Chapter 26 - Intracellular Membrane Traffic at High Resolution

Correlative Imaging (CI) visualizes a single sample/region of interest with two or more imaging modalities. The technique seeks to elucidate information that may not be discernible by using either of the constituent techniques in isolation. Correlative Light Electron Microscopy (CLEM) can be employed to streamline workflows, i.e., using fluorescent signals in the light microscope (LM) to inform the user of regions which should be imaged with electron microscopy (EM). The efficacy of correlative techniques requires high spatial resolution of signals from both imaging modalities. Ideally, a single point should originate from both the fluorescence and electron density. However, many of the ubiquitously used probes have a significant distance between their fluorescence and electron dense portions. Furthermore, electron dense metal nanoparticles used for EM visualization readily quench any proximal adjacent fluorophores. Therefore, accurate registration of both signals becomes difficult. Here we describe fluorescent nanoclusters in the context of a CLEM probe as they are composed of several atoms of a noble metal, in this case platinum, providing electron density. In addition, their structure confers them with fluorescence via a mechanism analogous to quantum dots. The electron dense core gives rise to the fluorescence which enables highly accurate signal registration between epifluorescence and electron imaging modalities. We provide a protocol for the synthesis of the nanoclusters with some additional techniques for their characterization and finally show how they can be used in a CLEM set up.

The potential for increasing the application of Correlative Light Electron Microscopy (CLEM) technologies in life science research is hindered by the lack of suitable molecular probes that are emissive, photostable, and scatter electrons well. Most brightly fluorescent organic molecules are intrinsically poor electron-scatterers, while multi-metallic compounds scatter electrons well but are usually non-luminescent. Thus, the goal of CLEM to image the same object of interest on the continuous scale from hundreds of microns to nanometers remains a major challenge partially due to requirements for a single probe to be suitable for light (LM) and electron microscopy (EM). Some of the main CLEM probes, based on gold nanoparticles appended with fluorophores and quantum dots (QD) have presented significant drawbacks. Here we present an Iridium-based luminescent metal complex (Ir complex 1) as a probe and describe how we have developed a CLEM workflow based on such metal complexes.

The cardiovascular system acts like a well-trained orchestra, where exosomes play a role in paracrine and autocrine signaling. Exosomes derived from various cell subtypes, ensure the cell-to-cell communication in the cardiovascular and cerebrovascular systems. These exosomes, found in all biological fluids, are involved in the stimulation of endothelial cell migration, angiogenesis and cardiac/blood vessels repair upon injury in physiological and pathological conditions. This chapter reviews the role of exosomes in a variety of cardiovascular and vascular neurodegenerative diseases. These roles vary from biomarkers to endogenous cargos and therapeutical bioengineered transfer systems. Possible benefits conferred by exosomes in the cardiac regeneration and repair are also discussed.

Cargo following the retrograde trafficking are sorted at endosomes to be targeted the trans-Golgi network (TGN), a central receiving organelle. Though molecular requirements and their interaction networks have been somewhat established, the complete understanding of the intricate nature of their action mechanisms in every step of the retrograde traffic pathway remains unachieved. This review focuses on elucidating known functions of key regulators, including scission factors at the endosome and tethering/fusion mediators at the receiving dock, TGN, as well as a diverse range of cargo.

Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms. We show the applicability of the protocol to localize and image damaged mitochondria marked by the presence of Parkin, a protein involved in initiating mitophagy. We discuss how this extension to previously published fiducial-based correlation procedures has potential to both allow identifying very rare events and assess the quality of preservation in high-pressure frozen samples.

Cell–cell communication between cardiac and vascular cells and from stem and progenitor cells to differentiated cardiovascular cells is both an important and complex process, achieved through a diversity of mechanisms that have an impact on cardiovascular biology, disease and therapeutics. In recent years, evidence has accumulated suggesting that extracellular vesicles (EVs) are a new system of intercellular communication. EVs of different sizes are produced via different biogenesis pathways and have been shown to be released and taken up by most of known cell types, including heart and vascular cells, and stem and progenitor cells. This review will focus on exosomes, the smallest EVs (up to 100 nm in diameter) identified so far. Cells can package cargoes consisting of selective lipids, proteins and RNA in exosomes and such cargoes can be shipped to recipient cells, inducing expressional and functional changes. This review focuses on exosomes and microRNAs in the context of cardiovascular disease and repair. We will describe exosome biogenesis and cargo formation and discuss the available information on in vitro and in vivo exosomes-based cell-to-cell communication relevant to cardiovascular science. The methods used in exosome research will be also described. Finally, we will address the promise of exosomes as clinical biomarkers and their impact as a biomedical tool in stem cell-based cardiovascular therapeutics.