Specific Cellular Compartments Research Articles

Oxidised phosphatidylcholine induces sarcolemmal ceramide accumulation and insulin resistance in skeletal muscle.

Intracellular ceramide accumulation in specific cellular compartments is a potential mechanism explaining muscle insulin resistance in the pathogenesis of type 2 diabetes. Muscle sarcolemmal ceramide accumulation negatively impacts insulin sensitivity in humans, but the mechanism explaining this localised accumulation is unknown. Previous reports revealed that circulating oxidised LDL is elevated in serum of individuals with obesity and type 2 diabetes. Oxidised phosphatidylcholine, which is present in oxidised LDL, has previously been linked to ceramide pathway activation, and could contribute to localised ceramide accumulation in skeletal muscle. We hypothesised that oxidised phosphatidylcholine inversely correlates with insulin sensitivity in serum, and induces sarcolemmal ceramide accumulation and decreases insulin sensitivity in muscle. We used LC-MS/MS to quantify specific oxidised phosphatidylcholine species in serum from a cross-sectional study of 58 well-characterised individuals spanning the physiological range of insulin sensitivity. We also performed in vitro experiments in rat L6 myotubes interrogating the role of specific oxidised phosphatidylcholine species in promoting sarcolemmal ceramide accumulation, inflammation and insulin resistance in skeletal muscle cells. Human serum oxidised phosphatidylcholine levels are elevated in individuals with obesity and type 2 diabetes, inversely correlated with insulin sensitivity, and positively correlated with sarcolemmal C18:0 ceramide levels in skeletal muscle. Specific oxidised phosphatidylcholine species, particularly 1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC), increase total ceramide and dihydroceramide and decrease total sphingomyelin in the sarcolemma of L6 myotubes by de novo ceramide synthesis and sphingomyelinase activation. POVPC also increases inflammatory signalling and causes insulin resistance in L6 myotubes. These data suggest that circulating oxidised phosphatidylcholine species promote ceramide accumulation and decrease insulin sensitivity in muscle, help explain localised sphingolipid accumulation and muscle inflammatory response, and highlight oxidised phosphatidylcholine species as potential targets to combat insulin resistance.

Biological Barriers for Drug Delivery and Development of Innovative Therapeutic Approaches in HIV, Pancreatic Cancer, and Hemophilia A/B.

Biological barriers remain a major obstacle for the development of innovative therapeutics. Depending on a disease's pathophysiology, the involved tissues, cell populations, and cellular components, drugs often have to overcome several biological barriers to reach their target cells and become effective in a specific cellular compartment. Human biological barriers are incredibly diverse and include multiple layers of protection and obstruction. Importantly, biological barriers are not only found at the organ/tissue level, but also include cellular structures such as the outer plasma membrane, the endolysosomal machinery, and the nuclear envelope. Nowadays, clinicians have access to a broad arsenal of therapeutics ranging from chemically synthesized small molecules, biologicals including recombinant proteins (such as monoclonal antibodies and hormones), nucleic-acid-based therapeutics, and antibody-drug conjugates (ADCs), to modern viral-vector-mediated gene therapy. In the past decade, the therapeutic landscape has been changing rapidly, giving rise to a multitude of innovative therapy approaches. In 2018, the FDA approval of patisiran paved the way for small interfering RNAs (siRNAs) to become a novel class of nucleic-acid-based therapeutics, which-upon effective drug delivery to their target cells-allow to elegantly regulate the post-transcriptional gene expression. The recent approvals of valoctocogene roxaparvovec and etranacogene dezaparvovec for the treatment of hemophilia A and B, respectively, mark the breakthrough of viral-vector-based gene therapy as a new tool to cure disease. A multitude of highly innovative medicines and drug delivery methods including mRNA-based cancer vaccines and exosome-targeted therapy is on the verge of entering the market and changing the treatment landscape for a broad range of conditions. In this review, we provide insights into three different disease entities, which are clinically, scientifically, and socioeconomically impactful and have given rise to many technological advancements: acquired immunodeficiency syndrome (AIDS) as a predominant infectious disease, pancreatic carcinoma as one of the most lethal solid cancers, and hemophilia A/B as a hereditary genetic disorder. Our primary objective is to highlight the overarching principles of biological barriers that can be identified across different disease areas. Our second goal is to showcase which therapeutic approaches designed to cross disease-specific biological barriers have been promising in effectively treating disease. In this context, we will exemplify how the right selection of the drug category and delivery vehicle, mode of administration, and therapeutic target(s) can help overcome various biological barriers to prevent, treat, and cure disease.

