analysis-fluorescent-stained-lipid-droplets-with-3d-reconstruction

Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment

Lipid droplets (LDs) are specialized organelles that mediate lipid storage and play a very important role in suppressing lipotoxicity and preventing ...

University of Coimbra

Clinical Academic Center of Coimbra (CACC)

Gap junctions (GJs) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct intercellular communication between eukaryotic cells. Although Cx43 has long been known to be a substrate for ubiquitination, the reversal of this modification by deubiquitylases (DUBs) has never been described. Here we report that the DUB-associated molecule with the SH3 domain of STAM (AMSH) interacts with Cx43 and mediates its deubiquitination. In this study, we demonstrate that Cx43 is modified with lysine 63-linked polyubiquitin chains and that these increase the interaction between Cx43 and AMSH. We also show that AMSH is recruited to GJ plaque sites at the plasma membrane, where it mediates the deubiquitination of Cx43. Using siRNA depletion or overexpression of a catalytically inactive mutant of AMSH, we show that by decreasing Cx43 deubiquitination, both the internalization and degradation rate of Cx43 are increased. Overall, these data strongly suggest that AMSH-mediated deubiquitination of Cx43 protects GJs from degradation.

AMSH-mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions.

Efficient electric activation and action potential propagation in the heart largely depends on gap junction (GJ) channels, formed by connexins (Cx) localized at the intercalated discs (IDs). Therefore, fine-tuning and maintenance of GJ in cardiomyocytes is essential for normal heart function. Several mechanisms have been implicated in the regulation of the amount of Cx43 at the plasma membrane. Results from our lab demonstrated that Nedd4-mediated ubiquitination of Cx43 signals internalization and degradation of GJ. However, the pathophysiological relevance of this mechanism has never been addressed before. The main objective of this study was to evaluate the involvement of ubiquitination on GJ remodeling, in the ischemic heart. To address this, we used the rat heart Langendorff model and evaluated the ubiquitination profile of Cx43 and its interaction with Nedd4, after 30 min of no-flow ischemia. By confocal microscopy, we show that ischemia induces extensive co-localization of ubiquitin and Nedd4 with Cx43 localized at IDs. Moreover, by subcellular fractionation and co-immunoprecipitation assays, we demonstrate an increased interaction with Nedd4 and ubiquitination of Cx43 localized at IDs. Altogether, these results suggest that ubiquitin is involved in the remodeling of GJ during myocardial ischemia, which requires the recruitment of Nedd4.

Heart ischemia results in connexin43 ubiquitination localized at the intercalated discs.

Connexins (Cxs) are transmembrane proteins that form channels which allow direct intercellular communication (IC) between neighbouring cells via gap junctions. Mechanisms that modulate the amount of channels at the plasma membrane have emerged as important regulators of IC and their de-regulation has been associated with various diseases. Although Cx-mediated IC can be modulated by different mechanisms, ubiquitination has been described as one of the major post-translational modifications involved in Cx regulation and consequently IC. In this review, we focus on the role of ubiquitin and its effect on gap junction intercellular communication.

Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive.

Sprouting angiogenesis, where new blood vessels grow from pre-existing ones, is a complex process where biochemical and mechanical signals regulate endothelial cell proliferation and movement. Therefore, a mathematical description of sprouting angiogenesis has to take into consideration biological signals as well as relevant physical processes, in particular the mechanical interplay between adjacent endothelial cells and the extracellular microenvironment. In this work, we introduce the first phase-field continuous model of sprouting angiogenesis capable of predicting sprout morphology as a function of the elastic properties of the tissues and the traction forces exerted by the cells. The model is very compact, only consisting of three coupled partial differential equations, and has the clear advantage of a reduced number of parameters. This model allows us to describe sprout growth as a function of the cell-cell adhesion forces and the traction force exerted by the sprout tip cell. In the absence of proliferation, we observe that the sprout either achieves a maximum length or, when the traction and adhesion are very large, it breaks. Endothelial cell proliferation alters significantly sprout morphology, and we explore how different types of endothelial cell proliferation regulation are able to determine the shape of the growing sprout. The largest region in parameter space with well formed long and straight sprouts is obtained always when the proliferation is triggered by endothelial cell strain and its rate grows with angiogenic factor concentration. We conclude that in this scenario the tip cell has the role of creating a tension in the cells that follow its lead. On those first stalk cells, this tension produces strain and/or empty spaces, inevitably triggering cell proliferation. The new cells occupy the space behind the tip, the tension decreases, and the process restarts. Our results highlight the ability of mathematical models to suggest relevant hypotheses with respect to the role of forces in sprouting, hence underlining the necessary collaboration between modelling and molecular biology techniques to improve the current state-of-the-art.

The Force at the Tip--Modelling Tension and Proliferation in Sprouting Angiogenesis.

