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Mechanobiology of the cellular circadian clock

Periodic Reporting for period 1 - MECHADIAN (Mechanobiology of the cellular circadian clock)

Reporting period: 2018-06-29 to 2020-06-28

Almost all organisms on Earth are subjected to a 24-hour oscillatory behaviour due to their adaptation to the daily pattern of sunlight. In mammals, those oscillations emerge from a molecular clock contained in almost every cell of their body, which can work independently but is controlled by the suprachiasmatic nucleus (SCN), a part of the brain that is sensitive to light. Recent studies have estimated that around the 43% of the mouse protein coding genes have a time-dependent expression somewhere in the organism and that this is cell-type dependent. Our project aims to study until what extent the clock of different cell types can be set on time and maintained beyond the instructions received by the SCN.

MECHADIAN aims to study the impact of the cell mechanical environment into their circadian clock. Until recently, it was believed that in peripheral tissues the clock is set on time by the pace dictated by the SCN which, although autonomous in its ability to oscillate, is regulated by the reception of light by the optical nerves. The SCN then would send signals to the rest of the cells of the body in either endocrine or neurocrine ways. However, studies have demonstrated that changes in the activity of some tissues are able to affect the timing of other cells bypassing the SCN: the liver is set in time not only by the SCN but also by the intake of food and the circadian timing of the muscular system gets affected by exercise.

We hypothesise that the clock of cells in peripheral tissues may be sensitive to, not only chemical but also mechanical, changes in their environment; and we aimed to a) study the impact of the mechanical microenvironment on the cellular clock, b) discover a possible influence of circadian oscillations on the mechanical behaviour of the cells and c) set the basis to characterise the interplay between mechanics and the clock during stem cell differentiation. We focused on the intracellular master clock of NIH3T3 fibroblasts, taking as a reference REVERBA, one of the main proteins that direct the circadian clock at the cell level.

We have developed an automatized method to measure population-wise properties of microscopy images of cells displaying REVERBA oscillations and observed that NIH3T3 fibroblasts show a robust circadian expression of REVERBA in a cell density-dependent manner. We have demonstrated that the effect of cell density on the circadian clock does not reside in differences in biochemical signalling but in mechanosensing. This mechanosensing ultimately controls the localization of certain transcriptional coactivators, whose enrichment in the nucleus in their active form breaks the fine regulation of the circadian clock.

This study could have a big impact in the field of chronomedicine, which addresses the treatment of pathologies considering the timing factor, given both the circadian behaviour and the importance of mechanosensing in the regulation of stem cells and their derived organs and tissues.
MECHADIAN consisted of two main phases: 1) create a technical framework to observe under the microscope the oscillatory behaviour of the NIH3T3 fibroblasts and perform quantitative analyses on populations of cells grown at different conditions, and 2) find those molecular pathways that regulate the circadian clock triggered by mechanosensing.

We created a cell line containing a circadian fluorescent construct reporting REVERBA transcriptional expression and established a strictly reproducible and controlled way to culture the cells. By performing experiments in polyacrylamide gels of specific stiffness and fibronectin concentration, cells were imaged under a confocal microscope every 15 minutes for 3-4 days and analysed with ImageJ and MATLAB so the intensity over time of every single cell could be tracked. We set up a toolkit to determine how circadian REVERBA expression is and to measure possible changes in velocity, nuclear size and the forces exerted in the substrate. To distinguish between biochemical and mechanical signalling we adapted existing microfabrication and micropatterning protocols in the lab. We used the novel PRIMO system to pattern fibronectin on desired shapes so we could attach individual cells on them and measure the effect of spreading area, shape, or exact cell number on the circadian oscillations. We also fabricated magnetic PDMS gaskets to culture cells in two contiguous monolayers separated by barriers of different widths and perform wound healing assays after the gasket removal. To alter the pathways cells normally use to transduce mechanical signals into their transcriptional program (mechanotransduction), we used drugs that alter the actin cytoskeleton dynamics. We optimised the concentration of those drugs to cause motility arrest and/or traction force deprivation in our cells and study the possible effect on the clock. All the techniques and protocols led us to very interesting results:

- REVERBA basal levels and oscillatory behaviourare dependent on cell density.

- That dependence is not caused by biochemical signalling but rather to changes in the mechanical context of the cells.

- Cell-cell adhesions are not essential for REVERBA circadian oscillations.

- The dynamic arrest of individual free cells (at low density) whose actin dynamics was blocked also rescued the REVERBA circadian expression.

- A screening of mechanosensitive factors revealed a strong correlation between nuclear enrichment of some of them and loss of REVERBA circadian expression.

- The overexpression of those mechanotransduction factors provoke the disappearance of REVERBA circadian oscillations.

These results have been already shared in an international conference and two symposiums and we expect to publish them in a manuscript before the end of the year.
The discovery that individual cells of virtually all the tissues in our body have their own circadian clock and that the regulation of these clocks is way more complex than what it was thought revolutioned chronobiology at the takeoff of this century, opening a rich variety of research lines. Coetaneous with that is the complex fastly-changing scientific field of mechanobiology, which aims to study how cells depend on and respond to the mechanical properties of their environment. Our work’s aim was to study how cells of peripheral tissues regulate their own circadian clock and to reveal the mechanobiological pathways participating in that regulation. The big potential of this project resides thus in the combination of two yet unexplored and unconnected fields. Only few recent publications have shown a correlation between the increase of stiffness in breast cancer and the loss of circadian rhythms. However, we still miss mechanistical explanations that link both processes.

Our work is pioneer: it is the first study of circadian rhythms at the cell level in micropatterned cells and shows for the first time a link between the mechanotransduction pathways that regulate the localization of transcriptional coactivators and the regulation of the circadian clock. We believe these results will impact -by connecting them in unprecedented ways- on both the chronobiology and the mechanobiology communities.
Figure 1.