Periodic Reporting for period 3 - BRIDGING (The function of membrane tethering in plant intercellular communication)
Berichtszeitraum: 2021-06-01 bis 2022-11-30
Intercellular communication is critical for multicellularity and evolution gave rise to distinct mechanisms to facilitate this process. Plants have evolved remarkable cellular machines -the Plasmodesmata (PD) pores- which interconnect virtually all cells within the plant body, establishing direct membrane and cytoplasmic continuity, a situation unique to plants. PD are decisive for development, environmental adaptation, defence signalling, and spreading of viruses, yet their mode of action remains elusive.
A striking feature of PD organisation, setting them apart from animal cell junctions, is a strand of endoplasmic reticulum (ER) running through the pore, tethered extremely tight (~10nm) to the plasma membrane (PM) by unidentified “spokes”. To date, the function of ER-PM contacts at PD remains a complete enigma. We don’t know how and why the two organelles come together at PD cellular junctions.
The focus of the ERC-BRIDGING is to understand the function and molecular mechanisms governing ER-PM organelle contacts at PD. We are using multidisciplinary approaches from lipidomics/proteomics, super-high-resolution microscopy, biophysics, molecular dynamics and cell biology and plant genetics to address the following objectives:
• Identify the mechanisms of PD membrane-tethering at the molecular level
• Elucidate the dynamics and 3D architecture of ER-PM contact sites at PD
• Uncover the function of ER-PM apposition for plant intercellular communication.
A striking feature of PD organisation, setting them apart from animal cell junctions, is a strand of endoplasmic reticulum (ER) running through the pore, tethered extremely tight (~10nm) to the plasma membrane (PM) by unidentified “spokes”. To date, the function of ER-PM contacts at PD remains a complete enigma. We don’t know how and why the two organelles come together at PD cellular junctions.
The focus of the ERC-BRIDGING is to understand the function and molecular mechanisms governing ER-PM organelle contacts at PD. We are using multidisciplinary approaches from lipidomics/proteomics, super-high-resolution microscopy, biophysics, molecular dynamics and cell biology and plant genetics to address the following objectives:
• Identify the mechanisms of PD membrane-tethering at the molecular level
• Elucidate the dynamics and 3D architecture of ER-PM contact sites at PD
• Uncover the function of ER-PM apposition for plant intercellular communication.
The protein machinery acting at the ER-PM interface within PD has remained unknown despite more than 40 years of intense research. Combining proteomics, live cell imaging, super-resolution microscopy, molecular dynamics, plant genetics and yeast complementation, we identified the Multiple C2 domains and Transmembrane region Protein (MCTPs) family as the main ER-PM tethers of specialised PD membrane contact sites. We showed show that MCTPs 1) are core PD components and enriched in PD with extremely tight membrane tethering, 2) are critical to plant development and cell-to-cell trafficking 3) insert in the ER through a C-terminal transmembrane region and dock to the PM through several N-terminal C2 domains, through anionic lipid interaction. Our work unveils, for the first time, the molecular identity of PD ER-PM tethers (Brault, Petit et al. 2019, EMBO reports).
How are PD built and organised within the narrow cytoplasmic sleeve between the ER and the PM, and the impact on cell-cell trafficking remained little understood. We used electron tomography to provide the first data of PD ER-PM contact site 3D organisation. Our data showed that within the PD pores, ER-PM contacts undergoes substantial modification and mature from very tight contacts to intermembrane gaps of about 10 nm and spanned by tethers, setting apart two PD morphotypes (Type I and Type II). In collaboration with the team of Yrjö Helariutta’s, we showed that this transition is related to lipid metabolism (Yan et al. 2019, Nature Plants). Phloem Unloading Modulator (plm) is involved in sphingolipid metabolism and plm loss-of-function Arabidopsis mutants present higher phloem unloading capacity in the roots. This increase in PD connectivity was linked with a defect in the remodelling of ER-PM junction from Type I to Type II. This work presents a new paradigm by showing that PD with tight ER-PM contacts may be more conductive than ‘open-sleeved’ ones, challenging current models of how plant cell-cell communication is regulated.
We demonstrated that, similar to other eukaryotic membrane contact sites, PD can rapidly change their molecular composition in response to abiotic stresses to induce cellular responses. From a PD proteomic screen, we identified two LRR-receptor-like kinases, Qian Shou Kinase 1 and Inflorescence Meristem Kinase, which upon osmotic and ionic stresses rapidly re-organised from the PM to PD ER-PM junctions. This process happens remarkably fast, within less than two minutes and is influenced by QSK1 phosphorylation status (Grison et al. 2019 Plant Physiology)
we are currently preparing two papers for submission from work directly from the ERC BRIDGING grant:
1-Title: Incomplete cytokinesis and cytoplasmic bridges formation in plants:
Abstract: Intercellular bridges arising from incomplete cell division act as structural mediators of multicellularity. In plants, all somatic cells divide following an incomplete
cytokinesis scheme, leading to the creation of hundreds of plasmodesmata (PD) bridges between two daughter cells. Here, we addressed the question of how plant cells connect while dividing. Using advanced microscopy and modeling, we found that PD formation requires the integration of endoplasmic reticulum (ER) across introgressing cell
plate fenestrae. ER marks the positions of and stabilises nascent PD against abscission, but requires constriction and physical tethering. This occurs through the action of MCTP3, 4, and 6 (Multiple C2 domains and transmembrane domain proteins) which shape and anchor the ER inside the nascent membrane bridges. By forming a protective shell,
MCTPs inhibit local ER severing, maintaining cell-cell membrane continuity and stable bridge insertion. Our work uncovers the molecular mechanisms and basic principles of how plant cells incompletely divide to promote communication.
