While nearly all cells of the body can experience stress caused by extrinsic or intrinsic factors (changes in nutrient availability, infection, etc), some cell types such as photoreceptors, adipose tissues, and hepatocytes (liver cells) operate under elevated levels of basal cellular stress due to the nature of their function. Unsurprisingly, organisms have evolved multiple stress response pathways to alleviate the effects of external stress which are coopted by specialized cells (photoreceptors, adipocytes, etc) to maintain cellular homeostasis under normal conditions. However, even in healthy individuals, the ability to cope with cellular stress generally declines with age, which manifests as progressive loss in vision, metabolic capacity and other age-related defects. The presence of disease-causing mutations further exacerbates cellular stress in these cell types, resulting to more rapid decline in tissue health and functionality.
Our broad research goal is to figure out:
How can we improve stress tolerance and sustain tissue homeostasis with age and disease?
Why do some cells die in response to stress while their neighbors are still alive? What makes a cell more resistant to degeneration than its neighbors?
Does prior exposure to stress make a cell more or less proficient in dealing with new stressors?
To answer these questions, Drosophila (fruit flies) are an unmatched model as far as availability of genetic tools and ease of husbandry go, which makes it our favorite discovery platform. We also do a fair bit of cell culture and collaborate with other labs that work on vertebrate models to corroborate our findings.
A schematic of the Integrated Stress Response (ISR) signaling pathway. The example here shows two ISR kinases, PERK and GCN2, responding to stress inflicted by misfolded Rhodopsin (Rh1). The kinsaes alter translation dynamics by affecting availability of initiator methionine (carried by the eIF2 complex). Some mRNAs, such as those encoding the stress response transcription factor ATF4, are translated even under these challenging conditions to help alleviate stress.
Metazoans have evolved a variety of different stress responsive signaling pathways that help minimize cellular damage and restore homeostasis. A majority of these pathways engage transcription programs to alleviate stress. Of course, it is not sufficient for an mRNA to be transcribed but it must also be translated for it to exert its effect. Turns out, some pathways, such as the Integrated Stress Response (ISR), regulate both transcription and translation in response to stress. Specifically, they reduce the availability of certain factors require for initiation of mRNA translation, such that only mRNAs with certain features are effectively translated under these conditions. We recently found that there are non-canonical translation factors that aid the translation of these 'select' mRNAs, such as the one encoding the master stress response transcription factor ATF4 (PMC7495428).
A surprising number of mRNAs, including many encoding stress response proteins, have unconventional 5' leader or 3' trailer structures. These mRNAs are regulated in at least one of the four steps of translation - cap recognition, initiation, elongation, and termination. A genetic screen revealed a whole host of non-canonical translation factors that likely mediate such translation regulation. We are trying to figure out the molecular mechanisms of these non-canonical translation factors, what mRNAs they affect, and which cell types they are required in.
We are particularly invested in understanding how these factors impact cells like photoreceptors that have a high basal level of cellular stress due to their function (your photoreceptors are working quite hard to be able to read this!). Read on for more on this.
mRNA translation can be explained in four broad steps: recognition of the 5' cap structure, recruitment of the translation initiation complex bearing the first methionine, elongation of the polypeptide chain, and finally the recognition of termination signals resulting in ribosome disassembly. Non-canonical translation factors such as those shown in purple blobs can regulate any of these steps.
The fly eye is comprised of ~800 individual units called ommatida. Each ommatidium has a cluster of photoreceptors and other cells require for vision processing, organized in uniform arrays. Photoreceptors can be easily visualized in this system by marking vision proteins such as Rhodopsin with fluorescent tags (RFP here). This allows us to monitor degeneration in vision caused by aging, disease mutations or phototoxicity since these conditions typically lead to loss in photoreceptor integrity or array organization.
The activation of the ISR signaling by PERK, which responds to perturbations in ER homeostasis, has different outcomes in different contexts: in some ophthalomogical disorders such as retinitis pigmentosa where vision degrades with age, the activation of PERK has a protective effect (PMC6583901, PMC7495428); in other neurodegenerative disorders inhibition of PERK leads to better outcomes in terms of cell viability (PMC3033190, PMC5010237). The fly eye is a great platform to study this because both protective and degenerative effects can be modeled in the same tissue. We're hoping to make a dent in our understanding of this dual nature of PERK activation using this model.
Interestingly, this is not the only cell type in which ISR signaling shows such duality; metabolically active cells like adipocytes (fat) or hepatocytes (liver) rely on ISR signaling for their homeostatic function but yet metabolic diseases like obesity are frequently associated with hyperactive ISR signaling.
Our current understanding of ISR signaling in homeostatic functioning of metabolically active tissues largely comes from phenotypic observations in loss-of-function mutants. Both mouse and Drosophila Atf4 null mutants show reduced lower lipid content (e.g. PMC2768187) but yet hyperlipidemia is associated with elevated ATF4 levels (e.g. PMC5000098). These data indicate that ATF4 regulates a certain set of genes required for fat tissue homeostasis that are distinct from those induced in response to excess lipid uptake (i.e. stress). However, the molecular mechanism by which ATF4 achieves these distinct responses remains unknown- we favor a co-factor switch model wherein ATF4 swaps interaction partners between homeostasis and stress conditions. We are figuring out these interaction partners out using Drosophila fat tissues and cultured hepatocytes.
Fat tissues in Drosophila (affectionately and officially called the 'fat body') show basal levels of ISR signaling during homeostasis- this is demonstrated in the control image here showing fat body cells marked with GFP and an ISR activity with DsRed. Predictably, ISR activity goes down when we deplete ATF4 using tissue-specific RNAi. We have exciting data on candidate interaction partners of ATF4 that also seem to affect ISR activity similar to ATF4.
Bright field images showing the mature oocytes in Drosophila ovary. Depleting ISR signaling factors, Perk and Gcn2, specifically in the fat tissue results in aberrant oocyte accumulation.
In addition to cell-autonomous effects on lipid metabolism, we've found that ISR signaling in fat tissues have non-autonomous effects on other metabolically-intense functions such as oogenesis (PMC8938396). We are dissecting how ISR signaling impacts inter-organ communication in this context and have found that ISR signaling acts as a metabolic sensor to inform whether the current state of the organism is suitable for gametogenesis and other reproductive behaviors such as ovulation. Read our latest on this subject in this preprint!