Final Report Summary - ESCRT HIGHRES (Employing quantitative high-resolution imaging to understand mechanistic principles of ESCRT-mediated membrane fission in dividing cells)
The ESCRT machinery, and specifically the ESCRT-III complex, has been associated with the membrane fission machinery that generates membrane budding away from the cytosol in several essential intracellular processes, such as multivesicular body (MVB) formation and virus budding. Experimental evidence has indicated that the ESCRT machinery is also involved in cytokinetic abscission, the final event of cell division, leading to the physical separation of two daughter cells, i.e. ESCRT proteins localize to the intercellular bridge where cytokinetic abscission occurs, are essential for abscission, and are known to mediate cell division in Archea.
How the ESCRT-III complex drives membrane fission in cells is still not known. The relatively large dimensions of the cytokinetic intercellular bridge (>1 µm), its slow kinetics (~ 2 hours) offer an ideal system to study the spatial and temporal behaviour of ESCRTs by using newly available imaging tools.
The goal of this work was to map out the regulatory basis for the spatiotemporal organization observed for ESCRT in mammalian cytokinesis with the aim to understand how such spatiotemporal organization is utilized to drive membrane fission in cells. The innovative nature of this project lies in its potential to provide detailed mechanistic information – which is normally limited to in vitro studies – in a physiologically relevant biological system utilizing the ESCRT machinery for its function. To reach this goal state-of-the-art imaging technologies and molecular tools had to be developed and optimized. In the past 4 years we have developed these tools and have utilized them to explore the spatiotemporal dynamics of ESCRTs in cytokinesis. The tools that we have developed include: 1) a new approach for inhibition of the ESCRT III complex in cytokinesis (Goliand et al. 2014), 2) a correlative live-super resolution assay to visualize rare intermediate organizations of ESCRT proteins can be visualized at high resolution (Sherman et al. 2015), 3) a correlative Live-Soft X-Ray microscopy approach to resolve new ultrastructural features of the intercellular bridge during different stages of abscission (Sherman et al. 2016).
Using the new set of tools we have developed, we found that:
1. Cytokinetic abscission is an event that occurs in the G1 phase of the following cell cycle. This finding raise the possibility that the regulators of abscission are factors that are expressed in the following cell cycle and are not classical regulators of cell division. This work was published in the peer-reviewed journal Cell Cycle (Gershony et al. 2014).
The results of this work opened up new research projects in the laboratory that are now being followed.
2. The ESCRT III component CHMP6 is a crucial component for ESCRT III mediated abscission. A truncated version of CHMP6, composed of its first 52 AA (CHMP6-N), arrives to the intercellular bridge, blocks abscission and subsequently leads to cell death in a specific manner. Taken together, these data suggest an active role for ESCRT-II and CHMP6 in ESCRT-mediated abscission. These findings were published in the peer-reviewed journal Molecular Biology of the Cell (Goliand et al. 2014). These results challenged the previous consensus in the field that ESCRT II and CHMP6 are dispensable for abscission and facilitate more publications by other groups that supported and expanded our findings by confirming that this indeed the case in abscission but that the same principles apply to viral budding as well. We are currently using the tools developed in these study to lean more about the nature of these interactions in cells.
3. The membrane is highly dynamic at the abscission sites and undergoes massive blebbing (figure 1). These new observations were in agreement with previously published work. Moreover, focusing on the changes that the membrane itself undergoes during abscission provided new information on the events that occur before and during membrane constriction, an information that is critical for understanding how ESCRTs induce membrane constriction and fission. These findings are reported in the peer-reviewed journal Scientific Reports (Sherman et al. 2016).
4. A complex array of helical filaments resides at the intercellular bridge during abscission. We Additionally, we have observed another spiral-like filaments that reside inside the bridge and do not interact with the membrane (figure 2). The existence of an inner helix raises the possibility that ESCRT filaments form nested helixes inside the intercellular bridge. This possibility, which was not considered before, is actually in agreement with nested helixes formation reported for ESCRT filaments in vitro. We are currently putting a lot of effort into finding the protein composition of the inner spiral and into substantiating this unexpected finding. These findings are reported in the peer-reviewed journal Scientific Reports (Sherman et al. 2016).
