-
Scientific Reports Jun 2024Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral...
Eukaryotic membranes are compartmentalized into distinct micro- and nanodomains that rearrange dynamically in response to external and internal cues. This lateral heterogeneity of the lipid bilayer and associated clustering of distinct membrane proteins contribute to the spatial organization of numerous cellular processes. Here, we show that membrane microdomains within the endoplasmic reticulum (ER) of yeast cells are reorganized during metabolic reprogramming and aging. Using biosensors with varying transmembrane domain length to map lipid bilayer thickness, we demonstrate that in young cells, microdomains of increased thickness mainly exist within the nuclear ER, while progressing cellular age drives the formation of numerous microdomains specifically in the cortical ER. Partitioning of biosensors with long transmembrane domains into these microdomains increased protein stability and prevented autophagic removal. In contrast, reporters with short transmembrane domains progressively accumulated at the membrane contact site between the nuclear ER and the vacuole, the so-called nucleus-vacuole junction (NVJ), and were subjected to turnover via selective microautophagy occurring specifically at these sites. Reporters with long transmembrane domains were excluded from the NVJ. Our data reveal age-dependent rearrangement of the lateral organization of the ER and establish transmembrane domain length as a determinant of membrane contact site localization and autophagic degradation.
Topics: Endoplasmic Reticulum; Autophagy; Saccharomyces cerevisiae; Membrane Microdomains; Cellular Senescence; Saccharomyces cerevisiae Proteins; Vacuoles; Membrane Proteins
PubMed: 38871812
DOI: 10.1038/s41598-024-64493-8 -
Biophysical Journal Jun 2024Annexin A2 (A2) induced microdomain formation is a key step in biological processes such as Ca-mediated exocytosis in neuroendocrine cells. In this work, a total of...
Annexin A2 (A2) induced microdomain formation is a key step in biological processes such as Ca-mediated exocytosis in neuroendocrine cells. In this work, a total of fifteen coarse-grained molecular dynamics simulations were performed on vesicle models having a diameter of approximately 250 Å for 15μs each using the Martini2 force field. Five simulations were performed in the presence of ten A2, five in the presence of A2 but absence of PIP, and five simulations in the absence of A2 but presence of PIP. Consistent results were generated among the simulations. A2-induced PIP microdomain formation was observed and shown to occur in three phases: A2-vesicle association, localized A2-induced PIP clustering, and A2 aggregation driving PIP microdomain formation. The relationship between A2 aggregation and PIP microdomain formation was quantitatively described using a novel method which calculated the variance among protein and lipid positions via the Fréchet mean. A large reduction in PIP variance was observed in the presence of A2 but not in its absence. This reduction in PIP variance was proportional to the reduction observed in A2 variance and demonstrates that the observed PIP microdomain formation is dependent upon A2 aggregation. The three-phase model of A2-induced microdomain formation generated in this work will serve as a valuable guide for further experimental studies and the development of novel A2-inhibitors. No microdomain formation was observed in the absence of A2 and minimal A2-membrane interaction was observed in the absence of PIP.
PubMed: 38859585
DOI: 10.1016/j.bpj.2024.06.006 -
BioRxiv : the Preprint Server For... May 2024Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly...
Fungal plasma membrane proteins represent key therapeutic targets for antifungal agents, yet their structure and spatial distribution in the native context remain poorly characterized. Herein, we employ an integrative multimodal approach to elucidate the structural and functional organization of plasma membrane protein complexes in , focusing on prominent and essential membrane proteins, the polysaccharide synthase β-(1,3)-glucan synthase (GS) and the proton pump Pma1. Cryo-electron tomography (cryo-ET) and live cell imaging reveal that GS and Pma1 are heterogeneously distributed into distinct plasma membrane microdomains. Treatment with caspofungin, an echinocandin antifungal that targets GS, alters the plasma membrane and disrupts the native distribution of GS and Pma1. Based on these findings, we propose a model for echinocandin action that considers how drug interactions with the plasma membrane environment lead to inhibition of GS. Our work underscores the importance of interrogating the structural and dynamic characteristics of fungal plasma membrane proteins to understand function and facilitate precisely targeted development of novel antifungal therapies.
PubMed: 38854035
DOI: 10.1101/2024.05.29.596243 -
Plant Communications Jun 2024The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the...
