13 October 2014
By Alena Pance
Electron microscopy image of a clathrin-coated vesicle at the surface of the cell. Credit: Tony Jackson
The ability of a cell to communicate with its environment is crucial for its survival. One of the proteins involved in this process is clathrin.
This molecule forms little baskets on the plasma membrane that pinch off in the form of bubbles called coated vesicles which allow messengers from the outside into the cell. In order to understand how clathrin works, we needed to create cells that lack this vital protein.
However, clathrin is essential for the cell, so Tony Jackson’s group in Biochemistry at Cambridge generated a B-lymphocyte cell line in which clathrin can be turned off at will. This way, the cells can be happily maintained expressing clathrin and their behaviour can be observed when the off signal is triggered.
Upon clathrin deletion cells died rapidly, as expected, providing a system where the role of this protein could be assessed. However, growth differences became apparent whereby the cells were more or less sensitive to clathrin depletion. This meant that on occasions some cells were not dying as we expected.
Finding an answer to this problem was a lengthy process of elimination that eventually took us to testing the chicken serum that needs to be included in the culture medium. The differences in growth and in the response to the absence of clathrin were directly linked with particular serum batches. What was the difference between the batches of chicken sera?
While looking into this, we found that when we increased the concentration of our chicken serum in the medium, the cells survived better in the absence of clathrin. A biochemical analysis led to the identification of transferrin as the protein in the chicken serum that is responsible for cell survival. This protein captures iron from the medium and transports it into the cell by binding the transferrin receptor on the plasma membrane. The complex containing the iron is then internalised by clathrin-coated vesicles.
Cells resistant to iron deprivation express SDF-1, activating CXCR4. This stimulates the cellular pathways that strengthen cell survival and overcome cell death. Credit: Tony Jackson and Alena Pance
Iron is vital for cells because it is a key cofactor for the synthesis of DNA and generation of energy in the cell. This means that cells that propagate rapidly such as white blood cells or some cancers are heavily dependent on iron. In this context, the use of drugs or strategies to interfere with iron metabolism has proven an effective method to tackle these malignancies and a number of these therapeutic strategies are already on trial.
While playing with the culture conditions and clathrin expression, we noticed that some cells were more resistant to the iron deprivation caused by clathrin depletion than others. These cells were stable and could be isolated, expanded and kept in culture. In order to investigate the underlying processes of resistance, gene expression in the sensitive and resistant cell lines was compared using RNA microarrays through a collaboration with the Roslin Institute.
Since both cell lines derive from the same parental line, only a handful of genes are differentially expressed. Of these, a particular protein: CXCR4, drew our attention because it is a plasma membrane receptor that can activate survival and proliferation pathways in the cell. Furthermore, we found that the ligand for CXCR4, called SDF-1, is synthesised by the resistant cells but not by the sensitive ones. In fact SDF-1 is normally not expressed in immune cells but released in immunological niches such as the bone marrow to attract white blood cells expressing CXCR4.
We conclude that expression of SDF-1 by the resistant cells activates CXCR4 thereby stimulating cellular pathways that strengthen survival and overcome cell death in response to iron deficiency.
In fact the up-regulation of the CXCR4-SDF1 survival pathway has been reported in many human cancers and particularly in leukaemia. Our work led us, in an unsuspected way, to describe a mechanism of survival that can allow cells to escape from anti-cancer therapies, highlighting its components as important factors in the resistance to chemotherapy.
Yet another example of science driven by the unexpected.
Alena Pance is a staff scientist in the Malaria Programme, where she works with Julian Rayner to develop a stem cell-based system to study the host component of malaria infection. The work described in this blog was conducted in collaboration with Tony Jackson, senior lecturer at the University of Cambridge's Department of Biochemistry.
- Pance, A et al (2014). SDF-1 Chemokine Signalling Modulates the Apoptotic Responses to Iron Deprivation of Clathrin-Depleted DT40 Cells. PLOS ONE. DOI: 10.1371/journal.pone.0106278