Periodic Reporting for period 4 - ERCOPE (The ER located master regulation of endosomal positioning and further movements)
Período documentado: 2021-03-01 hasta 2022-08-31
The cell is a complex society of different organelles, macro- and microstructures that work in assembly for a viable life. Yet, the organelles are not randomly positions in the cytosol by have in general a defined organization. The endosomal pathway is located in a perinuclear cloud with some endosomes swiftly moving in the cell's periphery. How does the cell control this organization? 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. This is a study to the fundamentals of the cell's life. 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. The ERCOPE program started by the identification that endosomes are not free organelles but in fact controlled by the major intracellular compartment, the endoplasmic reticulum (ER). Using a more holistic cell biological approach in which we considered the endosomes in the context of the whole cell, we aimed 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), signal transduction and the host-pathogen interface.
In the ERCOPE program, we considered how the ER controls the positioning of the endosomal pathway and vice versa. This yielded new biology.
Long distance transport of organelles runs through the action of kinesin- and dynein-based motor proteins that is tightly controlled and can be dramatically remodeled on demand. We defined an ER-associated pathway that establishes and maintains a bilateral architecture of the entire endosomal system, consisting of static perinuclear as well as dynamic peripheral endosomes. Abdication of this spatiotemporal divide attenuates endosomal maturation and delays receptor downregulation (Jongsma et al., Cell 2016). The program considered various aspects of ER control of endosomal positioning.
1. ER-Endosome interactions in endosome positioning, motility and beyond:
We identified the ER-associated E3 ubiquitin ligase RNF26 and its cytosolic substrate SQSTM1/p62 as master controllers of perinuclear endosomal positioning. This work has prompted new investigations on how ER-endosome interactions affect the timing and directionality of endosomal movement. Exploring enzymatic regulation of RNF26 activity, we have shown that deubiquitination of RNF26 substrate SQSTM1 by USP15 liberated perinuclear vesicles for further transport (Jongsma et al., Cell 2016). If there is an E3 enzyme, there also should be an E2 enzyme. We identified UBE2J1 as the E2 enzyme for RNF26. Inactivation of UBE2J1 resulted in the loss of the control of perinuclear endosomes that now scatter and move around the cytosol, a phenotype similar to inactivation of RNF26 (Cremer et al, Cell Rep 2021). Both RNF26 and UBE2J1 are ER embedded cytosolic proteins that then should move around by diffusion. This then would be conflicting with the perinuclear location of RNF26 and by default the endosomal pathway. We performed a BioID experiment to show that the inactive RING domain of RNF26 interact with vimentin, the protein that makes the intermediate filaments. RNF26 is released by activation and then spatially constraint by the interactions between active RNF26 and the different endosomes. Because UBE2J1 is also involved in ER-associated degradation (ERAD), we showed that the close spatial connection between the ER and endosomes are coupled (Cremer et al., EMBO J in revision). This work explains how a diffusible protein complex (RNF26/UBEJ1) still locates in the perinuclear space and why ER and endo-lysosomes have to be closely located.
2. ER master regulator proteins for intraorganellar interactions:
VAP-A and VAP-B are ER proteins that interact with other proteins that have so-called FFAT motifs. As a consequence, virtually any compartment including endosomes and mitochondria directly interacts with the ER in so-called membrane contact sites. We identified another three family members: MOSPD1, 2 and 3. These cluster in two subdomains, one containing VAP-A, VAP-B and MOSPD2 and the other MOSPD1 and 3. By BioID experiments the VAPome was defined; a comprehensive atlas of proteins in different organelles that interact with the ER (Cabukusta et al, Cell Rep 2020). These data are used as a platform for understanding the different biochemical reactions occurring between ER and other compartments.
3. Endosomes and the ER architecture:
If the ER and endosomes interact, the complex ER network is in part be formed moving endosomes. We have shown that moving late endosomes can drag along pieces of ER to alter the ER morphology. Limiting late endosomal movement then results in a simpler ER architecture (Spits et al., EMBO Rep 2021). The ER not only controls endosomes but endosomes also control the ER architecture.
Long distance transport of organelles runs through the action of kinesin- and dynein-based motor proteins that is tightly controlled and can be dramatically remodeled on demand. We defined an ER-associated pathway that establishes and maintains a bilateral architecture of the entire endosomal system, consisting of static perinuclear as well as dynamic peripheral endosomes. Abdication of this spatiotemporal divide attenuates endosomal maturation and delays receptor downregulation (Jongsma et al., Cell 2016). The program considered various aspects of ER control of endosomal positioning.
