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Adaptive character of responses to signals from the environment is a fundamental property of all living organisms. At the cellular level, it is brought about by a highly integrated process of transmembrane and intracellular signal transduction. Although many signaling molecules have been identified, how exactly the dynamics of their interactions ensure the ultimate specificity and adaptive character of cellular responses remains poorly understood. How changes in flux of substrates via an enzyme affects signalling specificity? How intracellular localization of a protein is coupled to its signaling role? What is the function of a given protein in network regulation?

To address these questions, we employ a combination of biochemical and advanced imaging techniques. We are developing tools to visualize the functional state of signaling molecules in live cells to determine how spatial distribution, regulation of specific activity and the corresponding changes in the flux of substrates via individual enzymes determine their signaling function. Furthermore, to examine the physiological relevance of individual enzymes in signaling, we will be developing new tools to selectively manipulate their localization and activity in live cells.

RESEARCH HIGHLIGHT:
The new model of Akt activation in cells

In the recent paper in the journal Molecular Cell, Ebner et al show that binding to the membrane lipid PI(3,4,5)P3 allosterically activates Akt kinase by relieving an intramolecular autoinhibitory mechanism. Ebner et al further demonstrate that in cells active Akt is exclusively associated with PI(3,4,5)P3-containing cellular membranes, ensuring that Akt activity remains proportional to stimuli upstream of PI(3,4,5)P3.

Full text     PDF     PDF + Supplemental Materials

The constructs used in the publication are now available via AddGene

The primary data related to the publication are available at FigShare

Development of reporters to examine signaling dynamics in live cells

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.

Cellular regulatory mechanisms of protein kinase Akt

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.

Intracellular localization and regulation of mTOR complex 2

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.

Dynamics of T cell receptor signaling

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.

RESEARCH HIGHLIGHT:
Localization and regulation of the endogenous mTORC2 in cells

In the recent paper in the Journal of Cell Biology, Ebner et al report development of a reporter for the endogenous activity of mTORC2 complex on various cellular compartments and use it to examine how mTORC2 activity is regulated by PI3 kinase.

Full text     PDF     PDF + Supplemental Materials

The primary data related to the publication are available at FigShare

The constructs used in the paper are now available via AddGene (plasmids #72899-72909)

Resources

Primary data

References to primary data for published articles on FigShare:
JCB LocaTOR2 paper
Molecular Cell Akt paper

Protocols

Protocols and references

Materials

References to Yudushkin lab plasmids deposited at AddGene:
LocaTOR2 JCB paper
Mol Cell paper constructs
TcR reporters PNAS paper

Contact

Department of Structural and Computational Biology
Max F. Perutz Laboratories (MFPL)
Department of Medical Biochemistry
Medical University of Vienna

Vienna Biocenter, Campus Vienna Biocenter 5, Rm. 1.624
1030 Vienna AUSTRIA

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