Regulation of and challenges in targeting NAD+ metabolism.

Nicotinamide adenine dinucleotide, in its oxidized (NAD+) and reduced (NADH) forms, is a reduction-oxidation (redox) co-factor and substrate for signalling enzymes that have essential roles in metabolism. The recognition that NAD+ levels fall in response to stress and can be readily replenished through supplementation has fostered great interest in the potential benefits of increasing or restoring NAD+ levels in humans to prevent or delay diseases and degenerative processes. However, much about the biology of NAD+ and related molecules remains poorly understood. In this Review, we discuss the current knowledge of NAD+ metabolism, including limitations of, assumptions about and unappreciated factors that might influence the success or contribute to risks of NAD+ supplementation. We highlight several ongoing controversies in the field, and discuss the role of the microbiome in modulating theavailability of NAD+ precursors such as nicotinamide riboside (NR) and nicotinamide mononucleotide (NMN), the presence of multiple cellular compartments that have distinct pools of NAD+ and NADH, and non-canonical NAD+ and NADH degradation pathways. We conclude that a substantial investment in understanding the fundamental biology of NAD+, its detection and its metabolites in specific cells and cellular compartments is needed to support current translational efforts to safely boost NAD+ levels in humans.

Fatty Acid-Binding Protein 4-Mediated Regulation Is Pivotally Involved in Retinal Pathophysiology: A Review.

Fatty acid-binding proteins (FABPs), a family of lipid chaperone molecules that are involved in intracellular lipid transportation to specific cellular compartments, stimulate lipid-associated responses such as biological signaling, membrane synthesis, transcriptional regulation, and lipid synthesis. Previous studies have shown that FABP4, a member of this family of proteins that are expressed in adipocytes and macrophages, plays pivotal roles in the pathogenesis of various cardiovascular and metabolic diseases, including diabetes mellitus (DM) and hypertension (HT). Since significant increases in the serum levels of FABP4 were detected in those patients, FABP4 has been identified as a crucial biomarker for these systemic diseases. In addition, in the field of ophthalmology, our group found that intraocular levels of FABP4 (ioFABP4) and free fatty acids (ioFFA) were substantially elevated in patients with retinal vascular diseases (RVDs) including proliferative diabetic retinopathy (PDR) and retinal vein occlusion (RVO), for which DM and HT are also recognized as significant risk factors. Recent studies have also revealed that ioFABP4 plays important roles in both retinal physiology and pathogenesis, and the results of these studies have suggested potential molecular targets for retinal diseases that might lead to future new therapeutic strategies.

Tissue-specific and endogenous protein labeling with split fluorescent proteins

The ability to label proteins by fusion with genetically encoded fluorescent proteins is a powerful tool for understanding dynamic biological processes. However, current approaches for expressing fluorescent protein fusions possess drawbacks, especially at the whole organism level. Expression by transgenesis risks potential overexpression artifacts while fluorescent protein insertion at endogenous loci is technically difficult and, more importantly, does not allow for tissue-specific study of broadly expressed proteins. To overcome these limitations, we have adopted the split fluorescent protein system mNeonGreen21-10/11 (split-mNG2) to achieve tissue-specific and endogenous protein labeling in zebrafish. In our approach, mNG21-10 is expressed under a tissue-specific promoter using standard transgenesis while mNG211 is inserted into protein-coding genes of interest using CRISPR/Cas-directed gene editing. Each mNG2 fragment on its own is not fluorescent, but when co-expressed the fragments self-assemble into a fluorescent complex. Here, we report successful use of split-mNG2 to achieve differential labeling of the cytoskeleton genes tubb4b and krt8 in various tissues. We also demonstrate that by anchoring the mNG21-10 component to specific cellular compartments, the split-mNG2 system can be used to manipulate protein localization. Our approach should be broadly useful for a wide range of applications.

Divergent Molecular Pathways for Toxicity of Selected Mutant C9ORF72-derived Dipeptide Repeats.

