Chemical Biology Tools to Study Lipids and their Metabolism with Increased Spatial and Temporal Resolution

Lipids are important cellular components providing many essential functions. To fulfill these various functions evolution has selected for a diverse set of lipids and this diversity is seen at the organismal, cellular and subcellular level. Understanding how cells maintain this complex lipid organization is a very challenging problem, which for lipids, is not easily addressed using biochemical and genetic techniques. Therefore, chemical tools have an important role to play in our quest to understand the complexities of lipid metabolism. Here we discuss new chemical tools to study lipids, their distribution and metabolism with increased spatial and temporal resolution.

A Review of Niosomes as Novel Drug Delivery Systems

The aim of this review is to present the structure of niosome, benefits and drawbacks, fundamentals of niosome preparation and characterization as well as a description of their applications in drug delivery. This review will provide an overview on the increasing interest on niosomes in the field of drug delivery. Drug delivery systems are defined as formulations aiming for transportation of a drug to the desired area of action within the body. The basic component of drug delivery systems is an appropriate carrier that protects the drug from rapid degradation or clearance and thereby enhances drug concentration in target tissues. Drug targeting is a kind of phenomenon in which drug gets distributed in the body in such a manner that the drug interacts with the target tissue at a cellular or subcellular level to achieve a desired therapeutic response at a desire site without undesirable interactions at other sites. This can be achieved by modern methods of targeting the drug delivery system such as niosomes. Niosomes are the type of non-ionic surfactant vesicles, which are biodegradable, non-toxic, more stable and inexpensive, a new approach to liposomes. Their structure similar to liposome and hence they can represent alternative vesicular systems with respect to liposomes. The niosomes have the tendency to load different type of drugs.

Optogenetic relaxation of actomyosin contractility uncovers mechanistic roles of cortical tension during cytokinesis

Actomyosin contractility generated cooperatively by nonmuscle myosin II and actin filaments plays essential roles in a wide range of biological processes, such as cell motility, cytokinesis, and tissue morphogenesis. However, subcellular dynamics of actomyosin contractility underlying such processes remains elusive. Here, we demonstrate an optogenetic method to induce relaxation of actomyosin contractility at the subcellular level. The system, named OptoMYPT, combines a protein phosphatase 1c (PP1c)-binding domain of MYPT1 with an optogenetic dimerizer, so that it allows light-dependent recruitment of endogenous PP1c to the plasma membrane. Blue-light illumination is sufficient to induce dephosphorylation of myosin regulatory light chains and a decrease in actomyosin contractile force in mammalian cells and Xenopus embryos. The OptoMYPT system is further employed to understand the mechanics of actomyosin-based cortical tension and contractile ring tension during cytokinesis. We find that the relaxation of cortical tension at both poles by OptoMYPT accelerated the furrow ingression rate, revealing that the cortical tension substantially antagonizes constriction of the cleavage furrow. Based on these results, the OptoMYPT system provides opportunities to understand cellular and tissue mechanics.

Subcellular Localization of Connexin 26 in Cardiomyocytes and in Cardiomyocyte-Derived Extracellular Vesicles

Connexins (Cxs) are a family of membrane-spanning proteins, expressed in vertebrates and named according to their molecular weight. They are involved in tissue homeostasis, and they function by acting at several communication levels. Cardiac Cxs are responsible for regular heart function and, among them, Cx26 and Cx43 are widely expressed throughout the heart. Cx26 is present in vessels, as well as in cardiomyocytes, and its localization is scattered all over the cell aside from at the intercalated discs as is the case for the other cardiac Cxs. However, having been found in cardiomyocytes only recently, both its subcellular localization and its functional characterization in cardiomyocytes remain poorly understood. Therefore, in this study we aimed to obtain further data on the localization of Cx26 at the subcellular level. Our TEM immunogold analyses were performed on rat heart ventricles and differentiated H9c2 cardiac cell sections as well as on differentiated H9c2 derived extracellular vesicles. The results confirmed the absence of Cx26 at intercalated discs and showed the presence of Cx26 at the level of different subcellular compartments. The peculiar localization at the level of extracellular vesicles suggested a specific role for cardiac Cx26 in inter-cellular communication in an independent gap junction manner.

