OUR RESEARCH
BACKGROUND
It is widely accepted that the functional pleiotropy of cyclic AMP (cAMP) depends on its ability to generate intracellular messenger “signatures” that the cell “decodes” to specific functions. Indeed, it has been postulated that cAMP generating stimuli (e.g. hormones) result in the activation of spatially distinct effector moieties (microdomains) that are coupled to specific cellular responses. Despite decades of intense research, the molecular mechanisms by which cAMP achieves the spatio-temporal organization of its cascade remain poorly understood and hotly debated. The leading idea associates the selective activation of effector proteins to the local availability of the messenger. Specifically, the subcellular heterogeneity in cAMP levels has been attributed to the localization or activity of either adenylate cyclases (ACs) and phosphodiesterases (PDEs) in different cellular sub compartments. However, arguing against cAMP microdomains being solely dependent on the messenger’s distribution are computational models which consistently show that the distribution of cAMP cannot be sufficiently restricted by PDEs implicating the need for additional factors in the generation of functionally distinct cAMP microdomains. In order to investigate this issue my group generated a series of FRET-based sensors that allow the measurement in real time in living cells of both, the levels of cAMP and the actions of its main effector Protein Kinase A (PKA) (PKA-dependent phosphorylation). As proof of principle we targeted our sensors to the outer mitochondrial membrane (OMM), a well-recognised functional PKA microdomain, and compared the levels of cAMP and PKA-dependent phosphorylation in response to cAMP generating agonists between OMM and cytosol. We found that, at equivalent levels of cAMP, PKA located at the OMM responded differently than the cytosol and that was due to differential action of the terminators of the cAMP/PKA axis, the phosphatases. Our work unveiled phosphatases as major, hitherto unrecognized contributors to the compartmentalization of PKA activity. This work demonstrated that differences in local phosphatase activity fully explain functional data assessed using readouts of cAMP signalling in the cytosolic and OMM compartments.
WHAT WE DO
We approach the study of cAMP microdomains at multiple levels, integrating classic biochemistry, molecular biology and single cell real time fluorescence imaging techniques. We are developing and validating new methodologies and tools able to measure and/or manipulate the cAMP cascade with unprecedented specificity. Using this molecular toolbox we aim to study the signals that trigger sharply defined cAMP/PKA events. Finally by combining our tools to “omics” we plan to identify the molecular players through which each cAMP microdomain is transduced in cellular function.
Currently we have three research projects in the lab.
Project 1 | Identifying new regulators of cAMP compartmentalization
Project 2 | Investigate the involvement of Mitochondrial cAMP/PKA domains in mitochondrial processes
Project 3 | Studying the molecular mechanisms and players underpinning the ability of cAMP to influence cell fate.
Despite decades of intensive research on cAMP microdomains the molecular mechanisms by which cAMP achieves the spatio-temporal organization of its cascade remain poorly understood and hotly debated. The leading view is that the actions of phosphodiesterases (PDEs), the enzymes that hydrolyze cAMP, determine which microdomains will be activated (allowing high cAMP levels) or will remain inert (maintaining low cAMP levels) in response to a given cAMP-generating signal. Based on this model cAMP microdomains could be defined as “subcellular sites where the concentration of cAMP is distinguishable from that of the surrounding areas”. However, mathematical models argue against the ability of sole PDEs to create cAMP microdomains and clearly suggest the existence of additional unidentified mechanisms.
In order to better understand whether differences in local cAMP levels mirror the spatially restricted activation of its main effector protein kinase A (PKA) we developed a co-culture approach that allow us to simultaneously measure in real time in single living cells cAMP levels and PKA activity in response to a given stimuli (fig 1) (Burdyga A & Lefkimmiatis K. Methods in Molecular Biology; 2015; 1294:1-12). By using immortalized cell lines and primary neonatal ventricular rat cardiomyocytes we are dissecting the roles that each component of the cAMP/PKA axis has in the creation of cAMP microdomains.
While it well known that PKA at the outer mitochondrial membrane (OMM) regulates numerous mitochondria-related functions such as apoptosis and mitochondrial fission, little is known of whether this distinct cAMP/PKA microdomain is involved in other mitochondrial processes. For instance it has been proposed that generalized cAMP elevations could both protect depolarized mitochondria from being targeted by mitophagy (Akabane S et.al Mol.Cell 2016) and increase the retrograde movement of defective organelles in neurons (Ogawa et.al. ACS chemical neuroscience, 2016). Both these processes (mitophagy and trafficking) are highly specific and target exclusively distinct organelles, therefore we asked the question of whether recognizable cAMP/PKA signatures may define which process a mitochondrion would undertake.
Thanks to the extensive molecular toolbox we developed for the measure (fig 2) and manipulation of different aspects of the cAMP signaling cascade we are in the unique position to test this possibility. For instance thanks to sensitive FRET-based sensors we plan to measure cAMP and PKA phosphorylation levels in individual mitochondria undergoing retrograde or anterograde trafficking. In addition we will take advantage of targeted versions of a photoactivatable adenylyl cyclase (bPAC) to selectively increase cAMP and consequently PKA activity at the OMM of distinct mitochondria, while, on the contrary, a targeted version of a genetically encoded cAMP buffer will decrease the levels of messenger only at the mitochondrial surface. Using combinations of these tools we will investigate the molecular mechanisms through which the cAMP/PKA axis is involved in mitochondria trafficking and recycling.
Multicellular organisms maintain their cell number by balancing cellular proliferation with cell death. Each of these processes has essential roles, and their deregulation can result in severe disease. For example, cell proliferation and differentiation are crucial for wound healing and tissue renewal but, if out of control, can result in cancer and autoimmune disease. Equally important is cell death. Cells that are damaged and could compromise the homeostasis of the organism are eliminated through regulated (programmed) forms of cellular death. Until recently, apoptosis was the only form of cellular death considered as regulated; however lately, a novel form of programmed cell death, called necroptosis has been described. The discovery of necroptosis (which can be induced by tumor necrosis factor alpha, TNFα) bares significant clinical weight, as it becomes increasingly clear that this process is involved in human disease. Consequently a better understanding of the mechanisms underpinning necroptosis is of primary importance.
One peculiar characteristic of cAMP is that can induce or inhibit cellular death. While the relation of cAMP/PKA to apoptosis is well studied whether this pathway is involved in the pathways that regulate other forms of programmed cell death is unknown. The major goals of this project are to a) establish whether the cAMP/PKA axis is involved in non-apoptotic forms of cell death and b) identify the molecular players linking the cAMP pathway to programmed cell death. To achieve our goals we designed a multidisciplinary approach that combines bioinformatics to classical biochemistry and high throughput proteomics screenings.
