Nadia Elkhatib and colleagues have provided evidence to explain why cells in 3D show fewer integrin rich focal adhesions compared to cells on flat surfaces.
They propose a new role for clathrin/AP-2 lattice tubes in holding on to collagen fibres, which allows the cells to pull themselves through pores in a 3D matrix. This study suggests a new role for an old player in a form of cell movement known as mesenchymal migration: the dominant type of migration in many cells including metastatic cancers.
In a physiological structure the environments that cells generally move through is not fluid and clear, it is a tangle of fibres akin to a dense patch of blackberry bushes. These fibres are the extracellular matrix (ECM), a group of proteins that fill in the spaces between the cells. Cells can’t freely move through these tangles, but need to hold on to the fibres and pull themselves through gaps, or use special proteins to break down the tangles and make gaps themselves.
It has been shown previously that cells concentrate small areas of their cytoskeleton in to protrusions that deliver particular proteins known as integrins (which are specially adapted to bind to these ECM proteins) to the membrane of the cell, which help the cell attach to the ECM and create the pulling motion. These areas are called focal adhesions (FA).
Traditionally, migrating cells have been studied in a 2 dimensional environment such as on a coated plastic or glass surface. In these conditions many strong FAs can be seen. However, in more physiologically relevant 3 dimensional matrices, these FAs are smaller, fewer and weaker, and tend to predominate in the leading edge of the migrating cells. This phenomenon led the researchers to investigate other areas of the plasma membrane that have previously been shown to contain concentrations of integrins, specifically clathrin-coated structures (CCSs). CCSs are special structures on the membrane that involve several proteins including clathrin and adaptor protein 2 (AP2) to create a lattice cage at the membrane, forcing it to curve inwards and create a parcel of membrane bound structures to be delivered into the cell for further processing. This process in known as endocytosis and is key to signalling processes resulting in many functional endpoints including cell movement, division and death.
Here it is seen that the CCSs aligned along the collagen fibres in 3D. Using a mixture of spinning disk confocal and electron microscopy it was established that the cells created these CCS lattice structures around the collagen fibres in response to the bending of the plasma membrane upon contact with the stiff collagen fibres, following integrin activation at the sites. In the electron microscope images it is possible to visualise the tubular construction of the lattices, which wrap around and pinch the collagen fibres.
Here, it wasn’t actually clathrin itself but another protein called AP-2, which links the membrane bound protein to the clathrin cage during endocytosis. By disrupting the formation of these CCS lattice structures by depleting the cells of AP-2 the researchers were able to determine that this protein was the key to their creation.
The researchers conclude that these lattice tubes are anchoring the cell body to the ECM fibres, maintaining tension in the cells in order to counterbalance the high tension created by the FAs in the cell protrusions. They stabilise the system and allow for the growth of these long FA rich protrusions, allowing the whole cell to squeeze through the ECM.
This study proposes a role for CCSs beyond the traditional endocytic processes in cellular migration in 3D. It also tries to explain the reduction in FAs seen outside of the cell protrusions in cells in a 3D environment versus those seen on 2D surfaces. Lastly, it shows the importance of developing physiologically relevant tools to investigate these cellular processes, as researchers are frequently reporting these significant differences between results seen in 2D versus more complex models.
Read the full paper here
Written by Brooke Lumicisi