Characterization of cell uptake and intracellular trafficking of exosomes by quantitative live cell imaging: towards biomimetic delivery vehicles of therapeutic RNA

Exosomes are biological nanoparticles which play a role in long distance cell-to cell communication. These 40-100 nm sized vesicles are released by virtually all cells and derive from the multivesicular bodies within their parent cells. They modulate their target cell fate by induction of cell signaling as well as RNA and protein cargo transfer. Exosomes have also moved into the spotlight of clinical research, with potential use as biomarkers or next generation therapeutic delivery agents. Exosomes are thought to be highly efficient intercellular messengers but quantitative characterization is lacking. Also, their routes of cell uptake and subcellular fate within recipient cells remain elusive. This work introduces an in depth and quantitative characterization of exosome cargo, physicochemical properties, labeling, isolation and their recipient cell interaction at the single cell – single vesicle level. Basic protocols for exosome purification were revisited in order to allow for isolation of exosomes with sufficient yields and in as native state as possible to enable functional studies. Since exosome integrity and recovery yields after differential ultracentrifugation (UC), the most commonly used protocol for exosome isolation, turned out to be poor and unreproducible, we describe an alternative protocol based on ultrafiltration (UF) with subsequent gel filtration (GF) for recovering exosomes relatively selectively, with intact biophysical and functional properties and significantly higher yields. Next we establish methods for specific exosome labeling using fluorescent marker proteins transiently expressed in parent cells, which led to a focus on FP tagged CD63 constructs. CD63-emGFP labeled exosomes were extensively characterized and showed identical properties compared to unlabeled exosomes based on sucrose density gradient, CryoTEM microscopy and proteomics analysis. Furthermore, we successfully adapted fluctuation correlation spectroscopy for characterization of fluorescently labeled exosomes. In another part of this work we describe a high content screen for exosome uptake which we use to provide a first systematic and quantitative profiling of exosome uptake across a panel of exosome parent recipient cells, including HEK293, Huh7, B16F10 as parental cells and additional primary fibroblasts, primary keratinocytes, iPS derived motor neurons and HUVEC primary human endothelial cells as recipient cell lines. These quantitative profiling data reveals preferences in exosome internalization by different cell types and suggests that specific receptor ligand interactions may determine tissue specificity. Finally, we address one of the fundamental questions in the field of cellular communication: how exosomes released by one cell enter and interact with their recipient cell. Our data quantifies for the first time the cell uptake dynamics of exosomes at the single vesicle and single cell level and reveals a quantitative efficiency paralleling that of infective pathogens rather than artificial delivery vehicles. We demonstrate that exosome uptake is largely mediated by active recruitment and surfing on filopodia to reach endocytic hotspots for their internalization at the filopodia base. This provides a cell biological explanation for the remarkably high efficiency of exosomes in targeting recipient cells and discovers a new parallel to some viruses and other pathogens. We propose that the process of filopodia surfing may have evolved as a highway for exosomes into the cell, being hijacked by certain pathogens for host cell interaction. This data does not support the previously reported exosome uptake by vesicle fusion with the plasma membrane or cargo release by endosomal escape. Instead we observe intact exosome uptake to enter endocytic vesicles, which then scan along the endoplasmic reticulum (ER) and end up in lysosomes. Our data suggest a model of controlled cargo delivery to defined subcellular localizations like the ER, rather than vesicle fusion and free release into the cytoplasm

Development of the chick wing and leg neuromuscular systems and their plasticity in response to changes in digit numbers

The tetrapod limb has long served as a paradigm to study vertebrate pattern formation. During limb morphogenesis, a number of distinct tissue types are patterned and subsequently must be integrated to form coherent functional units. For example, the musculoskeletal apparatus of the limb requires the coordinated development of the skeletal elements, connective tissues, muscles and nerves. Here, using light-sheet microscopy and 3D-reconstructions, we concomitantly follow the developmental emergence of nerve and muscle patterns in chicken wings and legs, two appendages with highly specialized locomotor outputs. Despite a comparable flexor/extensor-arrangement of their embryonic muscles, wings and legs show a rotated innervation pattern for their three main motor nerve branches. To test the functional implications of these distinct neuromuscular topologies, we challenge their ability to adapt and connect to an experimentally altered skeletal pattern in the distal limb, the autopod. Our results show that, unlike autopod muscle groups, motor nerves are unable to fully adjust to a changed peripheral organisation, potentially constrained by their original projection routes. As the autopod has undergone substantial morphological diversifications over the course of tetrapod evolution, our results have implications for the coordinated modification of the distal limb musculoskeletal apparatus, as well as for our understanding of the varying degrees of motor functionality associated with human hand and foot malformations

