Advanced Method Available for Detecting Dividing Cells

Adrian Salic, HMS assistant professor of cell biology, and Timothy Mitchison, HMS professor and deputy chairman of systems biology, have developed a chemical technique for detecting proliferation in cells and tissues that improves on commonly used methods. This tool will be useful to investigators wishing to evaluate the cell cycle to gain insight into cell metabolism, physiology, or pathology.

Courtesy Adrian Salic

Seeing cell proliferation. This whole-mount stain of EdU-labeled cells in mouse intestine reveals many intestinal vili containing labeled cells as indicators of cell division.

Their study, published in the Feb. 19 Proceedings of the National Academy of Sciences, involves the incorporation of a thymidine DNA precursor analogue, 5-ethynyl-2'deoxyuridine (EdU) into replicating DNA, followed by detection with a fluorescent azide using a copper-promoted chemical reaction.

Typically measuring proliferation in cells and tissues involves the incorporation of labeled deoxynucleosides into cellular DNA as it is replicating. This is followed by a detection method for quantifying or visualizing the newly synthesized DNA. The most frequently used labeled DNA precursors are tritiated thymidine, measured through audoradiography, and 5-bromo-2'deoxyuridine (BrdU), which utilizes an antibody detection system.

Each approach works well, but has limitations. Tritium is radioactive, making it cumbersome to work with. The detection method is labor intensive, and microscopic images have poor resolution. Though BrdU labeling is faster and allows for better imaging, it requires harsh denaturing agents for antibody detection that degrade the specimen. Furthermore, the size of the sample is limited by the ability of the antibody to penetrate fixed tissues, so the tissues must be sectioned. These restrictions are overcome by the use of EdU.

“It’s a method based on chemical detection rather than using an antibody, and it is more sensitive, much faster, and without some of the variables that BrdU and tritiated thymidine have,” explained Salic. “The unexpected advantage is that you can detect the EdU in tissues without harsh fixation and preparation conditions, such that the tissues you are going to look at will retain their structure and pristine form, unlike other methods, where the tissues take a beating during sample preparation.”

To test the chemical method, both live cells and mice were given EdU. Cells incorporated it into their DNA during replication, peppering the genetic material with this chemical group. Harvested cells and organs were reacted with a fluorescent azide that formed a covalent bond with EdU in the presence of a copper sulfate catalyst. When visualized under the microscope, the fluorescent azide-reacted EdU provided a stronger signal than the commonly used BrdU-labeled antibody method. And more of the EdU-containing molecules in the DNA could be detected. The technique also effectively visualized proliferating cells in the organs of the experimental mice.

“Because EdU is detected with a small chemical as opposed to a large antibody, it penetrates tissues rapidly and allows large chunks of tissue to be stained and examined under relatively low-power microscopes, giving an ensemble view of a tissue or organ that other methods cannot provide,” said Salic.

—Kafi Meadows

Gain-of-function Mutation Linked to Precocious Puberty

In its 125th anniversary edition several years ago, the journal Science identified “What triggers puberty?” as one of the top 100 questions for researchers over the next quarter century. A recent advance in understanding what happens when puberty goes awry has now illuminated the stimulus for the transition from childhood to adolescence.

Under normal conditions, puberty begins in girls between ages 8 and 13 and ages 9 and 14 in boys. However, in a small population of children, mostly girls, it is initiated earlier. Gonadotropin-dependent precocious puberty results from premature activation of the hypothalamic-pituitary-gonadal axis, which is responsible for the appearance of secondary sexual characteristics, accelerated growth, and ultimately the capacity for fertility. These changes in younger children can pose significant physical and emotional challenges.

“Precocious puberty is an interesting disorder to look at because, to date, there has not been any genetic cause identified. Although there were other genes implicated in failure of puberty, there had not been any for early puberty,” explained Ursula Kaiser, HMS associate professor of medicine at Brigham and Women’s Hospital. In the Feb. 14 New England Journal of Medicine, Kaiser and her colleagues report a study linking precocious puberty to an activating mutation in a G protein–coupled receptor, GPR54; the work was a collaboration with Ana Claudia Latronico and her lab at São Paulo University Medical School in Brazil.

