@article {154353, title = {Growth factor-triggered de-sialylation controls glycolipid-lectin-driven endocytosis.}, journal = {Nat Cell Biol}, year = {2025}, month = {2025 Feb 21}, abstract = {

Glycolipid-lectin-driven endocytosis controls the formation of clathrin-independent carriers and the internalization of various cargos such as β1 integrin. Whether this process is regulated in a dynamic manner remained unexplored. Here we demonstrate that, within minutes, the epidermal growth factor triggers the galectin-driven endocytosis of cell-surface glycoproteins, such as integrins, that are key regulators of cell adhesion and migration. The onset of this process-mediated by the Na/H antiporter NHE1 as well as the neuraminidases Neu1 and Neu3-requires the pH-triggered enzymatic removal of sialic acids whose presence otherwise prevents galectin binding. De-sialylated glycoproteins are then retrogradely transported to the Golgi apparatus where their glycan make-up is reset to regulate EGF-dependent invasive-cell migration. Further evidence is provided for a role of neuraminidases and galectin-3 in acidification-dependent bone resorption. Glycosylation at the cell surface thereby emerges as a dynamic and reversible regulatory post-translational modification that controls a highly adaptable trafficking pathway.

}, issn = {1476-4679}, doi = {10.1038/s41556-025-01616-x}, url = {https://pubmed.ncbi.nlm.nih.gov/39984654/}, author = {MacDonald, Ewan and Forrester, Alison and Valades-Cruz, Cesar A and Madsen, Thomas D and Hetmanski, Joseph H R and Dransart, Estelle and Ng, Yeap and Godbole, Rashmi and Shp, Ananthan Akhil and Leconte, Ludovic and Chambon, Val{\'e}rie and Ghosh, Debarpan and Pinet, Alexis and Bhatia, Dhiraj and Lombard, B{\'e}rang{\`e}re and Loew, Damarys and Larsen, Martin R and Leffler, Hakon and Lefeber, Dirk J and Clausen, Henrik and Blangy, Anne and Caswell, Patrick and Shafaq-Zadah, Massiullah and Mayor, Satyajit and Weigert, Roberto and Wunder, Christian and Johannes, Ludger} } @article {149407, title = {Concordant inter-laboratory derived concentrations of ceramides in human plasma reference materials via authentic standards.}, journal = {Nat Commun}, volume = {15}, year = {2024}, month = {2024 Oct 03}, pages = {8562}, abstract = {

In this community effort, we compare measurements between 34 laboratories from 19 countries, utilizing mixtures of labelled authentic synthetic standards, to quantify by mass spectrometry four clinically used ceramide species in the NIST (National Institute of Standards and Technology) human blood plasma Standard Reference Material (SRM) 1950, as well as a set of candidate plasma reference materials (RM 8231). Participants either utilized a provided validated method and/or their method of choice. Mean concentration values, and intra- and inter-laboratory coefficients of variation (CV) were calculated using single-point and multi-point calibrations, respectively. These results are the most precise (intra-laboratory CVs <= 4.2\%) and concordant (inter-laboratory CVs \< 14\%) community-derived absolute concentration values reported to date for four clinically used ceramides in the commonly analyzed SRM 1950. We demonstrate that calibration using authentic labelled standards dramatically reduces data variability. Furthermore, we show how the use of shared RM can correct systematic quantitative biases and help in harmonizing lipidomics. Collectively, the results from the present study provide a significant knowledge base for translation of lipidomic technologies to future clinical applications that might require the determination of reference intervals (RIs) in various human populations or might need to estimate reference change values (RCV), when analytical variability is a key factor for recall during multiple testing of individuals.

