Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation

Background:

Which came first, the chicken or the egg? A question everyone has heard and pondered (it was probably the egg by the way). This is a, potentially overused, paradox to highlight the difficulty of determining the cause and effect of an event. It applies well to the situation in our cells: a lot of cellular processes are very difficult to untangle, as they happen on such a small scale in a very short window of time.

One such application of the ‘chicken and egg’ problem (in the context of a cell) revolves around changes in nuclear morphology and gene expression. It is widely acknowledged that a range of cells are sensitive to forces, and some cellular components have the ability to sense external forces and change a cell’s properties accordingly. However, it’s also known that different cells have different morphologies as a result of unique gene expression patterns (e.g. a neuron looks very different to a fibroblast). This then begs the question, which is the cause, and which is the effect when considering cellular (and nuclear) morphology, and changes in gene expression? Is there a force being applied to the nucleus that causes a shift in gene expression? Or is there a change in gene expression that causes the cell (and the nucleus) to acquire different properties and deform? Untangling these two events can be very difficult to do experimentally, and so, the chicken-and-egg question remains.

This preprint focuses on ependymal cells lining the fluid-filled cavities in the brain and looks at nuclear deformation by actin, a major component in the ‘scaffolding’ of the cell, during the differentiation of these cells. More specifically, the authors sought to determine how actin accumulation, nuclear deformation, and cell differentiation were interlinked during this process.

 

Main Findings:

How does nuclear shape change during ependymal cell differentiation?

The authors found that during ependymal cell differentiation, nuclear shape changes drastically, going from a relatively round structure to a much flatter and longer shape with an overall increase in volume (as depicted in Figure 1). The nucleus also relocated towards the plasma membrane of the cell.

 

 

How does actin accumulation affect nuclear deformation?

The authors next investigated the effect of disrupting actin polymerisation on the cells’ ability to differentiate. They found that this disruption affected the nuclear deformation during differentiation, as the nuclei did not flatten as much as observed in control cells. Inhibiting myosin, a molecular motor that associates with actin, also affected nuclear deformation. The authors found that depleting branched actin (via inhibition of the Arp2/3 complex) led to decreased actin accumulation in differentiating cells. They also found that inhibition of this complex led to a defect in ependymal cell differentiation in in vivo mouse models, and that the nuclei in these cells migrated less towards the plasma membrane. Conversely, promoting actin polymerisation led to cell differentiation of ependymal cells at an earlier timepoint than observed in controls.

Actin has a lot of roles in a cell, so globally altering actin polymerisation is bound to have a range of off-target effects which may affect differentiation and nuclear deformation/movement. To overcome this, the authors severed the link between actin and the nucleus, through the LINC complex, to see how actin specifically interacting with the nucleus would affect differentiation. They found that severing these links led to a decrease of nuclear flattening as observed in the controls.

 

Does exerting force to the cell/nucleus induce differentiation?

And now back to the chicken and the egg paradox. To check whether nuclear deformation triggers ependymal cell differentiation, the authors manually applied a force to nuclei. For this, they used an in vitro cell line that undergoes similar nuclear changes during differentiation as observed in vivo (increase in nuclear volume, and nuclear flattening). After compressing the cells, the authors found an increased nuclear flattening and a higher rate of differentiation. The authors then investigated molecules that could be associated with the observed differentiation and identified RB1 phosphorylation as a potential link between actin accumulation and subsequent ependymal differentiation. And so, in ependymal cells at least, the chicken and egg problem has been solved. It is the force applied to the nuclear deformation that induces differentiation.

 

Why I am highlighting this preprint:

As part of my PhD project, I am using organoids that contain two distinct cell types. Although the role of morphogen signalling is pretty well researched, and clearly influences the fate of cells within the organoid, I am also interested in investigating the role of mechanical forces in the cell differentiation I observe within the organoid. This paper gives me a new angle to try and understand the patterning of the two cell types observed within the organoid.

 

Questions for the authors:

1. In in vivo development, is there a point where ependymal cells are exposed to a force that compresses them and could lead to subsequent differentiation?
2. Is the nuclear flattening due to constant force applied to it, or after initial force is it’s ‘resting’ shape flat? For example, if you were to remove the nucleus from the cell, would it retain its flatter shape, or would it revert to a rounder shape as seen in the non-differentiated cell?
3. Do you think nuclear deformation could be a factor that leads to differentiation in a lot of different cell types? Or do you think it’s more likely to be specific to a handful of cell types?

Tags: actin, cell differentiation, differentiation, ependymal, nuclear deformation

doi: https://doi.org/10.1242/prelights.38776

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