The soybean root system is complex. In addition to being composed of various cell types, the soybean root system includes the primary root, the lateral roots, and the nodule, an organ in which mutualistic symbiosis with the N-fixing rhizobia occurs. A mature soybean root nodule is characterized by a central infection zone where the atmospheric nitrogen is fixed and assimilated by the symbiont, resulting from the close cooperation between the plant cell and the bacteria. To date, the transcriptome of individual cells isolated from developing soybean nodules has been established, but the transcriptomic signatures of the cells of the mature soybean nodule have not yet been characterized. Applying single nucleus RNA-seq and Molecular Cartography technologies, we precisely characterized the transcriptomic signature of the soybean root and mature nodule cell types and revealed the co-existence of different sub-populations of B. diazoefficiens-infected cells in the mature soybean nodule including those actively involved in nitrogen fixation, and those engaged in senescence. The mining of the single cell-resolution nodule transcriptome atlas and associated gene co-expression network confirmed the role of known nodulation-related genes and identified new genes controlling the nodulation process. For instance, we functionally characterized the role of GmFWL3, a plasma membrane microdomain-associated protein controlling rhizobia infection. Our study reveals the unique cellular complexity of the mature soybean nodule and helps redefine the concept of cell types when considering the infection zone of the soybean nodule.
PubMed: 38845198
DOI: 10.1016/j.xplc.2024.100984 -
ELife Jun 2024The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these...
The organelles of eukaryotic cells maintain distinct protein and lipid compositions required for their specific functions. The mechanisms by which many of these components are sorted to their specific locations remain unknown. While some motifs mediating subcellular protein localization have been identified, many membrane proteins and most membrane lipids lack known sorting determinants. A putative mechanism for sorting of membrane components is based on membrane domains known as lipid rafts, which are laterally segregated nanoscopic assemblies of specific lipids and proteins. To assess the role of such domains in the secretory pathway, we applied a robust tool for synchronized secretory protein traffic (RUSH, etention sing elective ooks) to protein constructs with defined affinity for raft phases. These constructs consist solely of single-pass transmembrane domains (TMDs) and, lacking other sorting determinants, constitute probes for membrane domain-mediated trafficking. We find that while raft affinity can be sufficient for steady-state PM localization, it is not sufficient for rapid exit from the endoplasmic reticulum (ER), which is instead mediated by a short cytosolic peptide motif. In contrast, we find that Golgi exit kinetics are highly dependent on raft affinity, with raft preferring probes exiting the Golgi ~2.5-fold faster than probes with minimal raft affinity. We rationalize these observations with a kinetic model of secretory trafficking, wherein Golgi export can be facilitated by protein association with raft domains. These observations support a role for raft-like membrane domains in the secretory pathway and establish an experimental paradigm for dissecting its underlying machinery.
Topics: Protein Transport; Endoplasmic Reticulum; Golgi Apparatus; Membrane Microdomains; Secretory Pathway; Humans; Kinetics; Cell Membrane; Membrane Proteins; HeLa Cells
PubMed: 38837189
DOI: 10.7554/eLife.89306 -
BioRxiv : the Preprint Server For... May 2024Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates...
Calcineurin (CN), the only Ca -calmodulin activated protein phosphatase, dephosphorylates substrates within membrane-associated Ca microdomains. CN binds to substrates and regulators via short linear motifs (SLIMs), PxIxIT and LxVP. PxIxIT binding to CN is Ca independent and affects its distribution, while LxVP associates only with the active enzyme and promotes catalysis. 31 human proteins contain one or more composite 'LxVPxIxIT' motifs, whose functional properties have not been examined. Here we report studies of calcimembrin/C16orf74 (CLMB), a largely uncharacterized protein containing a composite motif that binds and directs CN to membranes. We demonstrate that CLMB associates with membranes via N-myristoylation and dynamic S-acylation and is dephosphorylated by CN on Thr44. The LxVP and PxIxIT portions of the CLMB composite sequence, together with Thr44 phosphorylation, confer high affinity PxIxIT-mediated binding to CN (KD∼8.9 nM) via an extended, LxVPxIxITxx(p)T sequence. This binding promotes CLMB-based targeting of CN to membranes, but also protects Thr44 from dephosphorylation. Thus, we propose that CN dephosphorylates CLMB in multimeric complexes, where one CLMB molecule recruits CN to membranes via PxIxIT binding, allowing others to engage through their LxVP motif for dephosphorylation. This unique mechanism makes dephosphorylation sensitive to CLMB:CN ratios and is supported by and analyses. CLMB overexpression is associated with poor prognoses for several cancers, suggesting that it promotes oncogenesis by shaping CN signaling.
PubMed: 38798520
DOI: 10.1101/2024.05.12.593783 -
Current Opinion in Cell Biology Jun 2024Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation... (Review)
Review
Caveolae are atypical plasma membrane invaginations that take part in lipid sorting and regulation of oxidative and mechanical plasma membrane stress. Caveola formation requires caveolin, cavin, and specific lipid types. The recent advances in understanding the structure and assembly of caveolin and cavin complexes within the membrane context have clarified the fundamental processes underlying caveola biogenesis. In addition, the curvature of the caveola membrane is controlled by the regulatory proteins EHD2, pacsin2, and dynamin2, which also function to restrain the scission of caveolae from the plasma membrane (PM). Here, this is integrated with novel insights on caveolae as lipid and mechanosensing complexes that can dynamically flatten or disassemble to counteract mechanical, and oxidative stress.
