Final Report Summary - ENDOCYTIC REGULATORS (A comprehensive analysis of essential protein regulators of clathrin-mediated endocytic pathway)
Clathrin-mediated endocytosis is a fundamental cellular process responsible for the uptake of nutrients and signals and for the maintenance of cell-limiting plasma membrane of every eukaryotic cell. It represents a primary entry route for many physiological molecules and stimuli, but also for multiple non-physiological and potentially deleterious agents like viruses and toxins, into the cell. To get a profound understanding of endocytic mechanisms is thus a critical task for current molecular cell biology in order to supply this knowledge to other biomedical disciplines and for potential therapeutic applications.
Though many important features of conserved endocytic process have been elucidated, several crucial aspects still await to be understood. Two of them were successfully investigated in the course of this project. At first, in accordance with the original proposal, we sought to characterise in yeast Saccharomyces cerevisie the functions of several essential endocytic proteins, which are difficult to study in other model organisms. To this end we depleted essential endocytic epsins Ent1 and Ent2, Eps15 homolog Pan1, protein Scd5, and ubiquitin ligase Rsp5 using transcriptional knock-down system (TetOn/Off) and followed the dynamics of endocytosis by a real-time fluorescence microscopy. The striking phenotype of epsin depletion gave us then a great opportunity to contribute to one of the main objectives of the current endocytic research, the characterisation of the role of actin cytoskeleton in this process.
The function of actin in endocytosis is under an intense research as it has recently become clear that the actin cytoskeleton is a key effector to reshape/remodel the plasma membrane when the endocytic proteins alone cannot provide enough energy for vesicle budding. In our project we described the underlying molecular mechanism of the coupling between the actin cytoskeleton and the membrane of the forming endocytic vesicle. This coupling is needed for transmitting force from the actin cytoskeleton to the budding vesicle. Understanding the coupling mechanism is essential for understanding how actin functions during endocytosis.
To transmit the energy of the actin cytoskeleton to the plasma membrane these two structures need to be coupled physically. Endocytic proteins of the Sla2/HIP1R family have been suggested to participate in this process as (i) they can bind both the membrane and actin and (ii) in their absence the endocytic vesicle budding is blocked with endocytic structures assembled on the membrane and accompanied by actin. However, endocytosis in yeast does not rely on the actin-binding properties of Sla2 protein. In the course of our depletion experiments we observed that similar endocytic block is induced in the absence of endocytic epsins Ent1 and Ent2. Epsin can also bind the membrane, but are not known to bind actin, leaving their role in membrane-actin coupling also unclear.
To reveal the roles of Sla2 and Ent1/2 in membrane-actin coupling we fluorescently-labeled these proteins and followed them in living yeast cells by a real-time two-color fluorescence microscopy. To detect their binding to membrane, actin and to each other we employed in vitro biochemical assays. Finally, using advanced tools of yeast genetics we created several mutated, truncated and chimeric versions of both proteins to corroborate their roles in the coupling process in vivo.
We showed that yeast epsin protein Ent1 and HIP1R homolog Sla2 work together to couple the plasma membrane and the actin cytoskeleton. If this link is disrupted actin cytoskeleton still polymerises at the endocytic site but membrane invagination and vesicle budding are inhibited. We found that the lipid-binding domains of Ent1 and Sla2 proteins (ENTH and ANTH domain, respectively) interact with each other in a ligand-dependent manner in order to bind the membrane cooperatively. While the lipid-binding domains of Ent1 and Sla2 are well known, the synergism in their membrane binding is novel and an unexpected feature of their function. We propose that the synergistic membrane binding leads to tight anchoring of Ent1 and Sla2 proteins to the membrane so that they can withstand (and therefore transmit) pulling forces from the actin cytoskeleton.
We next identified a novel actin-binding domain in epsin Ent1 and showed that this domain functions in parallel with Sla2's actin-binding domain to couple the actin cytoskeleton to the endocytic structures. Unlike the synergistic action of Ent1's and Sla2's membrane-binding domains, the actin-binding domains of these two proteins function largely in a redundant manner, providing a dynamic but robust coupling to the actin cytoskeleton. We also reveal that the interaction of epsin Ent1 with the actin cytoskeleton is negatively regulated by the phosphorylation of Prk1 kinase (homologous to human AAK). This is in a good agreement with the proposed role of Prk1 to dismantle protein-protein interactions of endocytic factors after the vesicle scission/during vesicle uncoating.
Hence, by synergistic binding to the membrane and redundant interaction with the actin cytoskeleton, Ent1 and Sla2 proteins form an essential molecular linker that transmits the pulling force from the actin cytoskeleton to the plasma membrane, leading to membrane invagination and endocytic vesicle budding.
Our study provides a molecular mechanism for energy transfer between the actin cytoskeleton and the plasma membrane of the endocytic site, which is probably essential for endocytic process in many eukaryotic organisms. The mechanism employs an unprecedented cooperative action between membrane-binding domains of two fundamental endocytic proteins epsin and Sla2/HIP1R and their redundant interaction with the actin cytoskeleton. We believe that similar mechanism (or its parts) could also be the basis for other membrane-remodelling processes, which occurs in eukaryotic cells and requires polymerising actin cytoskeleton. Our results will thus be of a great interest for many researchers working in fields of membrane traffic, cell signalling, cell morphogenesis, polarisation, differentiation etc. To this end our data could represent an important knowledgebase for many forthcoming research projects in several research fields and could potentially be a starting point for many biomedical applications.