Heart proteostasis relies on a complex and integrated network of molecular processes surveilling organ performance under physiological and pathological conditions. For this purpose, cardiac cells depend on the correct function of their proteolytic systems, such as the ubiquitin-proteasome system (UPS), autophagy and the calpain system. Recently, the role of protein SUMOylation (an ubiquitin-like modification), has emerged as important modulator of cardiac proteostasis, which will be the focus of this review.

Proteostasis and SUMO in the heart.

Under nitroxidative stress conditions, lipids are prone to be modified by reaction with reactive nitrogen species (RNS) and different modifications were reported to occur in fatty acids. However, in the case of phospholipids (PL) studied under nitroxidative stress conditions, only nitroalkene derivatives of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), were reported when using both in vitro biomimetic conditions and in vivo model system of type 1 diabetes mellitus. Therefore, in order to further explore other nitroxidative modifications of PL, a biomimetic model of nitroxidation combined with liquid chromatography mass spectrometry (MS) and MS/MS approaches were used to characterize the nitrated and nitroxidized derivatives of PCs and PEs. Single and multiple nitrated derivatives of phospholipids (PLs) such as nitroso and dinitroso, nitro, dinitro, and nitronitroso derivatives, together with nitroxidized derivatives were identified. Further, the specific MS/MS fragmentation pathways of these products were studied. Product ions arising from loss of HNO and HNO, from the combined loss of HNO (or HNO) and polar head groups, [NO-FA+O+H] and [NO-FA+O-H] (n=1-2) product ions corresponding to the modified fatty acyl chains were observed, depending on each modification. The knowledge obtained from the study of the MS/MS fragmentation pattern has allowed us to identify nitrated PCs, including NO-PC, (NO)-PCs, (NO)(NO)-PC, NO-PC; nitrated PEs, NO-PEs; and nitroxidized PCs, (NO)(2O)-PC in H9c2 cells under starvation, but not under ischemia or control conditions. The physiological relevance of this nitrated and nitroxidized PCs and PEs species observed exclusively in cardiomyoblast cells (H9c2) under starvation is still unknown but deserves to be explored.

Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach.

Myocardial infarction (MI) is the leading cause of morbidity and mortality worldwide and results from an obstruction in the blood supply to a region of the heart. In an attempt to replenish oxygen and nutrients to the deprived area, affected cells release signals to promote the development of new vessels and confer protection against MI. However, the mechanisms underlying the growth of new vessels in an ischaemic scenario remain poorly understood. Here, we show that cardiomyocytes subjected to ischaemia release exosomes that elicit an angiogenic response of endothelial cells (ECs).

Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis.

Communication is important to ensure the correct and efficient flow of information, which is required to sustain active social networks. A fine-tuned communication between cells is vital to maintain the homeostasis and function of multicellular or unicellular organisms in a community environment. Although there are different levels of complexity, intercellular communication, in prokaryotes to mammalians, can occur through secreted molecules (either soluble or encapsulated in vesicles), tubular structures connecting close cells or intercellular channels that link the cytoplasm of adjacent cells. In mammals, these different types of communication serve different purposes, may involve distinct factors and are mediated by extracellular vesicles, tunnelling nanotubes or gap junctions. Recent studies have shown that connexin 43 (Cx43, also known as GJA1), a transmembrane protein initially described as a gap junction protein, participates in all these forms of communication; this emphasizes the concept of adopting strategies to maximize the potential of available resources by reutilizing the same factor in different scenarios. In this Review, we provide an overview of the most recent advances regarding the role of Cx43 in intercellular communication mediated by extracellular vesicles, tunnelling nanotubes and gap junctions.

Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes.

Gap junctions (GJ) are specialized cell-cell contacts formed by connexins (Cxs), which provide direct communication between adjacent cells. Cx43 ubiquitination has been suggested to induce the internalization of GJs, as well as the recruitment of the autophagy receptor p62 to mediate binding to LC3B and degradation by macroautophagy. In this report, we describe a functional LC3 interacting region (LIR), present in the amino terminal of most Cx protein family members, which can mediate the autophagy degradation of Cx43 without the need of ubiquitin. Mutation of the LIR motif on Cx37, Cx43, Cx46 and Cx50 impairs interaction with LC3B and GABARAP without compromising protein ubiquitination. Through in vitro protein-protein interaction assays, we demonstrate that this LIR motif is required for the binding of Cx43 to LC3B and GABARAP. Overall, our findings describe an alternative mechanism whereby Cxs interact with LC3/GABARAP proteins, envisioning a new model for the autophagy degradation of connexins.

A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins.

