Over March break, I was lucky to have the opportunity to intern at the Westwood Lab, where I got to meet Dr. Westwood, who is currently the Associate Professor of the Cell & Systems Biology Department at University of Toronto — Mississauga. His lab is mainly studying the molecular mechanism of heat shock factor in Drosophila melanogaster, which is a type of fruit flies. By speaking with him, shadowing the lab, and doing some experiments myself, I learned a whole lot about genetics, and of course, fruit flies.
This is a two part article series. This article will focus on the basics of heat shock factor and fruit flies, as well as the research process. Future articles will go in more depth about the role of heat shock factors on fruit fly development.
Day 1 — My first encounter with fruit flies…
And we did not fall in love at first sight. When Professor Westwood sat me down for nearly six hours to talk about fruit flies and heat shock factor, I was quite confused. Then, I managed to distill my tangled thoughts into two basic questions: Why do we use Drosophila, and why do we need to care about heat shock factor (hsf) to begin with?
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Fruit flies have been used as a model in genetics for centuries, but it was only until T.H Morgan discovered the white mutation and its linkage to the X chromosome that scientists systematically used it to study genetics.
- Fruit flies are known to share 75% of the genes that cause disease in humans. In fact, the relationship between the human and fruit fly genes is so close that if there is a newly found gene, it is likely that we can find an equivalent gene in the fruit fly genome.
- The fruit fly genome is also known to have low gene redundancy. This means only one or few genes code for members of a protein class, whereas in higher organisms, several genes code for the same or closely related proteins, leading to some functional redundancy. While a mutation in one of these genes will have a lower effect on the function of that particular protein, gene redundancy usually complicates loss-of-function analysis.
- Fruit flies are cheap and easy to keep, facilitating stock management and high-throughput assay. Fruit flies also have a relatively short generation time that takes around 10 to 14 days, allowing researchers to map out the desired genetic outcome several months in advance. Due to the low capacity and size of fruit flies, we can use straightforward staining and fixation protocols to manipulate, observe, and study these flies.
The convenience in stock management, rapid research process, and large overlap with the human genes make the fruit flies (Drosophila melanogaster) an appealing model for studying biological mechanisms of heat shock responses.
Heat shock proteins — The hero against environmental stress
Cells of all kinds undergo different changes in the environment that cause stress, one example of which is elevated temperature. When temperature increases beyond the optimal point for proteins to function, they begin to lose their three-dimensional shape, or denature. Any changes in proteins structure will disable them from properly carrying out their function. In circumventing this situation, cells have something called the molecular chaperones.
Under normal, non-shock conditions, chaperones bind to newly created proteins to help them fold into their correct shape. In heat shock conditions, when there is an increase in misfolded proteins, the number of chaperones also needs to increase accordingly, a phenomenon known as a heat shock response (hsr).
The first step in this highly choreographed response is the activation of a transcription factor called the heat shock factor (HF). The type and number of HF varies depending on the species, such as a single HF1 in fruit flies and three HFs in humans. Nevertheless, the activation pathway remains similar the same across all species: the previously inactive monomers bind to one another to form an active complex of three monomers known as a homotrimer. The trimerization of HFs increase its DNA-binding affinity, facilitating its binding with the heat shock element, whose sequence is 5’-nGAAn-3’. Once bound, HF will initiate the unwinding of DNA and the transcription of heat shock proteins, which belong to a class of chaperones. The heat shock proteins will refold the misfolded proteins to its native shape, returning the to a healthy balanced state called homeostasis.
So, why does heat shock response matter?
They just prevent protein degradation, right? Wrong.
Dr. Westwood and his colleagues found that HF does not only bind to heat shock elements but also to 150 additional chromosomal sites, including development loci that is repressed during heat shock. Such findings suggest that heat shock factors also play a role in developmental processes, including embryogenesis, gametogenesis, and aging. Understanding how this works may give us new insights into developmental diseases, thus moving one step closer to treating them.
DAY 2 — I anesthetized and heat shocked fruit flies to study aging.
After understanding fruit flies and heat shock factor, I got the chance to work hands-on with fruit flies to study aging on my second day. Before jumping into the techniques, it is important to understand what leads to aging. You probably thought that aging was such a natural phenomenon that we should just let it be. Interestingly, there is a genetic regulatory network underlying aging, and by manipulating that network, we maybe able to prolong lifespan.