Specific ATPases drive compartmentalized glycogen utilization in rat skeletal muscle

Glycogen is a key energy substrate in excitable tissue and especially in skeletal muscle fibers it contributes with a substantial, but also local energy production. A heterogenic subcellular distribution of three distinct glycogen pools in skeletal muscle is proved by transmission electron microscopy (TEM), which is thought to represent the requirements for local energy stores at the subcellular level. Here, we show that the three main energy-consuming ATPases in skeletal muscles (Ca2+-, Na+,K+-, and myosin ATPases) utilize different local pools of glycogen. These results clearly demonstrate compartmentalized glycogen metabolism and emphasize that spatially distinct pools of glycogen particles act as energy substrate for separated energy requiring processes, suggesting a new paradigm for understanding glycogen metabolism in working muscles, muscle fatigue and metabolic disorders.

Localisation of cryptochrome 2 in the avian retina

Cryptochromes are photolyase-related blue-light receptors acting as core components of the mammalian circadian clock in the cell nuclei. One or more members of the cryptochrome protein family are also assumed to play a role in avian magnetoreception, but the primary sensory molecule in the retina of migratory birds that mediates light-dependent magnetic compass orientation has still not been identified. The mRNA of cryptochrome 2 (Cry2) has been reported to be located in the cell nuclei of the retina, but Cry2 localisation has not yet been demonstrated at the protein level. Here, we provide evidence that Cry2 protein is located in the photoreceptor inner segments, the outer nuclear layer, the inner nuclear layer and the ganglion cell layer in the retina of night-migratory European robins, homing pigeons and domestic chickens. At the subcellular level, we find Cry2 both in the cytoplasm and the nucleus of cells residing in these layers. This broad nucleic expression rather points to a role for avian Cry2 in the circadian clock and is consistent with a function as a transcription factor, analogous to mammalian Cry2, and speaks against an involvement in magnetoreception.

Highly resolved spatial transcriptomics for detection of rare events in cells

Single-cell spatial transcriptomics technologies leveraged the potential to transcriptionally landscape sophisticated reactions in cells. Current methods to delineate such complex interplay lack the flexibility in rapid target adaptation and are particularly restricted in detecting rare transcripts. We developed a multiplex single-cell RNA In-situ hybridization technique, called ‘Molecular Cartography’ (MC) that can be easily tailored to specific applications and, by providing unprecedented sensitivity, specificity and resolution, is particularly suitable in tracing rare events at a subcellular level. Using a SARS-CoV-2 infection model, MC allows the discernment of single events in host-pathogen interactions, dissects primary from secondary responses, and illustrates differences in antiviral signaling pathways affected by SARS-CoV-2, simultaneously in various cell types.

Multimodal Metabolic Imaging Reveals Pigment Reduction and Lipid Accumulation in Metastatic Melanoma

Objective and Impact Statement. Molecular signatures are needed for early diagnosis and improved treatment of metastatic melanoma. By high-resolution multimodal chemical imaging of human melanoma samples, we identify a metabolic reprogramming from pigmentation to lipid droplet (LD) accumulation in metastatic melanoma. Introduction. Metabolic plasticity promotes cancer survival and metastasis, which promises to serve as a prognostic marker and/or therapeutic target. However, identifying metabolic alterations has been challenged by difficulties in mapping localized metabolites with high spatial resolution. Methods. We developed a multimodal stimulated Raman scattering and pump-probe imaging platform. By time-domain measurement and phasor analysis, our platform allows simultaneous mapping of lipids and pigments at a subcellular level. Furthermore, we identify the sources of these metabolic signatures by tracking deuterium metabolites at a subcellular level. By validation with mass spectrometry, a specific fatty acid desaturase pathway was identified. Results. We identified metabolic reprogramming from a pigment-containing phenotype in low-grade melanoma to an LD-rich phenotype in metastatic melanoma. The LDs contain high levels of cholesteryl ester and unsaturated fatty acids. Elevated fatty acid uptake, but not de novo lipogenesis, contributes to the LD-rich phenotype. Monounsaturated sapienate, mediated by FADS2, is identified as an essential fatty acid that promotes cancer migration. Blocking such metabolic signatures effectively suppresses the migration capacity both in vitro and in vivo. Conclusion. By multimodal spectroscopic imaging and lipidomic analysis, the current study reveals lipid accumulation, mediated by fatty acid uptake, as a metabolic signature that can be harnessed for early diagnosis and improved treatment of metastatic melanoma.

