Follow the Energy, See Where it Goes: Investigating the energetic expenditures of embryonic cleavage divisions

A fluorescent microscopy image of human kidney cells. Green dots are NoBody, Red dots are P-bodies, and yellow dots are where the proteins interact. Image courtesy of Yale University.

Energy is a tricky concept. The term is bandied around within and without science; even among scientists, “ener­gy” takes different meanings in different contexts. Though it is far from straight­forward to define what energy actually is, more important is what energy does. Energy is the potential to apply a force over a distance—otherwise known as work. It allows me to push a box across a floor or enables an enzyme to push two repelling electrons closer together. Elec­trical work in cells drives reactions away from equilibrium, or moves charges across membranes. In biology, energy is what allows an organism to live and grow. Living organisms are open sys­tems, sustained by a constant exchange of energy with the environment.

The Laws of Thermodynamics de­scribe the fundamental tenets of en­ergy, from cells of batteries to cells of zebrafish embryos. The Second Law of Thermodynamics states that the entro­py, or disorder, of the universe always increases. From an entropic standpoint, to be alive is to resist the tendency of every cell to collapse into disorder. Liv­ing things metabolize “high energy” nutrients and harness that energy to create high energy bonds, notably in ad­enosine triphosphate (ATP), commonly known as the “energy currency” in cells. The cells can then spend this currency to grow, reproduce, respond to their en­vironments, and maintain internal orga­nization.

But how is this energetic currency spent over the course of a cell’s lifetime? Jonathan Rodenfels, a postdoctoral as­sociate from the Neugebauer lab in the Department of Molecular Biophysics and Biochemistry at Yale, came across a cellular system that did not seem to be spending energy currency on growth. So he decided to follow the money.

The perfect system

The idea to investigate where energy goes in cells was sparked by a unique biological system. Zebrafish embryos, which develop outside of the mother’s body, are commonly used as a model organism to study development because they are easy to visualize and manipu­late. After a zebrafish egg is fertilized, the single large cell will undergo ten di­visions without growing in volume until it becomes 1,024 identical cells packed into the original cell volume. “These cleavage divisions are very unique from many points of view—energetic, meta­bolic, signaling—a very remarkable pro­cess,” said Joe Howard, Eugene Higgins Professor of Molecular Biophysics and Biochemistry and professor of physics, who was involved in the study.

This type of rapid, growth-free division in early development is also distinct from the rapid proliferation of cancer cells, which rely solely on an energy-inefficient process called glycolysis. Dividing em­bryos, however, use oxidative phosphor­ylation to capture energy, which is gener­ally much more efficient than glycolysis. “What’s not understood is how efficient­ly that energy is used for a diverse set of processes,” Rodenfels said.

Measuring energy as heat

With an intriguing model system to work with, the researchers then had to decide the best way to measure ener­gy flow through the system. “[Oxygen consumption] gives you an idea of how active the cell is, but it doesn’t really tell you how much energy the cell is using… so I thought we could measure heat,” Ro­denfels said. Thermodynamics, literally the “movement of heat,” was the key. An important implication of the Second Law is that the amount of usable energy con­stantly decreases. There can never be a perfectly energy-efficient process, because disorder has to increase. According to the First Law of Thermodynamics, however, energy is conserved. So, the disorder is manifest in heat released from the pro­cess—think friction, or resistors heating up. The rate of heat dissipation from the embryo reflects the rate of “spending” of high energy molecules like ATP to do work, or the net heat flow of all reactions in the embryo over a given time.

In biophysics, much like in chemistry, a calorimeter is used to measure the heat flow of a reaction. A calorimeter is a de­vice with a known heat capacity, in which chemical reactions are allowed to occur. The ideal calorimeter absorbs all the heat released by the reaction, and the change in its temperature can be measured to determine the heat flow of the reaction. “I thought maybe it would be possible to stick [a dividing embryo] in the calorim­eter and measure [its heat flow],” Roden­fels said. To the researchers’ surprise, the method worked incredibly well, and the results revealed an unexpected pattern.

