Periodic Reporting for period 2 - LiquOrg (Do liquid crystal-like phases of proteins organize membrane compartments?)
Berichtszeitraum: 2023-03-01 bis 2024-08-31
We are in the midst of a revolution in our understanding of the internal organization of cells. In the 1950s we learned that lipid bilayer-based membranes serve as containers (organelles) within the cytoplasm. Now we are learning that liquid-like “membrane-less” organelles i.e. without any container, self-assemble based on “liquidliquid” phase separations. We propose the seemingly radical idea that membrane-bounded organelles– like their membrane-less counterparts- are stabilized or even templated by analogous phase separations of their
surface proteins into largely planar liquids akin to liquid crystals. Our unique Synergy team is organized specifically to test this “liquid crystal hypothesis” on the cell’s secretory compartments - ER exit sites (ERES) and the Golgi stack - by employing our complementary skills in physics, physical chemistry, biochemistry and cell biology. We hypothesize based on pilot experiments evidence that the ERES and Golgi self-organize as a multi-layered series of adherent liquid crystal-like phases of “golgin” and similar proteins which surround and enclose their membranes. Their differential adhesion and repulsion would specify the topology and dynamics of the membrane compartments. If this is true, it will literally rewrite the history of cell biology. We will test the ‘liquid crystal’ hypothesis directly, systematically, and quantitatively on an unprecedented scale to either modify/disprove it or place it on a firm rigorous footing.
We will test the ‘liquid crystal’ hypothesis directly, systematically, and quantitatively on an unprecedented scale to either modify/disprove it or place it on a firm rigorous footing. Experiments (Aim 1) with 13 pure golgins in cis and trans pairwise combinations will establish their foundational physical chemistry. Surgically engineered changes in golgins/ERES proteins will alter the rank order (hierarchy) of their affinities for each other and link phase separation physics to cell biology (Aim 2) and be used to establish the structural basis of
phase separations and their specificity, and the potential for self-assembly of wholly synthetic biological organelles (Aim 3).
surface proteins into largely planar liquids akin to liquid crystals. Our unique Synergy team is organized specifically to test this “liquid crystal hypothesis” on the cell’s secretory compartments - ER exit sites (ERES) and the Golgi stack - by employing our complementary skills in physics, physical chemistry, biochemistry and cell biology. We hypothesize based on pilot experiments evidence that the ERES and Golgi self-organize as a multi-layered series of adherent liquid crystal-like phases of “golgin” and similar proteins which surround and enclose their membranes. Their differential adhesion and repulsion would specify the topology and dynamics of the membrane compartments. If this is true, it will literally rewrite the history of cell biology. We will test the ‘liquid crystal’ hypothesis directly, systematically, and quantitatively on an unprecedented scale to either modify/disprove it or place it on a firm rigorous footing.
We will test the ‘liquid crystal’ hypothesis directly, systematically, and quantitatively on an unprecedented scale to either modify/disprove it or place it on a firm rigorous footing. Experiments (Aim 1) with 13 pure golgins in cis and trans pairwise combinations will establish their foundational physical chemistry. Surgically engineered changes in golgins/ERES proteins will alter the rank order (hierarchy) of their affinities for each other and link phase separation physics to cell biology (Aim 2) and be used to establish the structural basis of
phase separations and their specificity, and the potential for self-assembly of wholly synthetic biological organelles (Aim 3).
During this first period we have consolidated the interactions of our synergetic group of four laboratories by organizing biweekly meetings with the four teams, weekly meetings with the in vitro teams involved in aims 1 and 3 (CNRS, UCL, UCM), regular meetings between CNRS and CRG and monthly meetings with the four PIs only. These meetings were all held by videoconference. In addition, we organized an “in-person” retreat of all groups with a few invited experts in September 2022 at CRG to evaluate our advances and define our strategy for the following year. Also, members of the UCL and Paris team went to the Rothman lab at Yale University to learn the difficult purification of golgins and how to handle these proteins without damaging them. These tight interactions ensured that we all work in a unified manner as swiftly and efficiently as possible and allowed us to proceed on the three aims of the proposals.
We can now efficiently and quickly produce several golgins at UCL. This process took time because training of the new members of staff has been conducted at Yale University to ensure continuity and uniformity of procedures and protocols, followed by a second training session at UCL to account for local conditions. This allowed UCL laboratory to master purification and functional characterization of proteins needed for collaborative project (various modifications of GM130, Golgin-160, Golgin-97, GMAP-210, Arf1 and ARNO) and to create plasmids for expression and purification of Golgin-84, Giantin and GM130 fragments. Working in close cooperation with CNRS and UCM labs, UCL staff has established logistical approaches to deliver proteins to them. Using these proteins on two main membrane platforms, supported bilayers and giant unilamellar vesicles, CNRS and UCM demonstrated that, as hypothesized in the proposal, golgins are able to self-assemble and form domains on membranes. A joint UCM, CNRS and UCL manuscript describing by AFM the formation of GM130 2D-condesates is currently under review in PNAS.We are now working on establishing the phase diagram of these domains determining their exact physical, chemical and molecular nature. This is exactly what was planned in aim 1.
