugation and immunoprecipitation to specifically enrich microsomes originating from the tubular ER in yeast. The ER tubule-derived microsomes can be further used for proteomic and lipidomic studies or other biochemical analyses.

Ribophagy, the cellular process wherein ribosomes are selectively self-digested through autophagy, plays a pivotal role in maintaining ribosome turnover. Understanding the molecular regulatory mechanisms governing ribophagy is pivotal to uncover its significance. Consequently, the establishment of methods for detecting ribophagy becomes important. In this protocol, we have optimized, enriched, and advanced existing ribophagy detection techniques, including immunoblotting, fluorescence microscopy, and transmission electron microscopy (TEM), to precisely monitor and quantify ribophagic events. Particularly noteworthy is the introduction of TEM technology for yeast ribophagy detection. In summary, the delineated methods are applicable for detecting ribophagy in both yeast and mammals, laying a solid foundation for further exploring the physiological importance of ribophagy and its potential implications in diverse cellular environments.

Determining correlations between molecules at various levels is an important topic in molecular biology. Large language models have demonstrated a remarkable ability to capture correlations from large amounts of data in the field of natural language processing as well as image generation, and correlations captured from data using large language models can also be applicable to solving a wide range of specific tasks, hence large language models are also referred to as foundation models. The massive amount of data that exists in the field of molecular biology provides an excellent basis for the development of foundation models, and the recent emergence of foundation models in the field of molecular biology has really pushed the entire field forward. We summarize the foundation models developed based on RNA sequence data, DNA sequence data, protein sequence data, single-cell transcriptome data, and spatial transcriptome data respectively, and further discuss the research directions for the development of foundation models in molecular biology.

ChatGPT explores a strategic blueprint of question answering (QA) to deliver medical diagnoses, treatment recommendations, and other healthcare support. This is achieved through the increasing incorporation of medical domain data via natural language processing (NLP) and multimodal paradigms. By transitioning the distribution of text, images, videos, and other modalities from the general domain to the medical domain, these techniques have accelerated the progress of medical domain question answering (MDQA). They bridge the gap between human natural language and sophisticated medical domain knowledge or expert-provided manual annotations, handling large-scale, diverse, unbalanced, or even unlabeled data analysis scenarios in medical contexts. Central to our focus is the utilization of language models and multimodal paradigms for medical question answering, aiming to guide the research community in selecting appropriate mechanisms for their specific medical research requirements. Specialized tasks such as unimodal-related question answering, reading comprehension, reasoning, diagnosis, relation extraction, probability modeling, and others, as well as multimodal-related tasks like vision question answering, image captioning, cross-modal retrieval, report summarization, and generation, are discussed in detail. Each section delves into the intricate specifics of the respective method under consideration. This paper highlights the structures and advancements of medical domain explorations against general domain methods, emphasizing their applications across different tasks and datasets. It also outlines current challenges and opportunities for future medical domain research, paving the way for continued innovation and application in this rapidly evolving field. This comprehensive review serves not only as an academic resource but also delineates the course for future probes and utilization in the field of medical question answering.

Calcium ions (Ca²⁺) are an archetypical and most versatile second messenger in virtually all cell types. Inspired by the discovery of Ca²⁺ sparks in the 1990s, vibrant research over the last three decades has unveiled a constellation of Ca²⁺ microdomains as elementary events of Ca²⁺ signaling and, more importantly, a digital-analog dualism as the system design principle of Ca²⁺ signaling. In this brief review, we present a sketchy summary on advances in the field of sparkology, and discuss how the digital subsystem can fulfill physiological roles otherwise impossible for any analog system. In addition, we attempt to address how the digital-analog dualism endows the simple cation messenger with signaling speediness, specificity, efficiency, stability, and unparalleled versatility.

Gap junction (GJ) intercellular communication is crucial in many physiological and pathological processes. A GJ channel is formed by head-to-head docking of two hexameric hemichannels from two neighboring cells. Heterotypic GJ channels formed by two different homomeric connexin hemichannels often display rectification properties in the current–voltage relationship while the underlying mechanisms are not fully clear. Here we studied heterotypic Cx46/Cx50 GJs at a single GJ channel level. Our data showed unitary Cx46/Cx50 GJ channel conductance (γ_j) rectification when 5 mmol/L Mg²⁺ was included in the patch pipette solution, while no γ_j rectification was observed when no Mg²⁺ was added. Including 5 mmol/L Mg²⁺ in pipette solution significantly decreased the γ_j of homotypic Cx46 GJ with little change in homotypic Cx50 γ_j. A missense point variant in Cx46 (E43F) reduced the Mg²⁺-dependent reduction in γ_j of Cx46 E43F GJ, indicating that E43 might be partially responsible for Mg²⁺-dependent decrease in γ_j of Cx46. A comprehensive understanding of Mg²⁺ modulation of GJ at the individual channel level is useful in understanding factors in modulating GJ-mediated intercellular communication in health and diseases.