Genes that control aging
The insulin/IGF-1 (insulin-like growth factor-1) pathway, which includes the DAF-2 transmembrane receptor, a series of intracellular kinases, and the DAF-16 family transcription factor, is known to be involved in aging. Studies have shown that the activation of a protein named protein kinase B by insulin/IGF-1 results in the phosphorylation of DAF-16, inhibiting DAF-16 from transcribing the DNA. On the other hand, the transcription of DAF-16 is indispensable for higher stress resistance, as it is related to the activation of other genes and pathways that produce more heat shock genes.
In short, more DAF-16 regulation leads to the fountain of youth.
Our lab so investigates: Is it possible to mutate a single gene that would result in more DAF-16?
The answer is yes. Two ways we can do so is (1) mutate the DAF-2 hormone receptor to prevent DAF-16 phosphorylation and (2) increase DAF-16 expression by limiting the degradation of DAF-16 that is naturally occurring in Drosophila. While the first step can be accomplished by using RNA interference or CRISPR to mutate the DAF-2 gene, we need to somehow intervene with DAF-16 degradation pathway to reach the second step.
Protein degradation pathway
Normally, proteins undergo a process called ubiquitination before being degraded by the proteasome. A crucial molecule that facilitates ubiquitination is ubiquitin — a highly conserved 76 amino acid polypeptide that can be covalently conjugated to the lysine sites of a target protein. Ubiquitin conjugation to a substrate requires a cascade of at least three different enzymatic reactions. First, ubiquitin is activated by E1, the ubiquitin-activating enzyme. Next, ubiquitin is transiently transferred to E2, the ubiquitin-conjugating enzyme. Finally, an E3 ubiquitin ligase transfers the activated ubiquitin molecule from the E2 enzyme to a lysine residue on the substrate.
Our enemy to youth — RLE-1
Here, the Westwood lab has identified E3 ubiquitin ligase, RLE-1, that functions as an E3 ubiquitin ligase for DAF-16. Overexpression of the RLE-1 gene promotes DAF-16 ubiquitination, while the disruption of rle-1 gene leads to longer lifespan. In fact, by turning off rle-1 gene and DAF-2 hormone receptor in the insulin/IGF pathway, the fruit flies live twice as long. The doubled lifespan simply by disrupting two genes to me was mind-boggling. It sounds like magic, but it’s not magic — it’s science.
Technique of the Day — Flies Pushing
To assist the research process, I was tasked with sorting male and female flies for future crossing. To do so, I first tipped the freshly enclosed flies from their vial onto a porous pad dispensing CO2. CO2 acts as a narcotic and is not harmful is exposure is kept for a few minutes. I then used a dissection microscope to distinguish male flies from female ones
(you may see the differences between them by referring to the image below).
Selected flies are added to fresh standard vials properly labelled with gender and genotype. The process repeats for following generations.
What does studying aging in fruit flies tell us about aging in general?
Remember, flies share 75% their genes with humans, and if there are genes in flies that carry out a certain function, it is likely that we can find equivalents of these genes in humans. The research also gives us a paradoxical yet very interesting insight: insulin/IGF signalling promotes growth and food storage, which all sound like good things, so why does lowering the signalling help us live longer? Well, the answer is lowering insulin/IGF pathway shift the physiology of our cells from maximizing growth to promoting cell maintenance (higher stress resistance). In retrospect, this happens in reality: in higher organisms, like dogs, the small, skinny chihuahua does have a longer lifespan than the chubby bulldog!
- Fruit fly is an ideal model to study genetics due to convenience in stock management, rapid research process, and large overlap with the human genes.
- Heat shock factors not only acts as a protein quality control to maintain homeostasis but are also involved in embryogenesis, gametogenesis, and aging.
- Lowering the IGF/insulin pathway by downregulating DAF-2 hormone receptor and overexpressing DAF-6 leads to prolonged lifespan. Another way to do so is by mutating rle-1 — the gene that is involved in the ubiquitination of DAF-6.
Curious about the role of heat shock factor in development, more laboratory techniques, and my personal takeaways? Stay tuned for my next article!