These two lines of images illustrate cell (top line) and cytoplast (bottom line) organisation in response to defined geometries. To produce cytoplast, cells have been attached and centrifuged in order to induce enuclation. The centrosome is tightly linked to the nucleus. Their physical association is determinant in the definition of the entire cell organisation and polarity. The centrosome-microtubules system is capable of self-centering as illustrated below in cytoplasts. The presence of the nucleus interfere with this behavior by off-centering the centrosome in these confined cells. The nucleus is a volume from which microtubules are excluded but on which they are anchored via the centrosome, their organizing centre. Thereby the nucleus/centrosome/microtubules system is an intrinsically polarised system. The mecanical interplay governing centrosome and nucleus positionings is a critical issue in the understanding of the definition of cell polarity.
While the mother centriole, which nucleates and anchors microtubules, is always found at the cell centre, the daughter centriole, which can't anchor them, can be seen far from the centre as it is the case here on the disc.
The polarised organisation of the microtubule network doesn't result only from what happens at the cell centre and its association with the nucleus, but is also regulated by what happens at the cell periphery where microtubule growing ("plus") ends interact with the cortical actin network. When microtubules reach the cell periphery they adopt different behavior depending on the type of actin structures they encounter there. If the cell edge is locally attached to the extra-cellular matrix, the formins nucleate a branched network of actin and microtubules stop growing when entering this region. On the opposite, if the cell edge is not attached, actin filaments form contractile bundles or stress fibers and microtubules keep growing along these actin bundles until they reach a cell adhesion site.
The movie below shows EB1-GFP decorating microtubule plus ends. The cell is attached on a fibronectin micropattern with a crossbow shape (blue picture). The two behaviors can be seen close to adhesive or non-adhesive edges.
EB1 Microtubule trajectories on Crossbow shaped micropattern
fibronectin micropattern actin stress fibers microtubule trajectories
(projection of EB1 time-lapse image sequence)
The geometry of cell microenvironment is replicated by the actin network at the cell surface
and transmitted by the microtubule network to the cell interior.
It is then not surprising to observe that the geometry of the cell adhesive microenvironment
accurately govern centrosome and nucleus positioning.
The centrosome maintains a central position in all cases. The nucleus is off-centred towards the cortical regions where actin is organized in contractile structures (red regions). The Golgi apparatus is compacted around the centrosome and faces the regions where actin is organized in a branched polymerising network (blue regions).
Therefore, in response to a define geometry of cell adhesion and actin architecture,
the nucleus/centrosome/Golgi/microtubules system adopts a specific orientation.
Interestingly, while the Golgi apparatus in cells cultured in classical conditions usually appears stretched and spread around the nucleus (left), it is always remarquably compact and focused around the centrosome in cells that are constrained on micropatterns (right). This spherical geometry doesn't seem to reflect only a mechanical equilibrium. The Golgi apparatus is the protein sorting and addressing factory, its specific spherical geometry could highlight a global reorganisation of this traffic. It also highlight ssome physical properties of its interaction with the centrosome/microtubule system that have been masked in the preceeding culturing method and deserve to be investigated.
Golgi apparatus in free cells Golgi apparatus in constrained and oriented cells
Cell actin cytoskeleton is not only sensitive to adhesion anisotropy, it is also highly sensitive to the level of spatial confinement due to the presence of extra-cellular matrix and neighboring cells. Spatial confinement has been shown to influence cell apoptosis, growth and differentiation. It acts directly on the architecture and dynamics of the actin network which further infuence centrosome positioning and thus primary cilium growth.
The proportion of ciliated cells is directly related to the extent of cell spreading.
In addition, in spread cells, cilia are also shorter than in confined cells.
The rigidity of cell microenvironment is also known to affect actin cytoskeleton architecture, contractility and dynamics. Logically, it also affects ciliogenesis. While very few cells are ciliated when cultured at low density on hard substrates, almost all become ciliated on soft substrates.
Use this link to download the paper dedicated to this work.
THE CENTROSOME, AN INTEGRATION CENTRE
The centrosome seems to play a key role in the definition of cell polarity. It is not only a mechanical linkage between the nucleus and the microtubule network. It is not only a converging point between cell periphery and cell nucleus. It seems to be an integration centre. Signals from different cell regions are collected here. They were spatially segregated at the cell periphery but they meet in this precise location. These signals can now be integrated all together. Here, some resulting activation can be transmitted to the nucleus. An adapted answer can be emitted and send back to the right place. Mechanics and biochemistry have rendez-vous here to discuss how the cell can adapt to various signals coming from different regions in a balanced, univocal and coherent way.