Microscopy and mass spectrometry (MS) are complementary techniques: The former provides spatiotemporal information in living cells, but only for a handful of recombinant proteins at a time, whereas the latter can detect thousands of endogenous proteins simultaneously, but only in lysed samples. Here, we introduce technology that combines these strengths by offering spatially and temporally resolved proteomic maps of endogenous proteins within living cells. Our method relies on a genetically targetable peroxidase enzyme that biotinylates nearby proteins, which are subsequently purified and identified by MS. We used this approach to identify 495 proteins within the human mitochondrial matrix, including 31 not previously linked to mitochondria. The labeling was exceptionally specific and distinguished between inner membrane proteins facing the matrix versus the intermembrane space (IMS). Several proteins previously thought to reside in the IMS or outer membrane, including protoporphyrinogen oxidase, were reassigned to the matrix by our proteomic data and confirmed by electron microscopy. The specificity of peroxidase-mediated proteomic mapping in live cells, combined with its ease of use, offers biologists a powerful tool for understanding the molecular composition of living cells.

Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

https://orbilu.uni.lu/bitstream/10993/21149/1/Advantages%20and%20challenges%20of%20microfluidic%20cell%20culture%20in%20polydimethylsiloxane%20devices.pdf

Advantages and challenges of microfluidic cell culture in polydimethylsiloxane devices.

Entry of tumor cells into the blood stream is a critical step in cancer metastasis. Although significant progress has been made in visualizing tumor cell motility in vivo, the underlying mechanism of cancer cell intravasation remains largely unknown. We developed a microfluidic-based assay to recreate the tumor-vascular interface in three-dimensions, allowing for high resolution, real-time imaging, and precise quantification of endothelial barrier function. Studies are aimed at testing the hypothesis that carcinoma cell intravasation is regulated by biochemical factors from the interacting cells and cellular interactions with macrophages. We developed a method to measure spatially resolved endothelial permeability and show that signaling with macrophages via secretion of tumor necrosis factor alpha results in endothelial barrier impairment. Under these conditions intravasation rates were increased as validated with live imaging. To further investigate tumor-endothelial (TC-EC) signaling, we used highly invasive fibrosarcoma cells and quantified tumor cell migration dynamics and TC-EC interactions under control and perturbed (with tumor necrosis factor alpha) barrier conditions. We found that endothelial barrier impairment was associated with a higher number and faster dynamics of TC-EC interactions, in agreement with our carcinoma intravasation results. Taken together our results provide evidence that the endothelium poses a barrier to tumor cell intravasation that can be regulated by factors present in the tumor microenvironment.

https://www.pnas.org/content/pnas/109/34/13515.full.pdf

Three-dimensional microfluidic model for tumor cell intravasation and endothelial barrier function

We might be at the turning point where research in microfluidics undertaken in academia and industrial research laboratories, and substantially sponsored by public grants, may provide a range of portable and networked diagnostic devices. In this Progress Report, an overview on microfluidic devices that may become the next generation of point-of-care (POC) diagnostics is provided. First, we describe gaps and opportunities in medical diagnostics and how microfluidics can address these gaps using the example of immunodiagnostics. Next, we conceptualize how different technologies are converging into working microfluidic POC diagnostics devices. Technologies are explained from the perspective of sample interaction with components of a device. Specifically, we detail materials, surface treatment, sample processing, microfluidic elements (such as valves, pumps, and mixers), receptors, and analytes in the light of various biosensing concepts. Finally, we discuss the integration of components into accurate and reliable devices.

Microfluidic Chips for Point‐of‐Care Immunodiagnostics

Several microRNAs have been identified that affect lymphocyte effector function and contribute to autoimmune inflammatory disease. Here Youcun Qian and his colleagues find that interleukin-17 (IL-17) suppresses miR-23b expression in nonimmune cells present in inflammatory lesions from individuals with rheumatoid arthritis, systemic lupus erythematosus or multiple sclerosis and in the respective mouse models. MiR-23b targets several signaling molecules downstream of IL-17, TNF-α and IL-1β signaling, thereby suppressing proinflammatory cytokine expression and inhibitng autoimmune pathogenesis in vivo.

