Tracking multiple single molecules in living cells
Most cellular processes are the result of multiple factors coming together at precise subcellular locations to form catalytic complexes. The cell orchestrates an intricate interplay between substrates, (DNA for replication and transcription, and messenger RNA (mRNA) for translation), enzymes (DNA and RNA polymerases and the ribosome), and accessory regulatory factors (histone modifications, and transcription and translation factors). Currently, few methodologies are available with temporal resolution high enough to image short-lived interactions of molecules in the cell, and this has seriously impaired the development of quantitative models for how fundamental biological processes are regulated. For instance, it is possible to image single molecules in the nucleus of living eukaryotic cells,1 but such experiments are limited to single-color imaging. To determine cause-and-effect relationship requires the ability to mark additional components.
We have developed a microscope to study when and where single molecules interact inside living cells, and how nuclear events and the environment regulate cellular activity and processes.2 Our multicolor single-cell imaging assay simultaneously follows three of the agents involved in cellular activity: the substrate or product, the enzyme, and the regulator. We built the microscope on the framework of the accessible and adaptable rapid automated modular microscope (Applied Scientific Instrumentation). It employs separate tube lenses for each of three electron-multiplying CCD cameras, to minimize chromatic aberrations. The multi-band optics enables detection of four open emission windows, three simultaneously (see Figure 1). Pulses of the three excitation lasers illuminate the cellular nucleus in a highly inclined and laminated optical sheet (HiLo) configuration. This compact design requires no relay lenses, shutters, or acousto-optic tunable filters, enabling optimal imaging with minimal photon loss. Furthermore, the microscope has no moving parts, which makes it inherently stable.
To operate the system, we strobe millisecond laser excitation pulses and synchronize them to the frame times of the cameras, such that detections from even fast-moving fluorophores are not motion-blurred during frame acquisitions.3 We have developed algorithms that accurately track and co-localize multiple interacting biomolecules. When we combine images obtained from the three-color microscope with these algorithms, we can image and track how labeled modifications to chromatin (an organizing structure composed of chromosomal DNA and proteins) precede transcription (using labeled nascent mRNA), or determine diffusion coefficients of mRNAs during translation (using labeled ribosomes). Such multiplexed single-molecule measurements at high spatiotemporal resolution inside living cells provide a major tool for testing models relating molecular interactions to biological dynamics.
To enable tracking, single molecules require bright emitters; thus, we used our microscope to characterize a variety of bright dyes for tracking experiments.4 The cell-permeable Janelia fluorophores (JF), developed at the Lavis Laboratory, have enabled live-cell multicolor tracking that would not otherwise have been possible. Moreover, this new palette of dyes is crucial to single-molecule tracking experiments in the nucleus of living cells. By selectively illuminating the nucleus using HiLo illumination, we can study how DNA-binding enzymes are regulated within the chromosomal environment, and how this affects transcriptional output.
With the advent of the clustered regularly interspaced short palindromic repeats (CRISPR/Cas) gene engineering system, it is possible to specifically label molecular enzymes and DNA5 with fluorescent tags in living cells; hence, we can extend the simultaneous multicolor imaging assay broadly to study other molecular processes in vivo. In collaboration with Ibrahim Cissé and Timothée Lionnet, we have used the three-camera microscope for simultaneous imaging of the local clustering dynamics of RNA polymerase molecules6 at the specific β-actin locus,7, 8 while simultaneously tracking its transcriptional output (the β-actin mRNA product) for tens of minutes. A novel cell-permeable near-IR dye (JF 700) enabled simultaneous imaging and tracking of JF 700-lableled β-actin mRNA and Dendra2-labeled polymerase6 molecules without cross-talk and cross-bleaching effects of the two fluorophores.9
In summary, we have developed a robust, multicolor quantitative imaging approach that will enable a wide range of studies probing nuclear organization and dynamics with unprecedented spatial and temporal resolution, directly in living cells. We emphasize that the multicolor super-resolution imaging technique reported here is general in nature: in principle, we can label and image any pair of interacting factors at the single-molecule level. In future work, we plan to develop a chromosomal guide-star system based on CRISPR/Cas,8 which would provide internal reference points as the chromosome is dynamically rearranging, and would ensure that the movement of many transcriptional loci can be accounted for simultaneously on the minute timescale. Chromatin crosslinking and sequencing techniques (such as 3C, 4C, 5C, ChIA-PET, and Hi-C) have provided important insight into how DNA is organized in chromosomal ‘neighborhoods’ for an ensemble of cells,10, 11 and our imaging-based single-cell approach provides a complementary assay.
The authors acknowledge Timothée Lionnet, Timothy Stasevich, Ibrahim Ciss#e, and Luke Lavis for ongoing collaborations. We thank David Grünwald for his technical expertise and helpful discussions. The Howard Hughes Medical Institute supported this work.
Brian P. English is a research scientist whose aim is to develop quantitative single-cell and single-molecule assays to study when and where molecules are interacting inside living cells and where enzymes are active.
Robert H. Singer is the Harold and Muriel Block Chair at the Department of Anatomy and Structural Biology, and co-director of the Gruss-Lipper Biophotonics Center at the Albert Einstein College of Medicine. He is also a senior fellow at the Howard Hughes Medical Institute Janelia Research Campus.