An intriguing question in microbiology revolves around bacteria that can adopt a dormant state and remain metabolically inactive for extended periods. One medically relevant example, Mycobacterium tuberculosis, can persist in a dormant state in tissues and regain virulence decades later by mechanisms incompletely elucidated.
Single-cell approaches may allow metabolically quiescent cells to be compared to their metabolically active counterparts and unveil genomic features that could explain the mechanisms of dormancy.
A large portion of marine microorganims, just like those populating the soil or the human body, are dormant and refractory to classical culturing methods. It is still not understood how they survive in that state without being quickly eaten up by grazers such as protists.
Dr. Stepanauskas and his colleagues are using single-cell approaches to compare bacteria isolated from the food vacuoles of bacterivorous protists to metabolically active and dormant members of the bacterioplankton, to better understand how bacterial metabolic activity correlates with the probability of being grazed, a concept with important implications for the plankton genetic diversity.
“I think that single-cell genomics provides a huge opportunity to better understand what makes microbes dormant, what enables them to survive in a dormant state, and what triggers them to become active again. This is a basic question in microbiology, with implications anywhere from marine ecology to human diseases,” explains Dr. Stepanauskas.
Investigators at the Bigelow Laboratory for Ocean Sciences established a microbial single-cell genomics core facility that recently started processing the first samples and will offer increased throughput and reduced per-sample costs. In its full capacity, which will be reached in 2010, the facility anticipates performing cell sorting, whole genome amplification, and PCR-based screening on tens of thousands of individual cells weekly.
As the importance of a few selected cells is increasingly recognized as driving tumor development, metastatic dissemination, and resistance to therapy, single-cell genomics emerges as a promising tool to understand tumor heterogeneity.
“Obtaining the whole transcriptome from single cells is invaluable, because every cancer cell is different, every cell is at a different developmental stage, and to truly understand carcinogenesis you really need to do it at a single-cell level,” states Kaiqin Lao, Ph.D., principal scientist in the molecular cell biology division at Applied Biosystems, a division of Life Technologies.
In collaboration with a group from the University of Cambridge, Dr. Lao used Applied Biosystems’ SOLiD system to perform digital expression profiling of a single mouse blastomere and identified 75% more genes than by microarray methodologies.
This approach unveiled 1,753 previously unknown splice junctions and, for the first time, unambiguously confirmed that 8–19% of the genes with multiple known transcript isoforms, expressed at least two isoforms within the same blastomere or oocyte. This finding will provide insight into biological processes relying on specific isoforms and facilitate their selective therapeutic manipulation.
“This is really intriguing and important,” states Dr. Lao. “With conventional transcriptome assays it was not possible to ascertain if the multiple transcript isoforms found in a tissue or organ coexist in the same cell, or if they are just expressed in different cell types or at different cell cycle stages of the same cell type. This knowledge is crucial to provide a real understanding of the transcriptome complexity within individual cells. Such single-cell technology would be very important to gain a greater understanding of the behavior cancer stem cells and solid tumors, such as breast cancer, exhibit.”
Measurements relying on genetic material extracted from entire organs or tissues do not reveal the heterogeneity of genomic information. “The ability to truly perform single-cell nucleic acid measurements, which retain the true, unbiased information coming from cells, is the gold mine in genome biology,” believes Patrice Milos, Ph.D., vp and CSO at Helicos BioSciences.
A recent innovative approach developed at Helicos relies on sequencing RNA from small numbers of cells by capturing the polyA tails of the cellular transcripts through hybridizing to an oligo-dT surface. The RNA-DNA hybrids formed as a consequence of this strong and stable interaction are then subjected to sequencing-by-synthesis by using Helicos’ True Single Molecule Sequencing (tSMS)™ technology. This approach does not require ligation or PCR amplification steps and thus offers an unbiased view of events at what will become single-cell nucleic acid levels, according to Dr. Milos.
Increasingly, new experimental tools become informative about discreet features that set individual cells apart and reveal their contribution to the population as a whole. In this context, Richard Feynman’s words, so eloquently illustrating the need to explore individual components that shape entire systems, become very relevant: “Nature uses only the longest threads to weave her patterns, so each small piece of her fabric reveals the organization of the entire tapestry.”