2-Title: A novel class of membrane contact sites sitting at cell-cell junctions regulates intercellular trafficking in plants
Abstract: Membrane contact sites (MCS) are key actors of intracellular communication. Through membrane tethering and molecular specialisation, they allow organelles to directly communicate and integrate their activities. While MCS function in intracellular processes is well documented, their role at intercellular interfaces remains unexplored.
Using plants as a complex multicellular model, we describe an unconventional MCS acting at cell-cell interface to regulate intercellular communication. These atypical MCS, called plasmodesmata, present a tubular ER-PM arrangement, the function of which had remained unknown for decades. Here we demonstrate that similar to other MCS, plasmodesmata regulate ER-PM apposition through the action of the protein tether-complex MCTP3, 4 and 6 (Multiple C2 domains and transmembrane domain proteins), in a PI4P lipid-dependant manner. Unlike other MCS however, regulation of ER-PM contacts primarily serves intercellular communication. Through the action of MCTP3, 4 and 6 and the anionic lipid PI4P, the ER-PM gap can be adjusted, similar to a flow-control valve, to regulate molecular transport from cell to cell. MCTP-tether driven mechanism bypasses the well-established plasmodesmata regulator callose, and is required for controlling brassinosteroid precursors trafficking, a hormone critical for plant development and growth. Our work indicates that during evolution, MCS have acquired diversified functions in the transmission of information ranging from intra- to intercellular cellular activities.
How are PD built and organised within the narrow cytoplasmic sleeve between the ER and the PM, and the impact on cell-cell trafficking remained little understood. We used electron tomography to provide the first data of PD ER-PM contact site 3D organisation. Our data showed that within the PD pores, ER-PM contacts undergoes substantial modification and mature from very tight contacts to intermembrane gaps of about 10 nm and spanned by tethers, setting apart two PD morphotypes (Type I and Type II). In collaboration with the team of Yrjö Helariutta’s, we showed that this transition is related to lipid metabolism (Yan et al. 2019, Nature Plants). Phloem Unloading Modulator (plm) is involved in sphingolipid metabolism and plm loss-of-function Arabidopsis mutants present higher phloem unloading capacity in the roots. This increase in PD connectivity was linked with a defect in the remodelling of ER-PM junction from Type I to Type II. This work presents a new paradigm by showing that PD with tight ER-PM contacts may be more conductive than ‘open-sleeved’ ones, challenging current models of how plant cell-cell communication is regulated.
We demonstrated that, similar to other eukaryotic membrane contact sites, PD can rapidly change their molecular composition in response to abiotic stresses to induce cellular responses. From a PD proteomic screen, we identified two LRR-receptor-like kinases, Qian Shou Kinase 1 and Inflorescence Meristem Kinase, which upon osmotic and ionic stresses rapidly re-organised from the PM to PD ER-PM junctions. This process happens remarkably fast, within less than two minutes and is influenced by QSK1 phosphorylation status (Grison et al. 2019 Plant Physiology)
we are currently preparing two papers for submission from work directly from the ERC BRIDGING grant:
1-Title: Incomplete cytokinesis and cytoplasmic bridges formation in plants:
Abstract: Intercellular bridges arising from incomplete cell division act as structural mediators of multicellularity. In plants, all somatic cells divide following an incomplete
cytokinesis scheme, leading to the creation of hundreds of plasmodesmata (PD) bridges between two daughter cells. Here, we addressed the question of how plant cells connect while dividing. Using advanced microscopy and modeling, we found that PD formation requires the integration of endoplasmic reticulum (ER) across introgressing cell
plate fenestrae. ER marks the positions of and stabilises nascent PD against abscission, but requires constriction and physical tethering. This occurs through the action of MCTP3, 4, and 6 (Multiple C2 domains and transmembrane domain proteins) which shape and anchor the ER inside the nascent membrane bridges. By forming a protective shell,
MCTPs inhibit local ER severing, maintaining cell-cell membrane continuity and stable bridge insertion. Our work uncovers the molecular mechanisms and basic principles of how plant cells incompletely divide to promote communication.
2-Title: A novel class of membrane contact sites sitting at cell-cell junctions regulates intercellular trafficking in plants
Abstract: Membrane contact sites (MCS) are key actors of intracellular communication. Through membrane tethering and molecular specialisation, they allow organelles to directly communicate and integrate their activities. While MCS function in intracellular processes is well documented, their role at intercellular interfaces remains unexplored.
Using plants as a complex multicellular model, we describe an unconventional MCS acting at cell-cell interface to regulate intercellular communication. These atypical MCS, called plasmodesmata, present a tubular ER-PM arrangement, the function of which had remained unknown for decades. Here we demonstrate that similar to other MCS, plasmodesmata regulate ER-PM apposition through the action of the protein tether-complex MCTP3, 4 and 6 (Multiple C2 domains and transmembrane domain proteins), in a PI4P lipid-dependant manner. Unlike other MCS however, regulation of ER-PM contacts primarily serves intercellular communication. Through the action of MCTP3, 4 and 6 and the anionic lipid PI4P, the ER-PM gap can be adjusted, similar to a flow-control valve, to regulate molecular transport from cell to cell. MCTP-tether driven mechanism bypasses the well-established plasmodesmata regulator callose, and is required for controlling brassinosteroid precursors trafficking, a hormone critical for plant development and growth. Our work indicates that during evolution, MCS have acquired diversified functions in the transmission of information ranging from intra- to intercellular cellular activities.
Within the frame of the ERC-BRIDGING project we expect to reveal how ER-PM membrane contact sites controlled plasmodesmata formation during cell division and how they contribute to the regulation of cell-cell communication in plants.