Sedimentation solid-state NMR is a novel method for sample preparation in solid-state NMR (ssNMR) studies. It involves the sedimentation of soluble macromolecules such as large protein complexes. By utilizing ultra-high centrifugal forces, the molecules in solution are driven into a high-concentrated hydrogel, resulting in a sample suitable for solid-state NMR. This technique has the advantage of avoiding the need for chemical treatment, thus minimizing the loss of sample biological activity. Sediment ssNMR has been successfully applied to a variety of non-crystalline protein solids, significantly expanding the scope of solid-state NMR research. In theory, using this method, any biological macromolecule in solution can be transferred into a sedimented solute appropriate for solid-state NMR analysis. However, specialized equipment and careful handling are essential for effectively collecting and loading the sedimented solids to solid-state NMR rotors. To improve efficiency, we have designed a series of loading tools to achieve the loading process from the solution to the rotor in one step. In this paper, we illustrate the sample preparation process of sediment NMR using the H1.4-NCP¹⁶⁷ complex, which consists of linker histone H1.4 and nucleosome core particle, as an example.

Alzheimer’s disease (AD) has been conceptualized as a syndrome of brain network dysfunction. Recent imaging connectomics studies have provided unprecedented opportunities to map structural and functional brain networks in AD. By reviewing molecular, imaging, and computational modeling studies, we have shown that highly connected brain hubs are primarily distributed in the medial and lateral prefrontal, parietal, and temporal regions in healthy individuals and that the hubs are selectively and severely affected in AD as manifested by increased amyloid-beta deposition and regional atrophy, hypo-metabolism, and connectivity dysfunction. Furthermore, AD-related hub degeneration depends on the imaging modality with the most notable degeneration in the medial temporal hubs for morphological covariance networks, the prefrontal hubs for structural white matter networks, and in the medial parietal hubs for functional networks. Finally, the AD-related hub degeneration shows metabolic, molecular, and genetic correlates. Collectively, we conclude that the brain-network-hub-degeneration framework is promising to elucidate the biological mechanisms of network dysfunction in AD, which provides valuable information on potential diagnostic biomarkers and promising therapeutic targets for the disease.

The whole heart decellularized extracellular matrix (ECM) has become a promising scaffold material for cardiac tissue engineering. Our previous research has shown that the whole heart acellular matrix possesses the memory function regulating neural stem cells (NSCs) trans-differentiating to cardiac lineage cells. However, the cell subpopulations and phenotypes in the trans-differentiation of NSCs have not been clearly identified. Here, we performed single-cell RNA sequencing and identified 2,765 cells in the recellularized heart with NSCs revealing the cellular diversity of cardiac and neural lineage, confirming NSCs were capable of trans-differentiating into the cardiac lineage while maintaining the original ability to differentiate into the neural lineage. Notably, the trans-differentiated heart-like cells have dual signatures of neuroectoderm and cardiac mesoderm. This study unveils an in-depth mechanism underlying the trans-differentiation of NSCs and provides a new opportunity and theoretical basis for cardiac regeneration.

Mitochondrial base editing tools hold great promise for the investigation and treatment of mitochondrial diseases. Mitochondrial DNA base editors (mitoBEs) integrate a programmable transcription-activator-like effector (TALE) protein with single-stranded DNA deaminase (TadA8e-V106W, APOBEC1, etc.) and nickase (MutH, Nt.BspD6I(C), etc.) to achieve heightened precision and efficiency in mitochondrial base editing. This innovative mitochondrial base editing tool exhibits a number of advantages, including strand-selectivity for editing, high efficiency, and the capacity to perform diverse types of base editing on the mitochondrial genome by employing various deaminases. In this context, we provide a detailed experimental protocol for mitoBEs to assist others in achieving proficient mitochondrial base editing.

Met1-linked ubiquitination (Met1-Ub), also known as linear ubiquitination, is a newly identified atypical type of polyubiquitination that is assembled via the N-terminal methionine (Met1) rather than an internal lysine (Lys) residue of ubiquitin. The linear ubiquitin chain assembly complex (LUBAC) composed of HOIP, HOIL-1L and SHARPIN is the sole E3 ubiquitin ligase that specifically generates Met1-linked ubiquitin chains. The physiological role of LUBAC-mediated Met1-Ub has been first described as activating NF-κB signaling through the Met1-Ub modification of NEMO. However, accumulating evidence shows that Met1-Ub is broadly involved in other cellular pathways including MAPK, Wnt/β-Catenin, PI3K/AKT and interferon signaling, and participates in various cellular processes including angiogenesis, protein quality control and autophagy, suggesting that Met1-Ub harbors a potent signaling capacity. Here, we review the formation and cellular functions of Met1-linked ubiquitin chains, with an emphasis on the recent advances in the cellular mechanisms by which Met1-Ub controls signaling transduction.