Regarding aim 2, at CRG, we have successfully expressed TANGO1-short and Sec23 bearing optogenetically controlled features at the endoplasmic reticulum exit site (ERES). We can trap these proteins at ERES by exposure of cells to blue light. Under these conditions, transport of secretory cargo from the ERES is blocked, but retrograde transport to the ER is unaffected. Upon shifting to white light, the TANGO1short-Sec23 complex is broken. The blue light induced assembly and disassembly upon shift to white light reactions are extremely rapid and allow us to monitor the dynamics of ERES proposed in our ERC synergy proposal objectives. We plan to use this set up to ask questions related to the ability of TANGO1 to undergo phase condensation at the ERES and test how this assembly affects its interaction with proteins of the export machinery in cis, and proteins of the early Golgi cisternae in trans. These studies are aimed to address the question of ER-Golgi interface in creating compartmental boundary and in cargo transport in the anterograde and retrograde transport.
In a related objective, we have preliminary data that a modified tagged TANGO1 can be expressed at the outer mitochondrial membrane. This would allow addressing whether TANGO1 has the property to assemble into LLPS, the role of other interactors in cis and in trans, and whether it can recruit Golgi stacks in trans because of its properties and location.
Regarding aim 3, we have tested a few approaches to generate Golgi-like structures in vitro. We tried to incorporate golgins in “sponge” phases. However, we discovered an incompatibility between the golgin we used, GM130, and one of the surfactants involved in the membrane assembly, -octadecyl glucoside. Alternatly, we tried to fuse small unilamellar vesicles to form deflated structures. We did not reach a sufficient yield of fusion to obtain large enough compartments. This type of hurdles was to be expected. We may continue in this direction by testing other surfactants (sponge phases) or by increasing our fusion efficiency (small unilamellar vesicles). Alternately, we may use other approaches such as deflating larger vesicles.
In spite of these advances each lab met several problems that slowed down the process.
We can now efficiently and quickly produce several golgins at UCL. This process took time because training of the new members of staff has been conducted at Yale University to ensure continuity and uniformity of procedures and protocols, followed by a second training session at UCL to account for local conditions. This allowed UCL laboratory to master purification and functional characterization of proteins needed for collaborative project (various modifications of GM130, Golgin-160, Golgin-97, GMAP-210, Arf1 and ARNO) and to create plasmids for expression and purification of Golgin-84, Giantin and GM130 fragments. Working in close cooperation with CNRS and UCM labs, UCL staff has established logistical approaches to deliver proteins to them. Using these proteins on two main membrane platforms, supported bilayers and giant unilamellar vesicles, CNRS and UCM demonstrated that, as hypothesized in the proposal, golgins are able to self-assemble and form domains on membranes. A joint UCM, CNRS and UCL manuscript describing by AFM the formation of GM130 2D-condesates is currently under review in PNAS.We are now working on establishing the phase diagram of these domains determining their exact physical, chemical and molecular nature. This is exactly what was planned in aim 1.
Regarding aim 2, at CRG, we have successfully expressed TANGO1-short and Sec23 bearing optogenetically controlled features at the endoplasmic reticulum exit site (ERES). We can trap these proteins at ERES by exposure of cells to blue light. Under these conditions, transport of secretory cargo from the ERES is blocked, but retrograde transport to the ER is unaffected. Upon shifting to white light, the TANGO1short-Sec23 complex is broken. The blue light induced assembly and disassembly upon shift to white light reactions are extremely rapid and allow us to monitor the dynamics of ERES proposed in our ERC synergy proposal objectives. We plan to use this set up to ask questions related to the ability of TANGO1 to undergo phase condensation at the ERES and test how this assembly affects its interaction with proteins of the export machinery in cis, and proteins of the early Golgi cisternae in trans. These studies are aimed to address the question of ER-Golgi interface in creating compartmental boundary and in cargo transport in the anterograde and retrograde transport.
In a related objective, we have preliminary data that a modified tagged TANGO1 can be expressed at the outer mitochondrial membrane. This would allow addressing whether TANGO1 has the property to assemble into LLPS, the role of other interactors in cis and in trans, and whether it can recruit Golgi stacks in trans because of its properties and location.
Regarding aim 3, we have tested a few approaches to generate Golgi-like structures in vitro. We tried to incorporate golgins in “sponge” phases. However, we discovered an incompatibility between the golgin we used, GM130, and one of the surfactants involved in the membrane assembly, -octadecyl glucoside. Alternatly, we tried to fuse small unilamellar vesicles to form deflated structures. We did not reach a sufficient yield of fusion to obtain large enough compartments. This type of hurdles was to be expected. We may continue in this direction by testing other surfactants (sponge phases) or by increasing our fusion efficiency (small unilamellar vesicles). Alternately, we may use other approaches such as deflating larger vesicles.
In spite of these advances each lab met several problems that slowed down the process.
A manuscript describing the role in anterograde trafficking of collagen is currently being prepared for submission. This is made possible by the joint effort of the CNRS and CRG groups, respectively. A first manuscript describing the self-assembly of GM130 proteins into 2D fluid-like domains has been submitted in February 2022.
Progress beyond the state of the art are indicated above.
Progress beyond the state of the art are indicated above.