Though many important features of conserved endocytic process have been elucidated, several crucial aspects still await to be understood. Two of them were successfully investigated in the course of this project. At first, in accordance with the original proposal, we sought to characterise in yeast Saccharomyces cerevisie the functions of several essential endocytic proteins, which are difficult to study in other model organisms. To this end we depleted essential endocytic epsins Ent1 and Ent2, Eps15 homolog Pan1, protein Scd5, and ubiquitin ligase Rsp5 using transcriptional knock-down system (TetOn/Off) and followed the dynamics of endocytosis by a real-time fluorescence microscopy. The striking phenotype of epsin depletion gave us then a great opportunity to contribute to one of the main objectives of the current endocytic research, the characterisation of the role of actin cytoskeleton in this process.
The function of actin in endocytosis is under an intense research as it has recently become clear that the actin cytoskeleton is a key effector to reshape/remodel the plasma membrane when the endocytic proteins alone cannot provide enough energy for vesicle budding. In our project we described the underlying molecular mechanism of the coupling between the actin cytoskeleton and the membrane of the forming endocytic vesicle. This coupling is needed for transmitting force from the actin cytoskeleton to the budding vesicle. Understanding the coupling mechanism is essential for understanding how actin functions during endocytosis.
To transmit the energy of the actin cytoskeleton to the plasma membrane these two structures need to be coupled physically. Endocytic proteins of the Sla2/HIP1R family have been suggested to participate in this process as (i) they can bind both the membrane and actin and (ii) in their absence the endocytic vesicle budding is blocked with endocytic structures assembled on the membrane and accompanied by actin. However, endocytosis in yeast does not rely on the actin-binding properties of Sla2 protein. In the course of our depletion experiments we observed that similar endocytic block is induced in the absence of endocytic epsins Ent1 and Ent2. Epsin can also bind the membrane, but are not known to bind actin, leaving their role in membrane-actin coupling also unclear.
To reveal the roles of Sla2 and Ent1/2 in membrane-actin coupling we fluorescently-labeled these proteins and followed them in living yeast cells by a real-time two-color fluorescence microscopy. To detect their binding to membrane, actin and to each other we employed in vitro biochemical assays. Finally, using advanced tools of yeast genetics we created several mutated, truncated and chimeric versions of both proteins to corroborate their roles in the coupling process in vivo.
We showed that yeast epsin protein Ent1 and HIP1R homolog Sla2 work together to couple the plasma membrane and the actin cytoskeleton. If this link is disrupted actin cytoskeleton still polymerises at the endocytic site but membrane invagination and vesicle budding are inhibited. We found that the lipid-binding domains of Ent1 and Sla2 proteins (ENTH and ANTH domain, respectively) interact with each other in a ligand-dependent manner in order to bind the membrane cooperatively. While the lipid-binding domains of Ent1 and Sla2 are well known, the synergism in their membrane binding is novel and an unexpected feature of their function. We propose that the synergistic membrane binding leads to tight anchoring of Ent1 and Sla2 proteins to the membrane so that they can withstand (and therefore transmit) pulling forces from the actin cytoskeleton.
We next identified a novel actin-binding domain in epsin Ent1 and showed that this domain functions in parallel with Sla2's actin-binding domain to couple the actin cytoskeleton to the endocytic structures. Unlike the synergistic action of Ent1's and Sla2's membrane-binding domains, the actin-binding domains of these two proteins function largely in a redundant manner, providing a dynamic but robust coupling to the actin cytoskeleton. We also reveal that the interaction of epsin Ent1 with the actin cytoskeleton is negatively regulated by the phosphorylation of Prk1 kinase (homologous to human AAK). This is in a good agreement with the proposed role of Prk1 to dismantle protein-protein interactions of endocytic factors after the vesicle scission/during vesicle uncoating.
Hence, by synergistic binding to the membrane and redundant interaction with the actin cytoskeleton, Ent1 and Sla2 proteins form an essential molecular linker that transmits the pulling force from the actin cytoskeleton to the plasma membrane, leading to membrane invagination and endocytic vesicle budding.
Our study provides a molecular mechanism for energy transfer between the actin cytoskeleton and the plasma membrane of the endocytic site, which is probably essential for endocytic process in many eukaryotic organisms. The mechanism employs an unprecedented cooperative action between membrane-binding domains of two fundamental endocytic proteins epsin and Sla2/HIP1R and their redundant interaction with the actin cytoskeleton. We believe that similar mechanism (or its parts) could also be the basis for other membrane-remodelling processes, which occurs in eukaryotic cells and requires polymerising actin cytoskeleton. Our results will thus be of a great interest for many researchers working in fields of membrane traffic, cell signalling, cell morphogenesis, polarisation, differentiation etc. To this end our data could represent an important knowledgebase for many forthcoming research projects in several research fields and could potentially be a starting point for many biomedical applications.