Endocytosis is the process by which cells control the composition of their cell surface, and thus their communication with the environment, by constantly internalising large amounts of cell surface, recycling it back to the cell surface, storing it inside the cell for various amounts of time, or degrading it in lysosomes. Endocytosis employs various mechanisms of internalisation, and utilises a number of different downstream organelles for the processing of internalized material. Given its essential role in cellular signalling and homeostasis, endocytosis is under extensive control by and continuously adapted to the physiological state of a cell.

While an exhaustive amount of knowledge has been gathered on molecular mechanisms of endocytosis in many cell types from various model organisms, little is known about how endocytosis integrates with cellular physiology . Also, how function emerges from the dynamic collective behaviour of large collections of vesicles and endosomes in single cells and in particular the role of cell-to-cell variability in this behaviour is unclear . A quantitative analysis of how this variability emerges and is controlled will be crucial to understand how endocytosis is adapted to the physiological state of a cell. To study these properties of the endocytic membrane system, we have been one of the first to apply systems biology approaches to endocytosis in mammalian cells (Pelkmans, Fava, Grabner, Hannus, Habermann, Krausz, Zerial, 2005), and we continue to further develop this approach.

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In a recent study, we report a novel approach to map functional regulatory interactions of a complex cellular system, applied to endocytosis. It uses 13 image-based RNAi screens of endocytic activities and organelles (browsable on endocytome.org) to collect a set of orthogonal readouts on the endocytic membrane system upon perturbing a pre-selected set of genes that include both potential regulators and core machinery components. Accounting for population context in single cells is performed to allow cross-comparison of the readouts. From this dataset, numerous unknown systems properties of the endocytic membrane system emerged, such as how complexity evolved and how sets of endocytic activities and organelle abundances are co-regulated by regulatory programs in the cell. Furthermore, by using a recently developed statistical method in the lab, termed the hierarchical interaction score (HIS), which harnesses subset effects in the data, we were able to infer, at a large-scale, regulatory functional interactions between genes involved in signalling, membrane trafficking, and the cytoskeleton. An interactive map of these interactions can be found here. Intriguingly, these hierarchical interactions were enriched in several types of interaction motifs, such as Fan-In, Fan-Out, Cascade, Feedback, and Feedforward motifs, suggesting the existence of generic regulatory principles in the endocytic membrane system. These motifs revealed a common use of integrating actin and membrane trafficking machinery to downstream kinases as well as the integration of two upstream kinases to downstream membrane trafficking machinery (Fan-In), of tyrosine kinases being upstream of both endomembrane and plasma membrane (Fan-Out), of plasma membrane proteins interacting via protein kinases with the Golgi complex, which in turn interact with the plasma membrane (Feedback), and of tyrosine kinases being both directly upstream of actin cytoskeleton components, as well as indirectly via endosome components, integrating these two annotation groups (Feedforward).

 

Current questions:

  • Can we develop approaches for automated long-term timelapse imaging of endocytic vesicles and organelles in large numbers of single cells to study dynamics of endocytosis at long timescales (hours-days) but with a time-resolution that allows single vesicle tracking (100 ms)?
  • What is the role of the cell cycle in determining cell-to-cell variability in endocytosis?
  • To which extent does the complexity of internalisation, recycling, and degradation pathways manifest itself within single cells, or across a population of cells, in which specific subsets of cells only display part of this complexity?
  • How does the directionality of hierarchical functional interactions in the endocytic membrane system relate to biochemical directionality?
  • Which systems properties do hierarchical interaction motifs give to the endocytic membrane system, and what are their roles in molecular terms?

 

Current lab members involved:

Prisca Liberali Yanic Heer Gabriele Gut

 

Some relevant publications from the lab:

1.
Pelkmans, L. et al. Genome-wide analysis of human kinases in clathrin- and caveolae/raft-mediated endocytosis. Nature 436, 78–86 (2005).