Genetically Encoded Calcium (Ca²⁺) indicators (GECIs) are indispensable tools for dissecting intracellular Ca²⁺ signaling and monitoring cellular activities. Mitochondrion acts as a Ca²⁺ sink and a central player for maintaining Ca²⁺ homeostasis. Accurately monitoring Ca²⁺ transients within the mitochondrial matrix that undergo constant pH fluctuations is challenging, as signals of most currently available GECIs suffer from artifacts induced by physiological pH variations. Multiplexed monitoring of optophysiology is also hindered by the limited availability of GECIs with cyan fluorescence. Based on the bright variant of cyan fluorescence protein (CFP), mTurquoise2, we developed a GECI designated as TurCaMP. Results from molecular dynamics simulations and ab initio calculations revealed that the deprotonation of the chromophore may be responsible for the Ca²⁺-dependent changes in TurCaMP signals. TurCaMP sensors showed inverse response to Ca²⁺ transients, and their responses were not affected by pH changes within the range of pH 6–9. The high basal fluorescence and insensitivity to physiological pH fluctuations enabled TurCaMP to faithfully monitor mitochondrial Ca²⁺ responses with a high signal-to-noise ratio. TurCaMP sensors allow simultaneous multi-colored imaging of intracellular Ca²⁺ signals, expanding the possibility of multiplexed monitoring of Ca²⁺-dependent physiological events.

CX-5461, also known as pidnarulex, is a strong G4 stabilizer and has received FDA fast-track designation for BRCA1- and BRCA2- mutated cancers. However, quantitative measurements of the unfolding rates of CX-5461-G4 complexes which are important for the regulation function of G4s, remain lacking. Here, we employ single-molecule magnetic tweezers to measure the unfolding force distributions of c-MYC G4s in the presence of different concentrations of CX-5461. The unfolding force distributions exhibit three discrete levels of unfolding force peaks, corresponding to three binding modes. In combination with a fluorescent quenching assay and molecular docking to previously reported ligand-c-MYC G4 structure, we assigned the ~69 pN peak corresponding to the 1:1 (ligand:G4) complex where CX-5461 binds at the G4’s 5'-end. The ~84 pN peak is attributed to the 2:1 complex where CX-5461 occupies both the 5' and 3'. Furthermore, using the Bell-Arrhenius model to fit the unfolding force distributions, we determined the zero-force unfolding rates of 1:1, and 2:1 complexes to be (2.4 ± 0.9) × 10⁻⁸ s⁻¹ and (1.4 ± 1.0) × 10⁻⁹ s⁻¹ respectively. These findings provide valuable insights for the development of G4-targeted ligands to combat c-MYC-driven cancers.

The sarcoplasmic reticulum (SR) primarily serves as the intracellular Ca²⁺ store in cardiac myocytes, mediating cellular function under cardiac physiology and diseases. However, the properties of cardiac SR Ca²⁺ have not yet been fully determined, particularly in rats and mice, which are the most commonly used experimental species in studies on cardiac physiology and diseases. Here, we developed a spatially detailed numerical model to deduce Ca²⁺ movements inside the junctional SR (jSR) cisternae of rat cardiomyocytes. Our model accurately reproduced the jSR Ca²⁺ kinetics of local and global SR Ca²⁺ releases reported in a recent experimental study. With this model, we revealed that jSR Ca²⁺ kinetics was mostly determined by the total release flux via type 2 ryanodine receptor (RyR2) channels but not by RyR2 positioning. Although Ca²⁺ diffusion in global SR was previously reported to be slow, our simulation demonstrated that Ca²⁺ diffused very quickly inside local jSR cisternae and the decrease in the diffusion coefficient resulted in a significant reduction of jSR Ca²⁺ depletion amplitude. Intracellular Ca²⁺ was typically experimentally detected with fluorescence dye. Our simulation revealed that when the dynamical characteristics of fluorescence dye exerted a minimal effect on actual Ca²⁺ mobility inside jSR, the reaction rate of the dye with Ca²⁺ could significantly affect apparent jSR Ca²⁺ kinetics. Therefore, loading a chemical fluorescence dye with fast kinetics, such as Fluo-5N, into SR is important for Ca²⁺ measurement inside SR. Overall, our model provides new insights into deciphering Ca²⁺ handling inside nanoscopic jSR cisternae in rat cardiomyocytes.