Adipose mTORC2 is essential for sensory innervation in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and possibly sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue are poorly characterized.; Taking advantage of recent progress in whole-mount three-dimensional imaging, we identified a network of calcitonin gene-related protein (CGRP)-positive sensory neurons in murine white adipose tissue (WAT). We found that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, is required for arborization of sensory, but not of sympathetic neurons. Time course experiments revealed that adipose mTORC2 is required for maintenance of sensory neurons. Furthermore, loss of sensory innervation in WAT coincided with systemic insulin resistance. Finally, we established that neuronal protein growth-associated protein 43 (GAP43) is a marker for sensory neurons in adipose tissue.; Our findings indicate that adipose mTORC2 is necessary for sensory innervation in WAT. In addition, our results also suggest that WAT may affect whole-body energy homeostasis via sensory neurons

Adipose mTORC2 is essential for arborization of sensory neurons in white adipose tissue and whole-body energy homeostasis

Adipose tissue, via sympathetic and sensory neurons, communicates with the central nervous system (CNS) to mediate energy homeostasis. In contrast to the sympathetic nervous system, the morphology, role and regulation of the sensory nervous system in adipose tissue is poorly characterized. Taking advantage of recent progress in whole-mount three-dimensional imaging of adipose tissue, we identified a neuronal network of calcitonin gene-related protein (CGRP)-positive sensory neurons in white adipose tissue (WAT). Furthermore, we show that adipose mammalian target of rapamycin complex 2 (mTORC2), a major component of the insulin signaling pathway, mediates sensory innervation in WAT. Based on visualization of neuronal networks, mTORC2-deficient WAT displayed reduced arborization of (CGRP)-positive sensory neurons, while sympathetic neurons were unaffected. This selective loss of sensory innervation followed reduced expression of growth-associated protein 43 (GAP43) in CGRP-positive sensory neurons. Finally, we found that loss of sensory innervation in WAT correlated with systemic insulin resistance. Our findings suggest that adipose mTORC2 is necessary for sensory innervation in WAT which likely contributes to WAT-to-CNS communication

Hypoxia Triggers the Intravasation of Clustered Circulating Tumor Cells

Circulating tumor cells (CTCs) are shed from solid cancers in the form of single or clustered cells, and the latter display an extraordinary ability to initiate metastasis. Yet, the biological phenomena that trigger the shedding of CTC clusters from a primary cancerous lesion are poorly understood. Here, when dynamically labeling breast cancer cells along cancer progression, we observe that the majority of CTC clusters are undergoing hypoxia, while single CTCs are largely normoxic. Strikingly, we find that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage, but it increases intra-tumor hypoxia, resulting in a higher CTC cluster shedding rate and metastasis formation. Conversely, pro-angiogenic treatment increases primary tumor size, yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC cluster generation

Using the NoiSee workflow to measure signal-to-noise ratios of confocal microscopes

Confocal microscopy is used today on a daily basis in life science labs. This "routine" technique contributes to the progress of scientific projects across many fields by revealing structural details and molecular localization, but researchers need to be aware that detection efficiency and emission light path performance is of major influence in the confocal image quality. By design, a large portion of the signal is discarded in confocal imaging, leading to a decreased signal-to-noise ratio (SNR) which in turn limits resolution. A well-aligned system and high performance detectors are needed in order to generate an image of best quality. However, a convenient method to address system status and performance on the emission side is still lacking. Here, we present a complete method to assess microscope and emission light path performance in terms of SNR, with a comprehensive protocol alongside NoiSee, an easy-to-use macro for Fiji (available via the corresponding update site). We used this method to compare several confocal systems in our facility on biological samples under typical imaging conditions. Our method reveals differences in microscope performance and highlights the various detector types used (multialkali photomultiplier tube (PMT), gallium arsenide phosphide (GaAsP) PMT, and Hybrid detector). Altogether, our method will provide useful information to research groups and facilities to diagnose their confocal microscopes

