Periodic Reporting for period 4 - BRIDGING (The function of membrane tethering in plant intercellular communication)
Periodo di rendicontazione: 2022-12-01 al 2024-05-31
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 have two major research work in revision in Cell and Science:
1-Title: Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants:
Abstract: Membrane contact sites (MCS) are fundamental for intracellular communication, but their role in intercellular communication remains unexplored. We show that in plants, plasmodesmata communication bridges function as atypical endoplasmic reticulum (ER)-plasma membrane (PM) tubular MCS, operating at cell-cell interfaces. Similar to other MCS, ER-PM apposition is controlled by a protein-lipid tethering complex, but uniquely, this serves intercellular communication. Combining high-resolution microscopy, molecular dynamics, pharmacological and genetic approaches, we show that cell-cell trafficking is modulated through the combined action of Multiple C2 domains and transmembrane domain proteins (MCTP) 3, 4, and 6 ER-PM tethers, and phosphatidylinositol-4-phosphate (PI4P) lipid. Graded PI4P amounts regulate MCTP docking to the PM, their plasmodesmata localization and cell-cell permeability. SAC7, an ER-localized PI4P-phosphatase, regulates MCTP4 accumulation at plasmodesmata and modulates cell-cell trafficking capacity in a cell-type specific manner. Our findings expand MCS's functions in information transmission, from intracellular to intercellular cellular activities.
2-Title: Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis
Abstract: Diverging from conventional cell division models, plant cells undergo incomplete division to generate plasmodesmata communication bridges between daughter cells. While fundamental for plant multicellularity, the molecular events leading to bridge stabilization, as opposed to severing, remain unknown. Using electron tomography, we mapped the transition from cell plate fenestrae to plasmodesmata. We show that the ER connects daughter cells across fenestrae, and as the cell plate matures, fenestrae contract, causing the PM to mold around constricted ER tubes. The ER's presence prevents fenestrae fusion, forming plasmodesmata, while its absence results in closure. The ER-PM tethers MCTP3, 4, and 6 further stabilize nascent plasmodesmata during fenestrae contraction. Genetic deletion in Arabidopsis reduces plasmodesmata formation. Our findings reveal how plants undergo incomplete division to promote intercellular communication.
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 have two major research work in revision in Cell and Science:
1-Title: Plasmodesmata act as unconventional membrane contact sites regulating inter-cellular molecular exchange in plants:
Abstract: Membrane contact sites (MCS) are fundamental for intracellular communication, but their role in intercellular communication remains unexplored. We show that in plants, plasmodesmata communication bridges function as atypical endoplasmic reticulum (ER)-plasma membrane (PM) tubular MCS, operating at cell-cell interfaces. Similar to other MCS, ER-PM apposition is controlled by a protein-lipid tethering complex, but uniquely, this serves intercellular communication. Combining high-resolution microscopy, molecular dynamics, pharmacological and genetic approaches, we show that cell-cell trafficking is modulated through the combined action of Multiple C2 domains and transmembrane domain proteins (MCTP) 3, 4, and 6 ER-PM tethers, and phosphatidylinositol-4-phosphate (PI4P) lipid. Graded PI4P amounts regulate MCTP docking to the PM, their plasmodesmata localization and cell-cell permeability. SAC7, an ER-localized PI4P-phosphatase, regulates MCTP4 accumulation at plasmodesmata and modulates cell-cell trafficking capacity in a cell-type specific manner. Our findings expand MCS's functions in information transmission, from intracellular to intercellular cellular activities.
2-Title: Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis
Abstract: Diverging from conventional cell division models, plant cells undergo incomplete division to generate plasmodesmata communication bridges between daughter cells. While fundamental for plant multicellularity, the molecular events leading to bridge stabilization, as opposed to severing, remain unknown. Using electron tomography, we mapped the transition from cell plate fenestrae to plasmodesmata. We show that the ER connects daughter cells across fenestrae, and as the cell plate matures, fenestrae contract, causing the PM to mold around constricted ER tubes. The ER's presence prevents fenestrae fusion, forming plasmodesmata, while its absence results in closure. The ER-PM tethers MCTP3, 4, and 6 further stabilize nascent plasmodesmata during fenestrae contraction. Genetic deletion in Arabidopsis reduces plasmodesmata formation. Our findings reveal how plants undergo incomplete division to promote intercellular communication.
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.