The microRNA miR-23b suppresses IL-17-associated autoimmune inflammation by targeting TAB2, TAB3 and IKK-α

microRNAs are known to be deregulated in cancer Using a screen for microRNAs that alter cell migration, Zhang et al show that mir-23b blocks cell migration in vitro and in vivo and is reduced in expression in human colon cancer, suggesting a therapeutic potential for this microRNA

/pdf/genome-wide-functional-screening-of-mir-23b-as-a-pleiotropic-3rakpv3vzm.pdf

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

The interaction between tumor and endothelial cells is crucial to cancer metastasis and angiogenesis. We developed a novel microfluidic device to assess the cell-cell interaction quantitatively at the single cell resolution. This integrated chip offers 16 coculture experiments in parallel with controllable microenvironments to study interactions between cells dynamically. We applied this approach to model the tumor invasion using Hela cells and human umbilical vein endothelial cells (HUVECs) and monitored the migration of both. We observed the retreatment of HUVECs upon the approach of Hela cells during coculture, indicating that the interaction between two cells was mediated by soluble factors. This interaction was further analyzed through quantitatively processing the phase-contrast microscopic time-lapse images of each individual coculture chamber. We also confirmed this paracrine effect by varying the frequency of medium change. This microfluidic technique is highly controllable, contamination free, fully automatic, and inexpensive. This approach not only offers a unique way to quantitatively study the interaction between cells but also provides accurate spatial-temporal tunability of microenvironments for cell coculture. We believe this method, intrinsically high-throughput and quantitative, will greatly facilitate the study of cell-cell interactions and communications.

Quantitative Study of the Dynamic Tumor–Endothelial Cell Interactions through an Integrated Microfluidic Coculture System

RNA molecules are highly compartmentalized in eukaryotic cells, with their localizations intimately linked to their functions. Despite the importance of RNA targeting, our current knowledge of the spatial organization of the transcriptome has been limited by a lack of analytical tools. In this study, we develop a chemical biology approach to label RNAs in live cells with high spatial specificity. Our method, called CAP-seq, capitalizes on light-activated, proximity-dependent photo-oxidation of RNA nucleobases, which could be subsequently enriched via affinity purification and identified by high-throughput sequencing. Using this technique, we investigate the local transcriptomes that are proximal to various subcellular compartments, including the endoplasmic reticulum and mitochondria. We discover that messenger RNAs encoding for ribosomal proteins and oxidative phosphorylation pathway proteins are highly enriched at the outer mitochondrial membrane. Due to its specificity and ease of use, CAP-seq is a generally applicable technique to investigate the spatial transcriptome in many biological systems.

Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling.

The subcellular organization of biomolecules such as proteins and nucleic acids is intimately linked to their biological functions. APEX2, an engineered ascorbate peroxidase that enables proximity-dependent labeling of proteins in living cells, has emerged as a powerful tool for deciphering the molecular architecture of various subcellular structures. However, only phenolic compounds have thus far been employed as APEX2 substrates, and the resulting phenoxyl radicals preferentially react with electron-rich amino acid residues. This narrow scope of substrates could potentially limit the application of APEX2. In this study, we screened a panel of aromatic compounds and identified biotin-conjugated arylamines as novel probes with significantly higher reactivity towards nucleic acids. As a demonstration of the spatial specificity and depth of coverage in mammalian cells, we applied APEX2 labeling with biotin-aniline (Btn-An) in the mitochondrial matrix, capturing all 13 mitochondrial messenger RNAs and none of the cytoplasmic RNAs. APEX2-mediated Btn-An labeling of RNA is thus a promising method for mapping the subcellular transcriptome, which could shed light on its functions in cell physiology.

Expanding APEX2 Substrates for Proximity-Dependent Labeling of Nucleic Acids and Proteins in Living Cells.

A high-throughput microfluidic device is developed to handle liquid dispensation in nanoliter range. The dispenser system shows no cross-contamination between the microwells, indicating its great potential in large-scale screening experiments. An array of 115 nl PCR reactions, as well as the single channel addressable chip demonstrate the high flexibility and wide applications of this novel system.

Ying Zhou

Papers

Genome-wide functional screening of miR-23b as a pleiotropic modulator suppressing cancer metastasis

Quantitative Study of the Dynamic Tumor–Endothelial Cell Interactions through an Integrated Microfluidic Coculture System

Mapping spatial transcriptome with light-activated proximity-dependent RNA labeling.

Expanding APEX2 Substrates for Proximity-Dependent Labeling of Nucleic Acids and Proteins in Living Cells.

A chip-to-chip nanoliter microfluidic dispenser.