Topics: Humans; Caveolae; Cell Membrane; Animals; Caveolins
PubMed: 38788266
DOI: 10.1016/j.ceb.2024.102371 -
The Journal of Clinical Investigation May 2024Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress...
Endothelial cells (ECs) in the descending aorta are exposed to high laminar shear stress, and this supports an anti-inflammatory phenotype. High laminar shear stress also induces flow-aligned cell elongation and front-rear polarity, but whether these are required for the anti-inflammatory phenotype is unclear. Here, we showed that Caveolin-1-rich microdomains polarize to the downstream end of ECs that are exposed to continuous high laminar flow. These microdomains were characterized by high membrane rigidity, filamentous actin (F-actin), and raft-associated lipids. Transient receptor potential vanilloid-type 4 (TRPV4) ion channels were ubiquitously expressed on the plasma membrane but mediated localized Ca2+ entry only at these microdomains where they physically interacted with clustered Caveolin-1. These focal Ca2+ bursts activated endothelial nitric oxide synthase (eNOS) within the confines of these domains. Importantly, we found that signaling at these domains required both cell body elongation and sustained flow. Finally, TRPV4 signaling at these domains was necessary and sufficient to suppress inflammatory gene expression, and exogenous activation of TRPV4 channels ameliorated the inflammatory response to stimuli both in vitro and in vivo. Our work revealed a polarized mechanosensitive signaling hub in arterial ECs that dampens inflammatory gene expression and promotes cell resilience.
PubMed: 38771648
DOI: 10.1172/JCI175057 -
Frontiers in Molecular Biosciences 2024Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to...
INTRODUCTION
Advances in molecular targeting of ion channels may open up new avenues for therapeutic approaches in cancer based on the cells' bioelectric properties. In addition to or models, models can provide deeper insight into the complex role of electrophysiology in cancer and reveal the impact of altered ion channel expression and the membrane potential on malignant processes. The A549 model is the first computational cancer whole-cell ion current model that simulates the bioelectric mechanisms of the human non-small cell lung cancer cell line A549 during the different phases of the cell cycle. This work extends the existing model with a detailed mathematical description of the store-operated Ca entry (SOCE) and the complex local intracellular calcium dynamics, which significantly affect the entire electrophysiological properties of the cell and regulate cell cycle progression.
METHODS
The initial model was extended by a multicompartmental approach, addressing the heterogenous calcium profile and dynamics in the ER-PM junction provoked by local calcium entry of store-operated calcium channels (SOCs) and uptake by SERCA pumps. Changes of cytosolic calcium levels due to diffusion from the ER-PM junction, release from the ER by RyR channels and IP3 receptors, as well as corresponding PM channels were simulated and the dynamics evaluated based on calcium imaging data. The model parameters were fitted to available data from two published experimental studies, showing the function of CRAC channels and indirectly of IP3R, RyR and PMCA via changes of the cytosolic calcium levels.
RESULTS
The proposed calcium description accurately reproduces the dynamics of calcium imaging data and simulates the SOCE mechanisms. In addition, simulations of the combined A549-SOCE model in distinct phases of the cell cycle demonstrate how Ca - dynamics influence responding channels such as KCa, and consequently modulate the membrane potential accordingly.
DISCUSSION
Local calcium distribution and time evolution in microdomains of the cell significantly impact the overall electrophysiological properties and exert control over cell cycle progression. By providing a more profound description, the extended A549-SOCE model represents an important step on the route towards a valid model for oncological research and supported development of novel therapeutic strategies.
PubMed: 38770217
DOI: 10.3389/fmolb.2024.1394398 -
Langmuir : the ACS Journal of Surfaces... May 2024Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral...
Diverse collections of lipids self-assemble into domains within biological membranes, and these domains are typically organized in both the transverse and lateral directions of the membrane. The ability of the membrane to link these domains across the membrane's interior grants cells control over features on the external cellular surface. Numerous hypothesized factors drive the cross-membrane (or transverse) coupling of lipid domains. In this work we seek to isolate these transverse lipid-lipid influences in a simple model system using droplet interface bilayers (DIBs) to better understand the associated mechanics. DIBs enable symmetric and asymmetric combinations of domain-forming lipid mixtures within a model bilayer, and the evolving energetics of the membrane may be tracked using drop-shape analysis. We find that symmetric distributions of domain-forming lipids produce long-lasting, gradual shifts in the DIB membrane energetics that are not observed in asymmetric distributions of the lipids where the domain-forming lipids are only within one leaflet. The approach selected for this work provides experimental measurement of the mismatch penalty associated with antiregistered lipid domains as well as measurements of the influence of rafts on DIB behaviors with suggestions for their future use as a model platform.
Topics: Lipid Bilayers; Membrane Microdomains; Phosphatidylcholines
PubMed: 38753461
DOI: 10.1021/acs.langmuir.4c00958