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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - ERCOPE (The ER located master regulation of endosomal positioning and further movements)

Teaser

Summary

The overarching focus of the ERCOPE program seeks to answer why and how do membrane compartments reside at defined sites in cells, and what allows them to become mobile in response to cellular demand. The cell biology of endosomes involves many transport and fusion steps necessary to enable delivery of proteins and other cell components to their correct destination. Failures in endosomal transport have profound consequences for the health of the organism, resulting in diverse malfunctions that range from neuronal diseases to obstructed immune responses. In the ERCOPE program, we aim to study how cells determine and manipulate the location of their dynamic vesicular systems in molecular terms, and what ramifications loss of such information poses to organelle biology, cellular order integrity (under steady state and in dividing cells) and the host-pathogen interface.

Work performed

Endosome maturation in the perinuclear cloud:
Long distance transport of organelles runs through the action of kinesin- and dynein-based motor protein complexes in a system whose organization is tightly controlled and can be dramatically remodeled on demand. We have recently found that an ER-associated pathway helps to establish and maintain a bilateral architecture of the entire endosomal system, consisting of relatively immobile perinuclear endosomes and highly dynamic peripheral endosomes. Abdication of this spatiotemporal divide attenuates endosomal maturation and delays receptor downregulation, as reported in Jongsma and Berlin et al., Cell 2016. Simultaneously, we found that autophagosome transport and maturation are also subject to ER-endosome contacts Wijdeven et al., Nat Commun 2016. Taken together with recent developments in the field, these studies imply that spatiotemporal regulation of the endolysosomal system by the ER streamlines the core physiological processes attributable to the former organelle. At present, we are zooming into the membrane dynamics within individual endosomes/lysosomes to understand how key ER-interacting proteins become exposed to the limiting membranes of these organelles. We have established a system to visualize back fusion or â€˜retrofusionâ€™ of intraluminal membranes in living cells and are exploring how this process is connected to exosome biology.

ER-Endosome interactions in endosome positioning, motility and beyond:
In Jongsma and Berlin et al., Cell 2016 we describe a mechanism of endosomal control by the ER-associated RNF26 and its cytosolic substrate SQSTM1/p62, which together attract specific endosomal adaptor proteins to form a molecular link between the ER and endosomes. Additionally, in a DUB depletion screen we identify USP32 as a potent regulator of endosome architecture and dynamics through deubiquitination of Rab7 Sapmaz and Berlin et al., Nat Commun 2019. This work has prompted new investigations on how ER-endosome interactions affect the timing and directionality of endosomal movement, including other modes of Rab7 regulation.
Exploring enzymatic regulation of RNF26 activity, we have shown that deubiquitination of RNF26 substrate SQSTM1 by USP15 liberates positioned vesicles for further transport (Jongsma and Berlin et al., Cell 2016). We now identified UBE2J1 as the ER-located E2 enzyme for RNF26. Because UBE2J1 is also involved in ER-associated degradation (ERAD), we are pursuing whether endosomal/autophagosomal positioning and ERAD are linked processes, and why.
As part of this work we are also probing the existence of a perinuclear endosome-interacting microdomain in the ER (particularly under ER stress conditions), and undertaking a systems-wide effort to define and characterize the cellular VAP-omeâ€”an interactome of VAP family ER-anchor proteins involved in the formation of membrane contact sites with other organelles in the cell.
Lastly, we are exploring the importance of these molecular insights at the host-pathogen interface. Here we are studying the impact of phagosome positioning on the intracellular lifestyle of Salmonella. This work is being further extended toward clinically relevant questions on the topic of bacteria and cancer (Mughini-Gras et al., PLoS One 2018; van Elsland and Neefjes, EMBO Rep 2018).

Final results

It is expected that the ERCOPE program will result in fruitful explorations of new and exciting directions, as well as a number of influential publications, in the coming years.
ER-Endosome interactions and mechanisms of endosome positioning and motility
Over the years we have built an extensive toolbox and acquired invaluable insights into various modes of ER-endosome interactions and how these affect endosomal location and motility determinants. A number of manuscripts are currently being prepared on these topics (see section above). Future work will focus on deciphering how positional information and directionality of vehicle movement following release from the perinuclear cloud is integrated at membrane contact sites between endosomes and the ER. Ultimately, these insights will be considered from the perspective of dividing cells as well as those confronted with a pathogen challenge to understand how cells reorganize themselves to attain ever changing functional states.
Retrofusion and exosome biology
We are currently preparing a manuscript on the retrofusion of intraluminal membranes, which we demonstrate for the first time to constitute part of the multivesicular body\'s normal lifecycle. Moving forward we will explore how this process influences exosome biology and transmission of materials, including infectious agents, between cells.

Collectively, these studies will give rise to new paradigms of how crucial physiological processes are controlled by cells at the molecular level, which will in turn provide new ideas for intervention into misregulated cellular states.