Bisphenol A Removal by the Fungus Myrothecium roridumIM 6482—Analysis of the Cellular and Subcellular Level

Bisphenol (BPA) is a key ingredient in the production of epoxy resins and some types of plastics, which can be released into the environment and alter the endocrine systems of wildlife and humans. In this study, the ability of the fungus M. roridumIM 6482 to BPA elimination was investigated. LC-MS/MS analysis showed almost complete removal of BPA from the growth medium within 72 h of culturing. Products of BPA biotransformation were identified, and their estrogenic activity was found to be lower than that of the parent compound. Extracellular laccase activity was identified as the main mechanism of BPA elimination. It was observed that BPA induced oxidative stress in fungal cells manifested as the enhancement in ROS production, membranes permeability and lipids peroxidation. These oxidative stress markers were reduced after BPA biodegradation (72 h of culturing). Intracellular proteome analyses performed using 2-D electrophoresis and MALDI-TOF/TOF technique allowed identifying 69 proteins in a sample obtained from the BPA containing culture. There were mainly structural and regulator proteins but also oxidoreductive and antioxidative agents, such as superoxide dismutase and catalase. The obtained results broaden the knowledge on BPA elimination by microscopic fungi and may contribute to the development of BPA biodegradation methods.

Sulforaphane downregulated fatty acid synthase and inhibited microtubule-mediated mitophagy leading to apoptosis

We previously demonstrated that sulforaphane (SFN) inhibited autophagy leading to apoptosis in human non-small cell lung cancer (NSCLC) cells, but the underlying subcellular mechanisms were unknown. Hereby, high-performance liquid chromatography-tandem mass spectrometry uncovered that SFN regulated the production of lipoproteins, and microtubule- and autophagy-associated proteins. Further, highly expressed fatty acid synthase (FASN) contributed to cancer malignancy and poor prognosis. Results showed that SFN depolymerized microtubules, downregulated FASN, and decreased its binding to α-tubulin; SFN downregulated FASN, acetyl CoA carboxylase (ACACA), and ATP citrate lyase (ACLY) via activating proteasomes and downregulating transcriptional factor SREBP1; SFN inhibited the interactions among α-tubulin and FASN, ACACA, and ACLY; SFN decreased the amount of intracellular fatty acid (FA) and mitochondrial phospholipids; and knockdown of FASN decreased mitochondrial membrane potential (ΔΨm) and increased reactive oxygen species, mitochondrial abnormality, and apoptosis. Further, SFN downregulated mitophagy-associated proteins Bnip3 and NIX, and upregulated mitochondrial LC3 II/I. Transmission electron microscopy showed mitochondrial abnormality and accumulation of mitophagosomes in response to SFN. Combined with mitophagy inducer CCCP or autophagosome–lysosome fusion inhibitor Bafilomycin A1, we found that SFN inhibited mitophagosome–lysosome fusion leading to mitophagosome accumulation. SFN reduced the interaction between NIX and LC3 II/I, and reversed CCCP-caused FA increase. Furthermore, knockdown of α-tubulin downregulated NIX and BNIP3 production, and upregulated LC3 II/I. Besides, SFN reduced the interaction and colocalization between α-tubulin and NIX. Thus, SFN might cause apoptosis via inhibiting microtubule-mediated mitophagy. These results might give us a new insight into the mechanisms of SFN-caused apoptosis in the subcellular level.