1. ER-Endosome interactions in endosome positioning, motility and beyond:
We identified the ER-associated E3 ubiquitin ligase RNF26 and its cytosolic substrate SQSTM1/p62 as master controllers of perinuclear endosomal positioning. This work has prompted new investigations on how ER-endosome interactions affect the timing and directionality of endosomal movement. Exploring enzymatic regulation of RNF26 activity, we have shown that deubiquitination of RNF26 substrate SQSTM1 by USP15 liberated perinuclear vesicles for further transport (Jongsma et al., Cell 2016). If there is an E3 enzyme, there also should be an E2 enzyme. We identified UBE2J1 as the E2 enzyme for RNF26. Inactivation of UBE2J1 resulted in the loss of the control of perinuclear endosomes that now scatter and move around the cytosol, a phenotype similar to inactivation of RNF26 (Cremer et al, Cell Rep 2021). Both RNF26 and UBE2J1 are ER embedded cytosolic proteins that then should move around by diffusion. This then would be conflicting with the perinuclear location of RNF26 and by default the endosomal pathway. We performed a BioID experiment to show that the inactive RING domain of RNF26 interact with vimentin, the protein that makes the intermediate filaments. RNF26 is released by activation and then spatially constraint by the interactions between active RNF26 and the different endosomes. Because UBE2J1 is also involved in ER-associated degradation (ERAD), we showed that the close spatial connection between the ER and endosomes are coupled (Cremer et al., EMBO J in revision). This work explains how a diffusible protein complex (RNF26/UBEJ1) still locates in the perinuclear space and why ER and endo-lysosomes have to be closely located.
2. ER master regulator proteins for intraorganellar interactions:
VAP-A and VAP-B are ER proteins that interact with other proteins that have so-called FFAT motifs. As a consequence, virtually any compartment including endosomes and mitochondria directly interacts with the ER in so-called membrane contact sites. We identified another three family members: MOSPD1, 2 and 3. These cluster in two subdomains, one containing VAP-A, VAP-B and MOSPD2 and the other MOSPD1 and 3. By BioID experiments the VAPome was defined; a comprehensive atlas of proteins in different organelles that interact with the ER (Cabukusta et al, Cell Rep 2020). These data are used as a platform for understanding the different biochemical reactions occurring between ER and other compartments.
3. Endosomes and the ER architecture:
If the ER and endosomes interact, the complex ER network is in part be formed moving endosomes. We have shown that moving late endosomes can drag along pieces of ER to alter the ER morphology. Limiting late endosomal movement then results in a simpler ER architecture (Spits et al., EMBO Rep 2021). The ER not only controls endosomes but endosomes also control the ER architecture.
The architecture of cells is a major cell biological question. Why are endosomes and the TGN positioned in a perinuclear region? And what controls the movement of endosomes? The ERCOPE project has uncovered the role of different ER processes in the control of this architecture. The ERCOPE program then provides understanding of this process. In addition, the program showed that this control is bidirectional in the sense that the endosomes also control the ER network. Finally, the ERCOPE program showed that the cell can best be considered as a large organelle-interacting network where metabolite exchange, signaling and many other processes occur in membrane contact sites. This realization provides a different view of the workings of a cell and a basis for a lot of new cell biology.
We have developed chemical biology tools to follow processes that could not be visualized before, such as the retrofusion of intraluminal vesicles in multivesicular bodies. Using this technology, we have been able to show that multivesicular bodies -which are in principle late endosomes- are in fact dynamic structures. Intraluminal vesicles not participating in retrofusion will become exosomes. This indicates that the different intraluminal vesicles in principle are different small organelles, again a concept that is entirely new.
While we have uncovered the basis for the control of the endosomal architecture in cells, the work performed within the ERCOPE program provides also a strong basis for exciting future work. We have started the studies on RNF26 targets that control mitotic processes. We do not understand the processes occurring at the membrane contract sites. The VAPome data will provide a start for understanding these processes. Also, the control endosomal movements in the cell periphery by the ER is another dilemma that requires further understanding. This also holds for the repositioning of the lysosomal system in cells from patients with lysosomal storage diseases as controlled by the ER. How does this work and can this be corrected? And with what type of implications?
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.
We have developed chemical biology tools to follow processes that could not be visualized before, such as the retrofusion of intraluminal vesicles in multivesicular bodies. Using this technology, we have been able to show that multivesicular bodies -which are in principle late endosomes- are in fact dynamic structures. Intraluminal vesicles not participating in retrofusion will become exosomes. This indicates that the different intraluminal vesicles in principle are different small organelles, again a concept that is entirely new.
While we have uncovered the basis for the control of the endosomal architecture in cells, the work performed within the ERCOPE program provides also a strong basis for exciting future work. We have started the studies on RNF26 targets that control mitotic processes. We do not understand the processes occurring at the membrane contract sites. The VAPome data will provide a start for understanding these processes. Also, the control endosomal movements in the cell periphery by the ER is another dilemma that requires further understanding. This also holds for the repositioning of the lysosomal system in cells from patients with lysosomal storage diseases as controlled by the ER. How does this work and can this be corrected? And with what type of implications?
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.