The Laboratory
We are a small, young laboratory interested in the cross-talk of important signalling proteins (PI3K, small GTPases) during complex cellular processes, such as cell migration. We are studying this two model systems, neutrophils and endothelial cells. We are part of the larger Inositide ISP and share space in open plan laboratory and bits of equipment. Moreover, we have weekly, shared laboratory meetings with the other members of the Inositide ISP.
Background to Research
Small GTPases of the Ras superfamily regulate many cellular functions including cell motility, adhesion, vesicular transport, transcription, cell division and survival. The superfamily is grouped into the Ras, Arf, Rho families of small GTPases. All of these have in common, that they are cycling between an active, GTP-bound and an inactive, GDP-bound form, effectively acting as molecular switches. Intrinsic hydrolysis of GTP to GDP is in some cases very slow and is enhanced by GTPase activating proteins (GAPs), whilst guanine nucleotide exchange factors (GEFs) catalyse exchange of GDP for GTP (Fig 1). Only the GTP-bound form of the small GTPase can bind to, and activate, effector proteins. GAPs, like effectors bind to the GTP-bound form of small GTPases. In some cases, a GAP can act as an effector and as a regulator of a small GTPase. Altogether our knowledge of GAPs is still limited. Large numbers of GAPs have been identified but only in few cases do we understand their substrate specificity, mode of regulation and physiological function fully.
Figure 1 (click to enlarge)
Cycling of a small GTPase. Only when GTP-bound can the small GTPase bind to and activate an effector. GAPs aid hydrolysis of GTP for GDP whilst GEFs catalyse exchange of GDP for GTP.
ARAP3 was identified in a screen for phosphoinositide 3-OH kinase (PI3K) effectors, by virtue of its binding to phosphatidylinositol-(3,4,5)-trisphosphate (PtdIns(3,4,5)P3), the lipid second messenger that PI3K produces. ARAP3 is a dual GTPase activating protein (GAP) for Arf6 and RhoA. It belongs to the ARAP family of dual GAPs. The acronym stands for Arf and Rho GAP with Ankyrin repeats and PH domains. All ARAP proteins share their unusual domain structure, comprising an N-terminal SAM domain, five PH domains, an Arf GAP and a Rho GAP domain as well as a poorly conserved Ras association domain.
Analysis of ARAP3’s catalytic activities identified it as a PtdIns(3,4,5)P3-dependent Arf6 GAP and a PI3K and Rap-GTP activated RhoA GAP (Fig 2). Transient-transfection based assays, and RNAi-mediated knock-down of ARAP3 indicated this protein might be involved in the regulation of an aspect of cell motility, such as polarisation or turnover of adhesive structures.
Figure 2 (click to enlarge)
ARAP3 is regulated by the PI3K lipid second messenger PtdIns(3,4,5)P3 by recruitment to the plasma membrane and catalytically as an Arf GAP. In addition, it is activated also by Rap-GTP as a Rho GAP.
Neutrophils
ARAP3 was initially identified from neutrophils, important cells of the innate immune system. Neutrophils reside in the blood stream, but when activated adhere to the vessel wall, extravasate and chemotax to a site of infection, where they produce reactive oxygen species, degranulate and phagocytose intruders. This needs to be tightly regulates, to avoid excessive inflammation whilst ensuring an adequate immune response. We are interested in how ARAP3 regulates these processes, in particular, how does it regulate chemotaxis?
Figure 3 (Click to enlarge)
Neutrophils that did or did not contain ARAP3 were seeded onto glass and allowed to chemotax to a point source of chemoattractant. Cells were time-lapse imaged to visualize cell movement. In the stills shown, the movement of the individual cells was tracked. Control cells moved readily to the micropipette, whilst ARAP3-deficient did not. Stars show the original positions of the micropipette in the control field; this had to be moved to avoid the cells from crawling into the micropipette.
Angiogenesis
Remodelling of an immature vascular network into a mature vascular tree is called angiogenesis. It ensures the adequate supply of oxygen to the tissue and therefore is vital for growth both during embryonic development, and in pathological situations, for example for tumour growth. Angiogenesis is a tightly controlled, complex biological process which requires co-ordination of cell division, differentiation and cell migration (Fig 4). We have recently shown that ARAP3 has a crucial role in developmental angiogenesis, where it signals immediately downstream of PI3K to regulate vessel sprouting. Current work in the laboratory is aimed to characterise cross-talk between PI3K and small GTPases in this vital, but complex process.
Figure 4 (Click to enlarge)
Developing vasculature in the mouse hindbrain at E11.5. Endothelial cells are labelled green.
Updated 24 August, 2011