GPR54 activation by its ligand kisspeptin turns on gonadotropin-releasing hormone (GnRH) secretion in the hypothalamus, causing the release of hormones that induce puberty. Based on previous studies reporting loss-of-function mutations in GPR54 among patients who failed to undergo puberty, the investigators hypothesized that a gain-of-function mutation could contribute to premature GnRH release, causing central precocious puberty. The team analyzed DNA isolated from 53 unrelated patients with the condition and 150 adults who underwent normal puberty. The results revealed a single activating mutation in GPR54, Arg386Pro, identified in an 8-year-old girl who displayed signs of puberty at 7.

To study the impact of this mutation on kisspeptin-mediated signaling, the researchers evaluated monkey kidney cells expressing Arg386Pro GPR54. The mutation disrupted normal receptor desensitization, causing a prolonged activation of the downstream inositol phosphate signal.

—Kafi Meadows

Molecular Calcium Sensors Support Development of Regulatory T Cells

Calcium release–activated calcium (CRAC) channels are the predominant gateway for calcium entry into immune cells. Their importance is underscored by the immunodeficiency found in patients with defects in CRAC channel function.
These plasma membrane conduits open in response to calcium depletion in the endoplasmic reticulum. Two calcium sensors in the endoplasmic reticulum, STIM1 and STIM2, together with ORAI1, a CRAC channel subunit, form the major components linking endoplasmic calcium depletion to CRAC channel opening.

The laboratory of Anjana Rao, HMS professor of pathology, generated conditional knockout mice with T cell–specific disruption of Stim1, Stim2 or both, and evaluated calcium influx through CRAC channels and subsequent cytokine production.

“We didn’t know the precise mechanism of how calcium efflux from the endoplasmic reticulum store was linked to the opening of calcium channels in the plasma membrane,” explained Masatsugu Oh-hora, HMS instructor in pediatrics at the Immune Disease Institute and first author of the study, which appears in the March 9 Nature Immunology. “We were interested in understanding the function of the regulatory molecules STIM1 and STIM2. We found both proteins to be positive regulators of store-operated calcium entry in T cells.”

STIM1-deficient T cells and fibroblasts had almost no calcium influx after depletion of endoplasmic reticulum calcium stores, and the T cells failed to produce cytokines after stimulation. By evaluating whether the calcium-regulated transcription factor NFAT moved to the nucleus, the authors determined that both STIM1 and STIM2 are required to start calcium influx, while STIM2 is important for sustained calcium influx.

The authors also generated mice in which both Stim1 and Stim2 were disrupted in T cells. Analysis of the double-knockouts revealed a surprising find. The spleen and lymph nodes of the mice became massively enlarged, and immune cells were found infiltrating the lung and other organs.

“When we knocked out Stim1 and Stim2, we expected profound immunodeficiency. What we got instead was lymphoproliferative disease,” said Rao. The researchers expected that the reduction in calcium influx would affect T cell development globally; however, only one population was affected, regulatory T cells, a specialized subpopulation of T cells that act to suppress the immune system. The loss of regulatory T cells allowed other immune cell populations to expand, resulting in an aberrant immune response resembling that seen in IPEX, an X-linked human autoimmune disease that results from loss or dysfunction of a transcription factor, FOXP3. Rao’s lab previously found that FOXP3 and NFAT cooperate, explaining why defects in calcium influx that prevent NFAT from going to the nucleus might have similar effects as defects in FOXP3.

These studies advance our understanding of the link between store-operated calcium entry, T cell activation, and regulatory T cells. Further research is needed to elucidate why regulatory T cells are particularly sensitive to STIM proteins compared to conventional T cells.

—Kafi Meadows

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