Dr. Alessandra Stangherlin

Research Area: Circadian Rhythms and Ion Homeostasis

Branches: BiochemistryCell BiologyPhysiology

Website: Stangherlin Lab

1. Research Background

Circadian rhythms are biological cycles in physiology and behaviour of approximately 24 h. Their role is to allow organisms to adapt and anticipate environmental changes of day and night. In humans, for example, the release of cortisol in the morning and melatonin in the evening regulate the sleep/wake cycle, whereas the morning increase in heart rate provides more cardiac output for daily activity. Misalignment with the external environment during jet lag or night shift work is associated with disease states, including cancer and neurological disorders.

It is now established that circadian rhythms originate at the cellular level via molecular timekeeping mechanisms or cellular clocks. Many cellular processes are under circadian control, from DNA replication to protein translation, as well as physiological outputs, including cell division and motility. Recently, we have shown that the abundance of many metal ions exhibits 24-h rhythms, with significant biological consequences. For instance, Mg2+ oscillations regulate ATP availability and energy balance, whereas Na+, K+, and Cl- rhythms compensate for the osmotic changes elicited by circadian variation in cytosolic protein abundance. Yet, very little is known about the impact of these rhythms on other functions, such as regulation of electrochemical gradients, modulation of protein rhythms, and mitochondrial function, which greatly depends on ion exchange.

2. Research questions addressed by the group:

We recently found that in mammalian cells, mTORC1 activity sustains circadian rhythms in cytosolic protein and leads to daily changes in cytosolic crowding. To maintain equilibrium with the ionic composition of the extracellular milieu, cells rhythmically import and export Na+, K+ and Cl-, with an opposite phase to cytosolic protein. Ion fluxes are modulated by the electroneutral cotransporters of the SLC12A family via the rhythmic activity of WNK and OXSR1 kinases, which are sensitive to the intracellular concentration of Cl- and macromolecules. This study reveals that the intracellular concentration of ions is not constant during the day, as generally assumed, and suggests a link between osmoregulation and protein homeostasis. Furthermore, we found that rhythmic changes in the intracellular level of Na+, K+ and Cl- impart daily variation in action potential firing rate in isolated cardiomyocytes. This cell-autonomous mechanism works independently of the central nervous system and anticipates the increased cardiac output required during the active phase of the day.

3. Possible project(s):

We offer several multidisciplinary projects to understand the relationship between intracellular ion dynamics and the timekeeping mechanism and how they modulate cellular functions. These include:

  • Investigate the reciprocal regulation of ion and protein rhythms
  • Explore ion rhythms at subcellular resolution
  • Analyse ion rhythms during ageing

4. Applied Methods and model organisms:

We use primary and immortalised mouse fibroblasts and mouse tissues. Our techniques range from cell culture and biochemistry to proteomics and bioluminescence-based assays. For example, we use a bioluminescence system to monitor circadian rhythms in gene expression. Our lab has also developed a state-of-the-art platform for measuring metal ions via atomic emission spectrometry. We aim to couple this technique with metabolomics to fully comprehend how osmolytes (organic and inorganic) regulate circadian rhythms and associated physiological functions.

5. Desirable skills and qualifications:

We are looking for curious and motivated individuals to join our group. Candidates should possess excellent organisational skills, including maintaining accurate and detailed experimental data and protocol records, a problem-solving attitude, and good command of spoken and written English. Candidates with a background in molecular biology, biochemistry, or in vivo mouse work are encouraged to apply.

6. References and key publications:

  1. Stangherlin, A. et al. Compensatory ion transport buffers daily protein rhythms to regulate osmotic balance and cellular physiology. Nat Commun 12, 6035, doi:10.1038/s41467-021-25942-4 (2021).
  2. Wong, D. C. S. et al. CRYPTOCHROMES promote daily protein homeostasis. EMBO J 41, e108883, doi:10.15252/embj.2021108883 (2022).
  3. Stangherlin, A., Day, J. & O'Neill, J. Inductively Coupled Plasma Mass Spectrometry for Elemental Analysis in Circadian Biology. Methods Mol Biol 2130, 19-27, doi:10.1007/978-1-0716-0381-9_2 (2021).
  4. Stangherlin, A., Seinkmane, E. & O'Neill, J. S. Understanding circadian regulation of mammalian cell function, protein homeostasis, and metabolism. Curr Opin Syst Biol 28, None, doi:10.1016/j.coisb.2021.100391 (2021).