Expansion of a hexanucleotide repeat in a noncoding region of the C9ORF72 gene is responsible for a significant fraction of Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) cases, but mechanisms linking mutant gene products to neuronal toxicity remain debatable. Pathogenesis was proposed to involve the production of toxic RNA species and/or accumulation of toxic dipeptide repeats (DPRs) but distinguishing between these mechanisms has been challenging. In this study, we first use complementary model systems for analyzing pathogenesis in adult-onset neurodegenerative diseases to characterize the pathogenicity of DPRs produced by Repeat Associated Non-ATG translation of C9ORF72 in specific cellular compartments: isolated axoplasm and giant synapse from the squid. Results showed selective axonal and presynaptic toxicity of GP-DPRs, independent of associated RNA. These effects involved a MAPK signaling pathway that affects fast axonal transport and synaptic function, a pathogenic mechanism shared with other mutant proteins associated with familial ALS, like SOD1 and FUS. In primary cultured neurons, GP but not other DPRs promote the "dying-back" axonopathy seen in ALS. Interestingly, GR- and PR-DPRs, which had no effect on axonal transport or synaptic transmission, were found to disrupt the nuclear membrane, promoting "dying-forward" neuropathy. All C9-DPR-mediated toxic effects observed in these studies are independent of whether the corresponding mRNAs contained hexanucleotide repeats or alternative codons. Finally, C9ORF72 human tissues confirmed a close association between GP and active P38 in degenerating motor neurons as well as GR-associated nuclear damage in the cortex. Collectively, our studies establish compartment-specific toxic effects of C9-DPRs associated with degeneration, suggesting that two independent pathogenic mechanisms may contribute to disease heterogeneity and/or synergize on disease progression in C9ORF72 patients with ALS and/or FTD symptoms.

Protein codes promote selective subcellular compartmentalization.

Cells have evolved mechanisms to distribute ~10 billion protein molecules to subcellular compartments where diverse proteins involved in shared functions must efficiently assemble. Here, we demonstrate that proteins with shared functions share amino acid sequence codes that guide them to compartment destinations. A protein language model, ProtGPS, was developed that predicts with high performance the compartment localization of human proteins excluded from the training set. ProtGPS successfully guided generation of novel protein sequences that selectively assemble in targeted subcellular compartments. ProtGPS also identified pathological mutations that change this code and lead to altered subcellular localization of proteins. Our results indicate that protein sequences contain not only a folding code, but also a previously unrecognized code governing their distribution in specific cellular compartments.

Real-Time FRET-Based Measurements of Ca2+/PKA Dynamics in Plasma Membrane Microdomains of Primary Hippocampal Neurons.

Both Ca2+ and protein kinase A (PKA) are multifaceted and ubiquitous signaling molecules, essential for regulating the intricate network of signaling pathways. However, their dynamics within specialized membrane regions are still not well characterized. By using genetically encoded fluorescent indicators specifically targeted to distinct plasma membrane microdomains, we have established a protocol that permits observing Ca2+/PKA dynamics in discrete neuronal microdomains with high spatial and temporal resolution. The approach employs a fluorescence microscope with a sensitive camera and a dedicated CFP/YFP/mCherry filter set, enabling the simultaneous detection of donor-acceptor emission and red fluorescence signal. In this detailed step-by-step guide, we outline the experimental procedure, including isolation of rat primary neurons and their transfection with biosensors targeted to lipid rafts or non-raft regions of plasma membrane. We provide information on the necessary equipment and imaging setup required for recording, along with highlighting critical parameters and troubleshooting guidelines for real-time measurements. Finally, we provide examples of the observed Ca2+ and PKA changes in specific cellular compartments. The application of this technique may have significant implications for studying cross-talk between second messengers and their alterations in various pathological conditions. © 2024 Wiley Periodicals LLC.