A cyclical surprise

The first thing the researchers noticed was an increase in heat flow from the em­bryo to the environment over time. This makes sense because the number of cells is increasing exponentially; even if the total volume is constant, the number of genomes synthesized and separated each cell cycle—or round of cell division—is increasing. More unexpected was a small oscillation pattern around the increasing trend that matched the fifteen-minute cell division cycle, with a peak at the be­ginning of mitosis (cell division) and a trough at the end, indicating some cycli­cal process requiring an additional ener­gy input. “We wanted to know if we could relate these oscillations to what’s happen­ing in these division cycles. The embryo is not growing, all it’s doing is replicating its DNA then segregating during mito­sis,” Rodenfels said.

The process responsible for the oscilla­tion had to be cyclical and accompany­ing the cell cycle. Three processes came to mind: DNA replication, mitosis, and cell cycle signaling. In three independent “perturbation experiments,” the research­ers added chemicals to the embryos that would inhibit key molecules for each pro­cess. The most popular prediction, which was popular among colleagues as well, was that setting up the mitotic spindles that organize chromosomes and pull cells apart was consuming the extra energy every division cycle. “That was the obvious thing to think…When am I sweating? When I’m at the gym and moving stuff around,” Neu­gebauer added.

But when the researchers blocked mito­sis, they observed the same oscillations. The same went for DNA synthesis, indicating that neither of these two processes required the energy input. As it turned out, the cell cycle oscillations did not disappear until they blocked the cycle itself by inhibiting the network of molecules that signal each cell to enter and exit mitosis. This network is known as the cell cycle oscillator.

Controlling the cycle

All cells, not just embryos, obey a strict cell cycle to make sure that the processes of DNA replication, mitosis, and division happen in the stated order. In most cells, the DNA replication phase (or S-phase) is sandwiched by two growth phases (termed G1 and G2), followed by mitosis (M-phase), after which the cell divides. A myriad of molecular checkpoints are in place to en­sure that each process ends correctly before the next one begins. During the ten reduc­tive cleavage divisions of the early zebraf­ish embryo, however, there are no growth phases and no checkpoints. Instead, egg fertilization kickstarts a time-controlled cycle—fifteen minutes per cell division.

The cell cycle oscillator functions as a central controller to regulate activating phosphorylation—attachment of a phos­phate group—and inhibiting dephosphory­lation of proteins involved in mitosis. The oscillation comes from the interplay of pos­itive and negative feedback loops around cyclin-dependent kinase 1 (Cdk1), a pro­tein that phosphorylates other proteins. At the beginning of the cycle, Cdk1 activates mitotic proteins in a positive feedback loop, starting mitosis. Eventually, a negative feed­back loop kicks in, whereby mitotic proteins and Cdk1 itself are deactivated as mitosis ends. After cell division, the cycle begins again. “This oscillator ensures that the cell doesn’t try to divide before it’s done repli­cating…it has to be accurate because these cleavage divisions are only fifteen minutes long,” Neugebauer said.

After the perturbation experiments, the researchers also used known kinetic and thermodynamic models from chemistry to calculate the predicted phase and am­plitude of heat oscillations for those three cellular processes. As expected, only the model for the cell cycle oscillator closely matched the observed phase and observed amplitude.

With this preliminary understanding of the energy allocation in embryonic divi­sions, the researchers then speculated why the dividing embryo would spend extra en­ergy on the cell cycle oscillator relative to other processes. “Without normal cell cy­cle checkpoints…the embryo would rather spend more energy than theoretically nec­essary to make sure that it doesn’t make a mistake early on,” Rodenfels said. Another idea is that synchronization of the divisions may be important. “These early embryos in fish and amphibian species are very vulner­able in the water, so that may be a reason for making this development as quick as possi­ble,” Howard said.

New ideas, new questions

Beyond the novelty of this experimental approach—measuring live embryos in a cal­orimeter—this type of research may be ex­tended to in vitro fertilization, where there may be a similar dependence of heat flow on the cell cycle. The heat dissipation pat­tern of healthy embryos could be a non-in­vasive metric of the quality of an embryo to help optimize growth conditions.

This study of how a developing organism uses energy begs broader questions. For example, disproportionate energy expendi­ture on cell cycle signaling indicates priority of information over mechanics. This study further distinguishes the metabolism of di­viding embryos from cancer cells, leading to the question of why and how unique cell types develop unique metabolic signatures. “We learn these nitty gritty details about metabolism, but we don’t learn the overall big questions like where this energy goes, and how much energy do you need to drive different processes in your cell,” Rodenfels said. Undoubtedly, exploring the energetics of cell development is crucial to understand why metabolism functions the way it does.