Although our research did not progress according to the proposed plan the overall goals were fully achieved. However, by obtaining new spatiotemporal information on ESCRTs in abscission we have opened up new questions and hypothesis that need to be answered before we can unlock the mechanism for ESCRT mediated membrane constriction and fission.
How the ESCRT-III complex drives membrane fission in cells is still not known. The relatively large dimensions of the cytokinetic intercellular bridge (>1 µm), its slow kinetics (~ 2 hours) offer an ideal system to study the spatial and temporal behaviour of ESCRTs by using newly available imaging tools.
The goal of this work was to map out the regulatory basis for the spatiotemporal organization observed for ESCRT in mammalian cytokinesis with the aim to understand how such spatiotemporal organization is utilized to drive membrane fission in cells. The innovative nature of this project lies in its potential to provide detailed mechanistic information – which is normally limited to in vitro studies – in a physiologically relevant biological system utilizing the ESCRT machinery for its function. To reach this goal state-of-the-art imaging technologies and molecular tools had to be developed and optimized. In the past 4 years we have developed these tools and have utilized them to explore the spatiotemporal dynamics of ESCRTs in cytokinesis. The tools that we have developed include: 1) a new approach for inhibition of the ESCRT III complex in cytokinesis (Goliand et al. 2014), 2) a correlative live-super resolution assay to visualize rare intermediate organizations of ESCRT proteins can be visualized at high resolution (Sherman et al. 2015), 3) a correlative Live-Soft X-Ray microscopy approach to resolve new ultrastructural features of the intercellular bridge during different stages of abscission (Sherman et al. 2016).
Using the new set of tools we have developed, we found that:
1. Cytokinetic abscission is an event that occurs in the G1 phase of the following cell cycle. This finding raise the possibility that the regulators of abscission are factors that are expressed in the following cell cycle and are not classical regulators of cell division. This work was published in the peer-reviewed journal Cell Cycle (Gershony et al. 2014).
The results of this work opened up new research projects in the laboratory that are now being followed.
2. The ESCRT III component CHMP6 is a crucial component for ESCRT III mediated abscission. A truncated version of CHMP6, composed of its first 52 AA (CHMP6-N), arrives to the intercellular bridge, blocks abscission and subsequently leads to cell death in a specific manner. Taken together, these data suggest an active role for ESCRT-II and CHMP6 in ESCRT-mediated abscission. These findings were published in the peer-reviewed journal Molecular Biology of the Cell (Goliand et al. 2014). These results challenged the previous consensus in the field that ESCRT II and CHMP6 are dispensable for abscission and facilitate more publications by other groups that supported and expanded our findings by confirming that this indeed the case in abscission but that the same principles apply to viral budding as well. We are currently using the tools developed in these study to lean more about the nature of these interactions in cells.
3. The membrane is highly dynamic at the abscission sites and undergoes massive blebbing (figure 1). These new observations were in agreement with previously published work. Moreover, focusing on the changes that the membrane itself undergoes during abscission provided new information on the events that occur before and during membrane constriction, an information that is critical for understanding how ESCRTs induce membrane constriction and fission. These findings are reported in the peer-reviewed journal Scientific Reports (Sherman et al. 2016).
4. A complex array of helical filaments resides at the intercellular bridge during abscission. We Additionally, we have observed another spiral-like filaments that reside inside the bridge and do not interact with the membrane (figure 2). The existence of an inner helix raises the possibility that ESCRT filaments form nested helixes inside the intercellular bridge. This possibility, which was not considered before, is actually in agreement with nested helixes formation reported for ESCRT filaments in vitro. We are currently putting a lot of effort into finding the protein composition of the inner spiral and into substantiating this unexpected finding. These findings are reported in the peer-reviewed journal Scientific Reports (Sherman et al. 2016).
Although our research did not progress according to the proposed plan the overall goals were fully achieved. However, by obtaining new spatiotemporal information on ESCRTs in abscission we have opened up new questions and hypothesis that need to be answered before we can unlock the mechanism for ESCRT mediated membrane constriction and fission.