Systems analysis of endocytosis
Our previous studies of Rab5 and endosomal fusion have provided information on the core molecular machinery underlying endosome biogenesis. This project aims at studying the endosomal network in a broader, systems biology context, since endocytosis is an essential process connected to multiple key cellular functions.
Systems survey of endocytosis by quantitative multi-parametric image analysis
We combined genome-wide RNAi, automated high-resolution confocal microscopy, quantitative multi-parametric image analysis and high-performance computing to accurately and quantitatively profile the activity of human genes with respect to Transferrin (Tfn) and Epidermal Growth Factor (EGF) endocytosis (Collinet et al., 2010). The screen identified novel regulators of endocytosis and endosome trafficking, including genes implicated in metabolism and signalling pathways (see: http://endosomics.mpi-cbg.de). A systems analysis by Bayesian networks revealed design principles for how the endosomal network operates. This systems analysis revealed that cells regulate the number, size, concentration of cargo and intracellular position of endosomes, thus uncovering novel properties of the endocytic system. Importantly, the screen yielded hypotheses and predictions regarding the role of pathways and quantitative parameters in the endosomal machinery that are now being tested experimentally.
A. Example of a three-colour high-resolution image collected with the automated spinning disk confocal OPERA microscope. Nuclei and cytoplasm are pseudo-coloured in blue, Alexa-488-labelled EGF is pseudo-coloured in red and Alexa-647-labelled Tfn is pseudo-coloured in green. B. Close-ups show the model structure of individual endosomes (see QMPIA LINK). C. Selection of quantitative parameters used to measure endosomal features.
Bayesian networks of the endocytic parameters for the genome-wide analysis of EGF and TF endocytosis. The nodes represent individual endocytic parameters measuring EGF or TF endocytosis and the numbers indicate the Pearson correlation values. In black the direct correlations shared by the two networks, in red (present) and grey (absent) correlations that present differences between the two networks.
A cut-out switch” regulates Rab5-Rab7 conversion
The Rab5-to-Rab7 conversion was modelled (in collaboration with Andreas Deutsch, TUD) as a cascade of functional modules of interacting Rab GTPases (Del Conte-Zerial et al., 2008). The inter-module interactions share similarities with the toggle switch described for the cell cycle. However, the Rab5-to-Rab7 conversion is rather based on a newly characterised cut-out switch analogous to an electrical safety-breaker. Both designs require cooperativity of auto-activation loops when coupled to a large pool of cytoplasmic proteins, but the cut-out switch is irreversible. The cut-out switch design could be a widespread engineering principle for Rab GTPases functionally linked in a pathway. In addition, it may underlie the integration of modules in regulatory cascades from a broad range of biological processes. The model predicted that a negative regulator of a Rab5 GEF or a Rab5 GAP would lead Rab7 to repress Rab5. The model has recently been supported by the identification of SAND-1/Mon1, a regulator of the localization of Rab5 and Rab7 GEFs (Poteryaev et al., Cell 2010).
During Rab5-to-Rab7 conversion, Rab5 can be switched off as a result of enhanced (e.g. cargo-dependent, pH-dependent) activation of Rab5, as Rab5 fosters Rab7 activity (upper green arrow) to pass a threshold above which Rab7 self-sustains (right green arrow) its own activation. Rab7 activation at supra-threshold values strongly suppresses Rab5 (red arrow) (adapted from Del Conte-Zerial et al., 2008).
The experimentally observed transition between two quasi steady states of Rab domains requires bistability and a parameter change across one of the hysteresis thresholds. During Rab5-to-Rab7 conversion, when the parameter passes the threshold value, the Rab5 shall switch from the high- to the low-density state, and Rab7 from the low- to the high-density state.
A general theoretical framework to infer endosomal network dynamics from quantitative image analysis
Early endosomes form a dynamic network of membranes undergoing fusion and fission, thereby continuously exchanging and redistributing cargo molecules. Parameters such as size, cargo content and intracellular position of endosomes are characteristic of the overall function of the network in cargo distribution and degradation (Systems survey of endocytosis by quantitative multi-parametric image analysis). In collaboration with the groups of Frank Juelicher (MPI-PKS) and Andreas Deutsch, TUD), we developed a new theory based on quantitative experimental data to study how the macroscopic kinetic properties of the endosomal network as a whole result from the collective behaviour of many individual endosomes (Foret et al., 2012) . We demonstrated that the theory can quantitatively describe the distributions of endocytosed cargo as a function of time. Remarkably, the theory allows the determination of microscopic kinetic parameters, such as the fusion rate between endosomes from still images of cargo distributions at different times of internalization. We are currently planning to extend this theoretical approach to the development of mathematical models that can describe the distribution of signaling cargo in endosomes on the basis of network parameters such as endosome fusion, fission, cargo concentration and degradation. We also plan to exploit information theory (cfAED) to elucidate the mechanisms underlying signal propagation in the cell, learning the design principles of the cellular computer from an engineering point of view.
The early endosomal network is structured as a funnel: The cell periphery contains many small Rab5-positive early endosomes that progressively fuse homotypically and move to the cell center. In the cell center, there are few large early endosomes that eventually undergo conversion into Rab7-positive late endosomes. Rab4 and Rab11-positive tubules remove recycling cargo back to the surface, whereas cargo destined for degradation progressively accumulates in the large endosomes. The shape of size distribution of the endosomal network primarily depends on two main processes, 1) homotypic fusion which reduces the number of endosomes and 2) homotypic fission which increases the number.
Genome-wide RNAi screen in C.elegans for genes maintaining epithelial membrane traffic and cell polarity
The structure and function of several organs such as the liver, kidney and intestine depend on the ability of their cells to polarize, i.e. to form distinct plasma membrane domains facing the blood or the external environment. To elucidate the regulatory networks that couple the organisation of the membrane traffic system to the establishment and maintenance of cell polarity, we performed a genomic RNAi screen in the C. elegans intestine (Winter et al., 2012). The screen uncovered novel candidate genes required for the maintenance of apical membrane transport and the positioning of apical recycling endosomes (AREs), and revealed an unexpected link between apical ARE enrichment and apico-basal F-actin asymmetry. These genes are likely to play an important role in all epithelial cells, including the liver hepatocytes. We are planning to explore the function of these genes in the context of liver physiology and clinically-relevant models of liver dysfunction.