Compartmental genomics in living cells revealed by single-cell nanobiopsy

Abstract

The ability to study the molecular biology of living single cells in heterogeneous cell populations is essential for next generation analysis of cellular circuitry and function. Here, we developed a single-cell nanobiopsy platform based on scanning ion conductance microscopy (SICM) for continuous sampling of intracellular content from individual cells. The nanobiopsy platform uses electrowetting within a nanopipette to extract cellular material from living cells with minimal disruption of the cellular milieu. We demonstrate the subcellular resolution of the nanobiopsy platform by isolating small subpopulations of mitochondria from single living cells, and quantify mutant mitochondrial genomes in those single cells with high throughput sequencing technology. These findings may provide the foundation for dynamic subcellular genomic analysis.

Figure 1. Schematic of single cell nanobiopsy

a) Illustration of automated approach to cell surface, penetration in the cell cytosol followed by controlled aspiration of cytoplasmic material by electrowetting, and delivery of the biopsied material into a tube for analysis. Scheme not to scale. b) Optical micrographs of the nanobiopsy procedure illustrating the approach, cell membrane penetration, aspiration via electrowetting, and retraction of the nanopipette tip. Scale bars 25µm. c) Chronoamperometry during nanobiopsy. A nanopipette filled with a 10mM solution of THATPBCl penetrates a cell in culture. Before the penetration, the nanopipette is biased at +100mV to prevent any aqueous solution from entering into the tip. After cell membrane penetration, the bias is switched to −500mV. The entry of intracellular solution in the nanopipette tip causes the ion current to increase. d) Calcium imaging during nanobiopsy procedure showing false color fluorescent micrographs of human BJ fibroblast cells stained with Fluo4 AM during nanobiopsy. Red arrows indicate where the nanopipette penetrated the cell. Scale bars 25µm, unless otherwise specified.

Figure 2. RNA Analysis post nanobiopsy

a) Scheme not to scale showing nanobiopsy followed by RNA analysis and SEM micrograph of a representative nanopipette tip b) Post biopsy analysis via qPCR targeting GFP RNA from HeLa cells showing a positive control of total RNA from a ~1000 cells lysate (red curve, C_q 20), a nanobiopsy taken from within a single cell (blue curve, C_q 30) and the negative control (black curve, C_q 33). c) Reproducibility of nanobiopsy protocols. qPCR targeting GFP RNA. Positive control (red curve) consists of RNA extracted for GFP HeLa cells spiked in the cDNA synthesis mix. Nanobiopsy (blue curve) represents the average of 4 nanobiopsies with 4 different nanopipettes performed in a PBS solution containing the lysate of ~10000 GFP HeLa cells following the protocol described in the manuscript. Negative control (black curve) where no input material was used for cDNA synthesis. d),e) Read coverage of RNA sequencing of aspirations are displayed in the UCSC genome browser. Genomic position is displayed along the X axis and depth of coverage is represented in the Y axis. d) Ensemble gene predictions are displayed in red, where lines represent introns and rectangles represent exons. These gene predictions are on the reverse strand and are displayed in the 3’ to 5’ direction. Alternative 3’ exon usage in the gene PABPC1 is seen in M3 and M7 vs M8. M8 shows coverage over the 3’ region of the longer isoform, whereas M3 and M7 show coverage over the shorter isoform. e) The gene MCCC2 is displayed in blue in the 5’ to 3’ direction. Read coverage extends across the entire length of the gene and demonstrates the feasibility of generating full length cDNA from transcripts isolated by nanobiopsy.

Figure 3. Mitochondrial nanobiopsy

a) Fluorescent micrograph of human BJ fibroblast cells stained with MitoTracker® Green before nanobiopsy (right panel) and after nanobiopsy (left panel). Red circles highlight a dark spot caused by mitochondria removal. Scale bars 15 µm. b) (left panel) Bright field image of the nanopipette tip (red circle) used for mitochondria nanobiopsy in a). (Right panel) Negative fluorescent micrograph (black areas indicate high fluorescence) of left panel showing fluorescence arising from the nanopipette tip which indicates the success of mitochondria nanobiopsy. Scale bars 15 µm. c) Sequencing results demonstrate variable conservation of heteroplasmic frequencies in aspirations. Heteroplasmic variants with estimated frequencies between 5% and 99% are displayed as circles where the area of the circle is proportional to the observed frequency. The nucleotide of the variant is specified by the color, where ‘A’ is represented in red, ‘C’ is represented in violet, ‘G’ is represented in blue and ‘T’ is represented in green. The 14713 A->T variant at shows similar frequencies across aspirations and population; whereas the 16278 C->T variant shows a greater variance of heteroplasmic frequencies in aspirations. Low frequency variants were also found in both aspirations, but not in the population.