The intracellular visualization of exogenous DNA in fluorescence microscopy

In the development of non-viral gene delivery vectors, it is essential to reliably localize and quantify transfected DNA inside the cell. To track DNA, fluorescence microscopy methods are commonly applied. These mostly rely on fluorescently labeled DNA, DNA binding proteins fused to a fluorescent protein, or fluorescence in situ hybridization (FISH). In addition, co-stainings are often used to determine the colocalization of the DNA in specific cellular compartments, such as the endolysosomes or the nucleus. We provide an overview of these DNA tracking methods, advice on how they should be combined, and indicate which co-stainings or additional methods are required to draw precise conclusions from a DNA tracking experiment. Some emphasis is given to the localization of exogenous DNA inside the nucleus, which is the last step of DNA delivery. We argue that suitable tools which allow for the nuclear detection of faint signals are still missing, hampering the rational development of more efficient non-viral transfection systems.Graphical abstractThe intracellular visualization of exogenous DNA in fluorescence microscopy. Created with BioRender.com.

A small-molecule probe to decipher stress-induced ER microenvironments and ER-Golgi communication.

Cellular stress is a crucial factor in regulating and maintaining both organismal and microenvironmental homeostasis. It induces a response that also affects the micropolarity of specific cellular compartments, which is essential for early disease diagnosis. In this contribution, we present a quantitative study of micropolarity changes inside the endoplasmic reticulum (ER) during the G1/S and G2/M phases, using a biocompatible small-molecule fluorophore called ER-Oct. This probe is selectively driven to the ER by its hydrophobicity, and it has the fastest diffusion properties among a series of analogous probes. We found that induced ER stress caused cell cycle arrests leading to an increase in ER micropolarity which is well supported by lambda scanning experiments and fluorescence lifetime imaging microscopy (FLIM) as well. ER-Oct is a versatile staining agent that could effectively stain the ER in various living/fixed mammalian cells, isolated ER, Caenorhabditis elegans, and mice tissues. Furthermore, we used this probe to visualize a well-known biological event, ER to Golgi transport, by live-cell fluorescence microscopy. Our exhaustive investigation of micropolarity using ER-staining dye provides a new way to study ER stress, which could provide a deeper understanding of proteostasis in model systems and even in fixed patient samples.

Albumin nanocapsules and nanocrystals for efficient intracellular drug release.

In order to achieve a therapeutic effect, many drugs have to reach specific cellular compartments. Nanoscale drug delivery systems extend the circulation time, reduce adverse effects and thus improve tolerability compared to systemic administration. We have developed two types of albumin-coated nanocarriers equipped with built-in dyes to track their cellular uptake and intracellular enzymatic opening. Using the approved antiprotozoal drug and STAT3 inhibitor Atovaquone (Ato) as prototype for a hydrophobic small molecule, we show that Ato-loaded ovalbumin-coated nanocapsules (Ato-nCap) preferentially enter human myeloid cells. In contrast, Ato nanocrystals coated with human serum albumin (Ato-nCry) distribute their cargo in all different immune cell types, including T and B cells. By measuring the effect of Ato nanocarriers on induced STAT3 phosphorylation in IL-10-primed human dendritic cells and constitutive STAT3 phosphorylation in human melanoma cells, we demonstrate that the intracellular Ato release is particularly effective from Ato nanocrystals and less toxic than equal doses of free drug. These new nanocarriers thus represent effective systems for intracellular drug delivery.

Examining Phosphatases Through Immunofluorescent Microscopy.

Immunofluorescent microscopy enables the examination of cellular expression and localization of proteins. Cellular localization can often impact protein function, as certain molecular interactions occur in specific cellular compartments. Here we describe in detail the processes necessary for identifying phosphatases in the cell through immunofluorescent microscopy. Identification of phosphatase expression and localization could lead to the discovery of protein function in disease states along with potential substrates and binding partners.

ADAMTS-1 has nuclear localization in cells with epithelial origin and leads to decreased cell migration

