Using a combination of optical microscopy techniques including widefield phase contrast/fluorescence time-lapse, LS confocal imaging, FRAP, TIRF, FRET and FLIM, the work of our group examines the dynamics of key proteins involved in forming and dissociating podosomes and invadopodia in living and migrating cells.
Podosomes are actin-rich adhesive structures found in myeloid cells. Red: f-actin core, also contains WASP and WIP. Green: vinculin as a ring component, also contains paxillin and talin with membrane integrins.
In collaboration with Dr Susan Cox (Imaging Section) we have also exploited the power of 3B image analysis to explore the kinetics of protein distribution within podosomes. I shall mention two of the projects ongoing in the laboratory at present.
Lizzy Foxall is studying the function of the serine-threonine kinase PAK4 in controlling podosome formation within macrophages. In collaboration with Dr Claire Wells (Cancer Division) we have identified that PAK4 resides in the outer ring of a podosome, its distribution coinciding with that of vinculin. Although conventional LS confocal imaging identified PAK4 as a component of podosomes, the differential localisation to the ring was most clearly demonstrated using 3B analysis.
Two forming podosomes within a larger cluster at the leading edge of a migrating THP-1 macrophage cell expressing eGFP-PAK4 and stained for vinculin. Our 3B analytical software clearly identifies PAK4 residing in the vinculin-rich ring of each podosome.
Lizzy is currently using a suite of in-house PAK4 inhibitors to test their effects on podosome formation.
She has also discovered that PAK4 interacts directly with vinculin and paxillin (another podosome ring protein). Work is in progress to identify a phosphorylation event or a RhoU-mediated action for PAK4 in the podosome complex.
Nisha Rafiq is studying how podosomes originate at the membrane-matrix interface of migrating cells. She has evidence that a novel ARNO-ARF-1 signalling cascade directs podosome initiation at sites of podosome development. She has shown that siRNA-mediated knockdown of ARF-1 abrogates the ability of macrophages to form podosomes.
We also found that the ARF-1 GEF known as ARNO or cytohesin-2 localises to the ring of forming podosomes.
We are currently undertaking a series of molecular and imaging experiments to explore the link between these two findings.