Some microbes are referred to as model organisms because they are easy to study in the laboratory and hold the ability to retain their characteristics during DNA replication, DNA transcription, and other fundamental processes. Studying these microbes in living cells via single-molecule imaging allows us to better understand these processes at highly improved spatiotemporal resolution. Single particle tracking photoactivated localization microscopy (sptPALM) is a robust tool for detecting the positions and motions of individual molecules with tens of nanometers of spatial and millisecond temporal resolution in vivo, providing insights into intricate intracellular environments that traditional ensemble methods cannot. With this approach, the fluorophores are photoactivated stochastically, a series of images are recorded, and the positions of fluorophores are identified in these images, and ultimately the locations are linked together to yield trajectories of individual molecules. Quantitative kinetic and spatial information, such as reaction rates, diffusion coefficients, and localization maps, can be obtained by further analysis. Here, we present a single-molecule tracking protocol that includes sample preparation, data acquisition and brief data processing. This protocol will enable researchers to directly unveil molecular and cellular mechanisms underlying the essential biological processes.
The purpose of this study was to examine the secretion of inflammatory cytokines by cultured monocytes/macrophages in patients with premature coronary artery disease (CAD). The study included 38 patients with premature CAD and 35 patients without CAD. A primary culture of CD14+ monocytes was obtained by immunomagnetic separation. The inflammatory response was induced by incubation of a cell culture with lipopolysaccharide (LPS) for 24 hours on Days 1 and 6. Basal and LPS-stimulated secretion of the cytokines, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) was assessed by enzyme immunoassay on Days 2 and 7 of cultivation. The level of basal secretion of TNF-α, IL-1β, IL-6, MCP-1 was higher in patients with CAD compared to patients in the control group. The levels of re-stimulated TNF-α secretion and the levels of LPS-stimulated and re-stimulated IL-1β secretion on the second and sixth days were also higher in patients with CAD. LPS-stimulated MCP-1 secretion on the second day did not differ in patients of both groups, but re-stimulated MCP-1 secretion was higher in patients with CAD. The results of logistic regression analysis showed that the basal secretion levels of IL-1β and IL-6 were independently associated with premature CAD, along with smoking, body mass index and serum HDL-cholesterol levels.
Chromatin contains not only heterochromatin (HC) and euchromatins (EC) but also facultative heterochromatin (fHC), which experience the dynamic remodeling between HCs and ECs by different regulators. The regulation of fHCs involves lots of different cell functions, like genomic stability and gene transcription. Heterochromatin protein 1 (HP1) recognizes methylated H3K9 and reshapes the chromatin into the fHCs through liquid–liquid phase separation (LLPS). Among the three members of the HP1 family, HP1α can condensate by itself and HP1β forms granules with the help of TRIM28, while the HP1γ cannot phase separation alone either and the coordinator is still unclear. So, in this study, we investigated the molecular mechanism of how HP1γ interacts with TRIM66 through PxVxL motif. Based on that, we examined the key regions that controlled the TRIM66-HP1γ co-phase separation behaviors both in vitro and in vivo. Furthermore, we proved that the liquid granules of TRIM66-HP1γ and chromatin highly correlated with H3K9me3 sites, which indicated the relationship with DNA damage response. Finally, combined with our previous study, we proposed the system for how TRIM66 remodeled the chromatin into compressed fHC through the TRIM66-HP1γ-H3K9me3 axis with liquid–liquid phase separation.
Viral epidemics pose major threats to global health and economies. A hallmark of viral infection is the reshaping of host cell membranes and cytoskeletons to form organelle-like structures, known as viral factories, which support viral genome replication. Viral infection in many cases induces the cytoskeletal network to form cage-like structures around viral factories, including actin rings, microtubule cages, and intermediate filament cages. Viruses hijack various organelles to create these replication factories, such as viroplasms, spherules, double-membrane vesicles, tubes, and nuclear viral factories. This review specifically examines the roles of cytoskeletal elements and the endomembrane system in material transport, structural support, and biochemical regulation during viral factory formation. Furthermore, we discuss the broader implications of these interactions for viral replication and highlight potential future research directions.
DNA double-strand breaks (DSBs) are the most severe form of DNA damage, primarily repaired by the non-homologous end joining (NHEJ) pathway. A critical step in this process is DNA synapsis, where the two broken ends are brought together to facilitate timely repair. Deficiencies in NHEJ synapsis can lead to improper DNA end configurations, potentially resulting in chromosomal translocations. NHEJ synapsis is a highly dynamic, multi-protein mediated assembly process. Recent advances in single-molecule techniques have led to significant progress in understanding the molecular mechanisms driving NHEJ synapsis. In this review, we summarize single-molecule methods developed for studying NHEJ synapsis, with a particular focus on the single-molecule fluorescence resonance energy transfer (smFRET) technique. We discuss the various molecular mechanisms of NHEJ synapsis uncovered through these studies and explore the coupling between synapsis and other steps in NHEJ. Additionally, we highlight the strategies, limitations, and future directions for single-molecule studies of NHEJ synapsis.
Advancements in molecular characterization technologies have accelerated targeted cancer therapy research at unprecedented resolution and dimensionality. Integrating comprehensive multi-omic molecular profiling of a tumor, proteogenomics, marks a transformative milestone for preclinical cancer research. In this paper, we initially provided an overview of proteogenomics in cancer research, spanning genomics, transcriptomics, and proteomics. Subsequently, the applications were introduced and examined from different perspectives, including but not limited to genetic alterations, molecular quantifications, single-cell patterns, different post-translational modification levels, subtype signatures, and immune landscape. We also paid attention to the combined multi-omics data analysis and pan-cancer analysis. This paper highlights the crucial role of proteogenomics in preclinical targeted cancer therapy research, including but not limited to elucidating the mechanisms of tumorigenesis, discovering effective therapeutic targets and promising biomarkers, and developing subtype-specific therapies.
