With the goal of examining regulation of enzymes in the complex cellular environment, we continuously develop optical and biochemical reporters, suitable for monitoring and manipulating the localization and activity of enzymes in live cells. We actively employ fluorescence lifetime imaging microscopy (FLIM), fluorescence correlation- and cross-correlation spectroscopy (FCS/FCCS), chemical and optogenetic probes and computer simulations to gain insight into operation and regulation of enzymes in cells.

Protein kinase B, also known as Akt is a central regulator of cellular metabolism, survival and proliferation. It integrates signals from cell surface receptors coupled to PI3 kinase and links them to downstream signalling pathways by phosphorylating multiple (>200) substrates at a conserved motif. When activity of Akt is not properly controlled, this could lead to uncontrolled cell division and enhanced survival, as observed in many human cancers.

Using a combination of imaging, biochemical reporters and in vitro studies with purified Akt, we characterize cellular and molecular mechanisms which restrain Akt activation and control its substrate specificity in cells.

Mechanistic target of rapamycin (mTOR) couples signalling from growth factor receptors with intracellular nutrient-sensing pathways, and thereby is a critical regulator of cellular anabolic functions, growth, survival and proliferation. In eukaryotic cells, mTOR exists in two distinct multisubunit complexes, mTORC1 and mTORC1, which have non-overlapping functions in cellular physiology. Specifically, mTORC2 is implicated in the control of cell proliferation, survival, cytoskeletal rearrangements and, indirectly, in regulation of mTORC1-controlled signalling.

Unlike mTORC1, little is known about localization and regulation of mTORC2. We use combination of superresolution imaging and specifically designed biochemical reporters to examine localization and regulation of mTORC2 activity inside cells.

T cell receptor complex (TcR/CD3) mediates physiological responses of lymphocytes to antigenic stimulation. Antigen binding induces phosphorylation of the cytoplasmic portion of the TcR/CD3 and subsequent recruitment and activation of the cytosolic kinase ZAP-70. Activated ZAP-70 in turn phosphorylates scaffold proteins LAT and SLP-76 resulting in recruitment of downstream effectors, which link TcR/CD3 activation with cytokine secretion, cytoskeletal rearrangement, mitogenic signalling and gene expression.

Similar to other transmembrane receptors, TcR/CD3 is constitutively internalised and recycled to the plasma membrane in T cells, although the exact role of its constant trafficking inside cells is unclear. We use imaging and biochemical approaches to investigate TcR/CD3 signalling from subcellular compartments and examine its role in T cell physiology.