A method for the detection of virus infectivity in single cells and real time: Towards an automated fluorescence neutralization test

ABSTRACTSpatial transcriptomics seeks to integrate single-cell transcriptomic data within the 3-dimensional space of multicellular biology. Current methods use glass substrates pre-seeded with matrices of barcodes or fluorescence hybridization of a limited number of probes. We developed an alternative approach, called ‘ZipSeq’, that uses patterned illumination and photocaged oligonucleotides to serially print barcodes (Zipcodes) onto live cells within intact tissues, in real-time and with on-the-fly selection of patterns. Using ZipSeq, we mapped gene expression in three settings: in-vitro wound healing, live lymph node sections and in a live tumor microenvironment (TME). In all cases, we discovered new gene expression patterns associated with histological structures. In the TME, this demonstrated a trajectory of myeloid and T cell differentiation, from periphery inward. A variation of ZipSeq efficiently scales to the level of single cells, providing a pathway for complete mapping of live tissues, subsequent to real-time imaging or perturbation.

Download Full-text

Enhancer priming enables fast and sustained transcriptional responses to Notch signaling

10.1101/497651 ◽

2018 ◽

Cited By ~ 1

Author(s):

Julia Falo-Sanjuan ◽

Nicholas C Lammers ◽

Hernan G Garcia ◽

Sarah Bray

Keyword(s):

Transcription Factors ◽

Dynamic Response ◽

Notch Signaling ◽

Real Time ◽

Single Cells ◽

Transcriptional Responses ◽

Sustained Activity ◽

Developmental Signaling ◽

Cellular Context ◽

Nascent Transcripts

SummaryInformation from developmental signaling pathways must be accurately decoded to generate transcriptional outcomes. In the case of Notch, the intracellular domain (NICD) transduces the signal directly to the nucleus. How enhancers decipher NICD in the real time of developmental decisions is not known. Using the MS2/MCP system to visualize nascent transcripts in single cells in Drosophila embryos we reveal how two target enhancers read Notch activity to produce synchronized and sustained profiles of transcription. By manipulating the levels of NICD and altering specific motifs within the enhancers we uncover two key principles. First, increased NICD levels alter transcription by increasing duration rather than frequency of transcriptional bursts. Second, priming of enhancers by tissue-specific transcription factors is required for NICD to confer synchronized and sustained activity; in their absence, transcription is stochastic and bursty. The dynamic response of an individual enhancer to NICD thus differs depending on the cellular context.

Download Full-text

Real-time dynamics of mutagenesis reveal the chronology of DNA repair and damage tolerance responses in single cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1801101115 ◽

2018 ◽

Vol 115 (28) ◽

pp. E6516-E6525 ◽

Cited By ~ 23

Author(s):

Stephan Uphoff

Keyword(s):

Dna Repair ◽

Real Time ◽

Molecular Mechanisms ◽

Damage Tolerance ◽

Single Cells ◽

Dna Alkylation ◽

Alkylation Damage ◽

Time Dynamics ◽

Dna Mismatches ◽

The Creation

Evolutionary processes are driven by diverse molecular mechanisms that act in the creation and prevention of mutations. It remains unclear how these mechanisms are regulated because limitations of existing mutation assays have precluded measuring how mutation rates vary over time in single cells. Toward this goal, I detected nascent DNA mismatches as a proxy for mutagenesis and simultaneously followed gene expression dynamics in single Escherichia coli cells using microfluidics. This general microscopy-based approach revealed the real-time dynamics of mutagenesis in response to DNA alkylation damage and antibiotic treatments. It also enabled relating the creation of DNA mismatches to the chronology of the underlying molecular processes. By avoiding population averaging, I discovered cell-to-cell variation in mutagenesis that correlated with heterogeneity in the expression of alternative responses to DNA damage. Pulses of mutagenesis are shown to arise from transient DNA repair deficiency. Constitutive expression of DNA repair pathways and induction of damage tolerance by the SOS response compensate for delays in the activation of inducible DNA repair mechanisms, together providing robustness against the toxic and mutagenic effects of DNA alkylation damage.

Download Full-text