In the study of tumorigenesis, the involvement of molecules within the extracellular matrix (ECM) is crucial. ADAMTSs (A Disintegrin and Metalloproteinase with Thrombospondin motifs), a group of secreted proteases known for their role in ECM remodeling, were primarily considered to be extracellular proteases. However, our research specifically detected ADAMTS-1, a member of this family, predominantly within the nucleus of mammary cells. Our main objective was to understand the mechanism of ADAMTS-1 translocation to the nucleus and its functional significance in this cellular compartment. Our investigation uncovered that nuclear ADAMTS-1 was present in cells exhibiting an epithelial phenotype, while cells of mesenchymal origin contained the protease in the cytoplasm. Moreover, disruption of ADAMTS-1 secretion, induced by Monensin treatment, resulted in its accumulation in the cytoplasm. Notably, our research indicated that alterations in the secretory pathways could influence the protease's compartmentalization. Additionally, experiments with conditioned medium from cells containing nuclear ADAMTS-1 demonstrated its internalization into the nucleus by HT-1080 cells and fibroblasts. Furthermore, heightened levels of ADAMTS-1 within the ECM reduced the migratory potential of mesenchymal cells. This highlights the potential significance of nuclear ADAMTS-1 as a critical component within the tumor microenvironment due to its functional activity in this specific cellular compartment.

Deterministic early endosomal maturations emerge from a stochastic trigger-and-convert mechanism

Endosomal maturation is critical for robust and timely cargo transport to specific cellular compartments. The most prominent model of early endosomal maturation involves a phosphoinositide-driven gain or loss of specific proteins on individual endosomes, emphasising an autonomous and stochastic description. However, limitations in fast, volumetric imaging long hindered direct whole cell-level measurements of absolute numbers of maturation events. Here, we use lattice light-sheet imaging and bespoke automated analysis to track individual very early (APPL1-positive) and early (EEA1-positive) endosomes over the entire population, demonstrating that direct inter-endosomal contact drives maturation between these populations. Using fluorescence lifetime, we show that this endosomal interaction is underpinned by asymmetric binding of EEA1 to very early and early endosomes through its N- and C-termini, respectively. In combination with agent-based simulation which supports a ‘trigger-and-convert’ model, our findings indicate that APPL1- to EEA1-positive maturation is driven not by autonomous events but by heterotypic EEA1-mediated interactions, providing a mechanism for temporal and population-level control of maturation.

Repeat RNA structure and protein interactions influence RAN translation and neurodegenerative phenotypes in models of FTD and ALS

AbstractBackgroundThe most common genetic cause of Frontotemporal dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS) is an intronic GGGGCC hexanucleotide repeat expansion in C9orf72 (C9FTD/ALS). Transcribed GGGGCC repeat RNAs sequester RNA‐binding proteins (RBPs) into nuclear and cytoplasmic foci, which inhibits their normal functions. GGGGCC repeat RNAs also support translation of toxic repeat peptides through a process known as repeat associated non‐AUG (RAN) translation. Here we sought to understand how repeat RNA secondary structure and RBP engagements that occur during RNA transcription, processing, and trafficking influence GGGGCC RAN translation and repeat elicited toxicity, with a goal of identifying novel therapeutic targets.MethodWe developed a cellular RNA‐capture methodology coupled to mass spectrometry to identify GGGGCC repeat RNA‐interacting proteins within specific cellular compartments. In parallel, we used chemical approaches to probe GGGGCC repeat RNA secondary structures inside cells and RAN translation assays to determine whether repeat RNA secondary structures and/or interacting RBPs influence RAN translation efficiency. We employed Drosophila and neuronal models of repeat expansions to evaluate potential RBPs as disease modifiers.ResultOur RNA structural studies revealed that GGGGCC repeat RNAs form primarily hairpin structures, rather than G‐quadruplex structures, in cells. Folding repat RNAs into such hairpins supports RAN translation whereas folding repeat RNAs into g‐quadruplex structures strongly inhibits their translation. We identified multiple RBPs enriching in the nuclear fraction of GGGGCC RNA‐interactome, including hnRNPH1 and SR (serine/arginine‐rich domain) proteins, which are known components of the nuclear foci found in C9FTD/ALS patient cells. Our comparative analysis found that lowering SR protein expression or modifying its phosphorylation modified toxicity in Drosophila models of multiple repeat expansion disorders. Currently we are investigating how repeat RNA structures influence RBP interactions and whether structure dependent RBP interactions selectively modulate RAN translation.ConclusionWe present a comprehensive approach to evaluate the roles of repeat RNA secondary structures and critical RBPs in RNA toxicity underlying dementia‐associated repeat expansion disorders.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

Cyclic adenosine monophosphate-elevating agents inhibit amyloid-beta internalization and neurotoxicity: their action in Alzheimer's disease prevention.