Photodynamic therapy (PDT) has demonstrated encouraging anticancer therapeutic results, but the current clinically approved photosensitizers (PSs) are not ideal in the treatment of bladder cancer. Conventional PSs have low selectivity to the bladder tumor tissue and induce toxicity or bystander effects on nontumor urothelium. Previous studies demonstrated that the use of galactose-photosensitizer (PS) conjugates is a more selective method of delivering PDT-mediated toxicity due to their ability to recognize carbohydrate-binding domains overexpressed in bladder tumors. Using patient-derived bladder tumor specimens cultured  and bladder cancer cell lines with different PDT sensitivity, we find that a galactose-phthalocyanine (PcGal) accumulates in bladder tumors expressing galactose-binding proteins and internalizes through an endocytic process. The endocytosis mechanism is cell line-dependent. In HT-1376 bladder cancer lines resistant to PDT, depletion of caveolin-1-the main structural protein of caveolae structures-increased the amount of sugar-binding proteins, i.e. GLUT1, at the cell membrane resulting in an improved PcGal uptake and PDT efficacy. These data show the potential of  cultures of bladder cancer, that ideally could mimic the original microenvironment, in screening galacto-PDT agents. Additionally, our studies demonstrate that PDT efficacy in bladder cancer depends on the endocytic mechanisms that regulate PS accumulation and internalization in cancer cells.

Caveolin-1 Modulation Increases Efficacy of a Galacto-Conjugated Phthalocyanine in Bladder Cancer Cells Resistant to Photodynamic Therapy.

Extracellular vesicles (EVs) are membranous structures that protect RNAs from damage when circulating in complex biological fluids, such as plasma. RNAs are extremely specific to health and disease, being powerful tools for diagnosis, treatment response monitoring, and development of new therapeutic strategies for several diseases. In this context, EVs are potential sources of disease biomarkers and promising delivery vehicles. However, standardized and reproducible EV isolation protocols easy to implement in clinical practice are missing. Here, a size exclusion chromatography-based protocol for EV-isolation from human plasma was optimized. We propose a workflow to isolate EVs for transcriptional research that allows concomitant analysis of particle number and size, total protein, and quantification of a major plasma contaminant. This protocol yields 7.54 × 10 ± 1.22 × 10 particles, quantified by nanoparticle tracking analysis, with a mean size of 115.7 ± 11.12 nm and a mode size of 83.13 ± 4.72 nm, in a ratio of 1.19 × 10 ± 7.38 × 10 particles/μg of protein, determined by Micro Bicinchoninic Acid (BCA) Protein Assay, and 3.09 ± 0.7 ng RNA, assessed by fluorescence-based RNA-quantitation, from only 900 μL of plasma. The protocol is fast and easy to implement and has potential for application in biomarkers research, therapeutic strategies development, and clinical practice.

Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research.

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for  autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field.

Article	Year
AMSH-mediated deubiquitination of Cx43 regulates internalization and degradation of gap junctions. FASEB journal : official publication of the Federation of American Societies for Experimental Biology\| PubMed ID: 25070368	2014
Heart ischemia results in connexin43 ubiquitination localized at the intercalated discs. Biochimie\| PubMed ID: 25748165	2015
Connexin 43 ubiquitination determines the fate of gap junctions: restrict to survive. Biochemical Society transactions\| PubMed ID: 26009193	2015
The Force at the Tip--Modelling Tension and Proliferation in Sprouting Angiogenesis. PLoS computational biology\| PubMed ID: 26248210	2015
Proteostasis and SUMO in the heart. The international journal of biochemistry & cell biology\| PubMed ID: 27662810	2016
Characterization of phospholipid nitroxidation by LC-MS in biomimetic models and in H9c2 Myoblast using a lipidomic approach. Free radical biology & medicine\| PubMed ID: 28219782	2017
Exosomes secreted by cardiomyocytes subjected to ischaemia promote cardiac angiogenesis. Cardiovascular research\| PubMed ID: 28859292	2017
Role of connexin 43 in different forms of intercellular communication - gap junctions, extracellular vesicles and tunnelling nanotubes. Journal of cell science\| PubMed ID: 29025971	2017
A Conserved LIR Motif in Connexins Mediates Ubiquitin-Independent Binding to LC3/GABARAP Proteins. Cells\| PubMed ID: 32272685	2020
Caveolin-1 Modulation Increases Efficacy of a Galacto-Conjugated Phthalocyanine in Bladder Cancer Cells Resistant to Photodynamic Therapy. Molecular pharmaceutics\| PubMed ID: 32339462	2020
Simple and Fast SEC-Based Protocol to Isolate Human Plasma-Derived Extracellular Vesicles for Transcriptional Research. Molecular therapy. Methods & clinical development\| PubMed ID: 32913880	2020
Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition). Autophagy\| PubMed ID: 33634751	2021

Teresa M. Ribeiro-Rodrigues

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