The rough endoplasmatic reticulum is the central site of siRNA-mediated RNA silencing

Despite the rapid advancement of our mechanistic understanding of the RNA interference (RNAi) pathways in the past years, the subcellular sites of RNA silencing still remain under debate. Here we show that a lion’s share of transfected small interfering RNA (siRNA) is cleared quickly and only few siRNA molecules are finally getting loaded into Ago2, with as little as 20 30 siRISC molecules per cell sufficient to promote 50 % mRNA knockdown. While the major RNAi pathway proteins are found in most subcellular compartments, the microRNA (miRNA)- and siRNA loaded Ago2 population as well as the RNAi mediated mRNA cleavage product co-sediment exclusively with the membranes of the rough endoplasmatic reticulum (rER) together with the RISC loading complex (RLC) factors Dicer, TRBP and PACT. Moreover, siRNA-loaded Ago2 associates with the cytosolic side of membranes through TRBP and PACT in an RNA-independent manner, potentially mediated through indirect interaction via Dicer. Our findings demonstrate that the outer membrane of the rER is the central site of RNA silencing, which explains the remarkable thermodynamic and kinetic efficiency of this mechanism

Exosomes surf on filopodia to enter cells at endocytic hot spots, and shuttle with endosomes to scan the endoplasmic reticulum – a highway to the cell.

Exosomes are nanovesicles released by virtually all cells which act as intercellular messengers by transfer of protein, lipid and RNA cargo. Their quantitative efficiency, routes of cell uptake and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake which saturates with dose and time and reaches near 100 % ‘transduction’ efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia, as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. Following internalization, exosomes shuttle with endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction and suggest unanticipated routes of subcellular cargo delivery. vonBueren S1, Graff-Meyer A4, Genoud C4, Martin K5, Voshol H1, Morrissey DV6, 7, EL Andaloussi S3,8, Wood MJ3, Meisner-Kober NC

Exosomes surf on filopodia to enter cells at endocytic hot spots and shuttle within endosomes to scan the ER

Exosomes are nanovesicles released by virtually all cells which act as intercellular messengers by transfer of protein, lipid and RNA cargo. Their quantitative efficiency, routes of cell uptake and subcellular fate within recipient cells remain elusive. We quantitatively characterize exosome cell uptake which saturates with dose and time and reaches near 100 % ‘transduction’ efficiency at picomolar concentrations. Highly reminiscent of pathogenic bacteria and viruses, exosomes are recruited as single vesicles to the cell body by surfing on filopodia, as well as filopodia grabbing and pulling motions to reach endocytic hot spots at the filopodial base. Following internalization, exosomes shuttle within endocytic vesicles to scan the endoplasmic reticulum before being sorted into the lysosome as their final intracellular destination. Our data quantify and explain the efficiency of exosome internalization by recipient cells, establish a new parallel between exosome and virus host cell interaction and suggest unanticipated routes of subcellular cargo delivery

High efficiency preparation of monodisperse plasma membrane derived extracellular vesicles for therapeutic applications

Abstract Extracellular vesicles (EVs) are highly interesting for the design of next-generation therapeutics. However, their preparation methods face challenges in standardization, yield, and reproducibility. Here, we describe a highly efficient and reproducible EV preparation method for monodisperse nano plasma membrane vesicles (nPMVs), which yields 10 to 100 times more particles per cell and hour than conventional EV preparation methods. nPMVs are produced by homogenizing giant plasma membrane vesicles following cell membrane blebbing and apoptotic body secretion induced by chemical stressors. nPMVs showed no significant differences compared to native EVs from the same cell line in cryo-TEM analysis, in vitro cellular interactions, and in vivo biodistribution studies in zebrafish larvae. Proteomics and lipidomics, on the other hand, suggested substantial differences consistent with the divergent origin of these two EV types and indicated that nPMVs primarily derive from apoptotic extracellular vesicles. nPMVs may provide an attractive source for developing EV-based pharmaceutical therapeutics