A modular cloning (MoClo) toolkit for reliable intracellular protein targeting in the yeast Saccharomyces cerevisiae

Modular Cloning (MoClo) allows the combinatorial assembly of plasmids from standardized genetic parts without the need of error-prone PCR reactions. It is a very powerful strategy which enables highly flexible expression patterns without the need of repetitive cloning procedures. In this study, we describe an advanced MoClo toolkit that is designed for the baker's yeast Saccharomyces cerevisiae and optimized for the targeting of proteins of interest to specific cellular compartments. Comparing different targeting sequences, we developed signals to direct proteins with high specificity to the different mitochondrial subcompartments, such as the matrix and the intermembrane space (IMS). Furthermore, we optimized the subcellular targeting by controlling expression levels using a collection of different promoter cassettes; the MoClo strategy allows it to generate arrays of expression plasmids in parallel to optimize gene expression levels and reliable targeting for each given protein and cellular compartment. Thus, the MoClo strategy enables the generation of protein-expressing yeast plasmids that accurately target proteins of interest to various cellular compartments.

Understanding FABP7 binding to fatty acid micelles and membranes

Fatty acid-binding proteins (FABPs) are chaperones that facilitate the transport of long-chain fatty acids within the cell and can provide cargo-dependent localization to specific cellular compartments. Understanding the nature of this transport is important because lipid signaling functions are associated with metabolic pathways impacting disease pathologies including cancer, autism, and schizophrenia. FABPs often associate with cell membranes to acquire and deliver their bound cargo as part of transport. We focus on brain FABP (FABP7), which demonstrates localization to the cytoplasm and nucleus, influencing transcription and fatty acid metabolism. We use a combined biophysical-computational approach to elucidate the interaction between FABP7 and model membranes. Specifically, we use multiple experiments to demonstrate that FABP7 can bind oleic acid and docosahexaenoic acid micelles. Data from NMR and multiscale molecular dynamics simulations reveal that the interaction with micelles is through FABP7’s portal region residues. Simulations suggest that binding to membranes occurs through the same residues as micelles. Simulations also capture binding events where fatty acids dissociate from the membrane and enter FABP7’s binding pocket. Overall, our data shed light on the interactions between FABP7 and OA or DHA micelles and provide insight into the transport of long-chain fatty acids.

A Mechanism of Double-Membrane Vesicle Formation from Liquid-Ordered/Liquid-Disordered Phase Separated Spherical Membrane.

Genome replication of coronaviruses takes place in specific cellular compartments, in so-called double-membrane vesicles (DMVs), formed from the endoplasmic reticulum (ER). An intensive production of DMVs is induced by non-structural viral proteins. Here, we proposed a possible mechanism of the DMV formation from ER-derived spherical vesicles where liquid-ordered and liquid-disordered lipid phases coexist. These vesicles are supposed to divide into two homogeneous liquid-ordered and liquid-disordered vesicles. The formation of two spherical vesicles constituting DMV requires a mechanical work to be performed. We considered the excess energy of the boundary between the coexisting lipid phases as the main driving force behind the division of the initial vesicle. Explicitly accounting for the energy of elastic deformations and the interphase boundary energy, we analyzed a range of physical parameters where the DMV formation is possible. We concluded that this process can principally take place in a very narrow range of system parameters. The most probable diameter of DMVs formed according to the proposed mechanism appeared to be approximately 220 nm, in an agreement with the average diameter of DMVs observed in vivo. Our consideration predicts the DMV size to be strongly limited from above. The developed analysis can be utilized for the production of DMVs in model systems.

Golgi Dysfunctions in Ciliopathies.

The Golgi apparatus (GA) is essential for intracellular sorting, trafficking and the targeting of proteins to specific cellular compartments. Anatomically, the GA spreads all over the cell but is also particularly enriched close to the base of the primary cilium. This peculiar organelle protrudes at the surface of almost all cells and fulfills many cellular functions, in particular during development, when a dysfunction of the primary cilium can lead to disorders called ciliopathies. While ciliopathies caused by loss of ciliated proteins have been extensively documented, several studies suggest that alterations of GA and GA-associated proteins can also affect ciliogenesis. Here, we aim to discuss how the loss-of-function of genes coding these proteins induces ciliary defects and results in ciliopathies.