}, keywords = {Calibration, Ceramides, Humans, Laboratories, Lipidomics, Mass Spectrometry, Reference Standards, Reproducibility of Results}, issn = {2041-1723}, doi = {10.1038/s41467-024-52087-x}, author = {Torta, Federico and Hoffmann, Nils and Burla, Bo and Alecu, Irina and Arita, Makoto and Bamba, Takeshi and Bennett, Steffany A L and Bertrand-Michel, Justine and Br{\"u}gger, Britta and Cala, M{\'o}nica P and Camacho-Mu{\~n}oz, Dolores and Checa, Antonio and Chen, Michael and Chocholou{\v s}kov{\'a}, Michaela and Cinel, Michelle and Chu-Van, Emeline and Colsch, Benoit and Coman, Cristina and Connell, Lisa and Sousa, Bebiana C and Dickens, Alex M and Fedorova, Maria and Eir{\'\i}ksson, Finnur Freyr and Gallart-Ayala, Hector and Ghorasaini, Mohan and Giera, Martin and Guan, Xue Li and Haid, Mark and Hankemeier, Thomas and Harms, Amy and H{\"o}ring, Marcus and Hol{\v c}apek, Michal and Hornemann, Thorsten and Hu, Chunxiu and H{\"u}lsmeier, Andreas J and Huynh, Kevin and Jones, Christina M and Ivanisevic, Julijana and Izumi, Yoshihiro and K{\"o}feler, Harald C and Lam, Sin Man and Lange, Mike and Lee, Jong Cheol and Liebisch, Gerhard and Lippa, Katrice and Lopez-Clavijo, Andrea F and Manzi, Malena and Martinefski, Manuela R and Math, Raviswamy G H and Mayor, Satyajit and Meikle, Peter J and Monge, Mar{\'\i}a Eugenia and Moon, Myeong Hee and Muralidharan, Sneha and Nicolaou, Anna and Nguyen-Tran, Thao and O{\textquoteright}Donnell, Valerie B and Ore{\v s}i{\v c}, Matej and Ramanathan, Arvind and Riols, Fabien and Saigusa, Daisuke and Schock, Tracey B and Schwartz-Zimmermann, Heidi and Shui, Guanghou and Singh, Madhulika and Takahashi, Masatomo and Thorsteinsd{\'o}ttir, Margr{\'e}t and Tomiyasu, Noriyuki and Tournadre, Anthony and Tsugawa, Hiroshi and Tyrrell, Victoria J and van der Gugten, Grace and Wakelam, Michael O and Wheelock, Craig E and Wolrab, Denise and Xu, Guowang and Xu, Tianrun and Bowden, John A and Ekroos, Kim and Ahrends, Robert and Wenk, Markus R} } @article {140659, title = {Immunogenicity of SARS-CoV-2 vaccines BBV152 (COVAXIN{\textregistered}) and ChAdOx1 nCoV-19 (COVISHIELD{\texttrademark}) in seronegative and seropositive individuals in India: a multicentre, nonrandomised observational study.}, journal = {Lancet Reg Health Southeast Asia}, volume = {22}, year = {2024}, month = {2024 Mar}, pages = {100361}, abstract = {

BACKGROUND: There are limited global data on head-to-head comparisons of vaccine platforms assessing both humoral and cellular immune responses, stratified by pre-vaccination serostatus. The COVID-19 vaccination drive for the Indian population in the age group 18-45 years began in April 2021 when seropositivity rates in the general population were rising due to the delta wave of COVID-19 pandemic during April-May\ 2021.

METHODS: Between June 30, 2021, and Jan 28, 2022, we enrolled 691 participants in the age group 18-45 years across four clinical sites in India. In this non-randomised and laboratory blinded study, participants received either two doses of Covaxin{\textregistered} (4 weeks apart) or two doses of Covishield{\texttrademark} (12 weeks apart) as per the national vaccination policy. The primary outcome was the seroconversion rate and the geometric mean titre (GMT) of antibodies against the SARS-CoV-2 spike and nucleocapsid proteins post two doses. The secondary outcome was the frequency of cellular immune responses pre- and post-vaccination.

FINDINGS: When compared to pre-vaccination baseline, both vaccines elicited statistically significant seroconversion and binding antibody levels in both seronegative and seropositive individuals. In the per-protocol cohort, Covishield{\texttrademark} elicited higher antibody responses than Covaxin{\textregistered} as measured by seroconversion rate (98.3\% vs 74.4\%, p\ \<\ 0.0001 in seronegative individuals; 91.7\% vs 66.9\%, p\ \<\ 0.0001 in seropositive individuals) as well as by anti-spike antibody levels against the ancestral strain (GMT 1272.1 vs 75.4 binding antibody units/ml [BAU/ml], p\ \<\ 0.0001 in seronegative individuals; 2089.07 vs 585.7 BAU/ml, p\ \<\ 0.0001 in seropositive individuals). As participants at all clinical sites were not recruited at the same time, site-specific immunogenicity was impacted by the timing of vaccination relative to the delta and omicron waves. Surrogate neutralising antibody responses against variants-of-concern including delta and omicron was higher in Covishield{\texttrademark} recipients than in Covaxin{\textregistered} recipients; and in seropositive than in seronegative individuals after both vaccination and asymptomatic infection (omicron variant). T cell responses are reported from only one of the four site cohorts where the vaccination schedule preceded the omicron wave. In seronegative individuals, Covishield{\texttrademark} elicited both CD4+ and CD8+ spike-specific cytokine-producing T cells whereas Covaxin{\textregistered} elicited mainly CD4+ spike-specific T cells. Neither vaccine showed significant post-vaccination expansion of spike-specific T cells in seropositive individuals.

INTERPRETATION: Covishield{\texttrademark} elicited immune responses of higher magnitude and breadth than Covaxin{\textregistered} in both seronegative individuals and seropositive individuals, across cohorts representing the pre-vaccination immune history of most of the vaccinated Indian population.

FUNDING: Corporate social responsibility (CSR) funding from Hindustan Unilever Limited (HUL) and Unilever India Pvt. Ltd. (UIPL).

}, issn = {2772-3682}, doi = {10.1016/j.lansea.2024.100361}, author = {Asokan, Mangaiarkarasi S and Joan, Roshni Florina and Babji, Sudhir and Dayma, Girish and Nadukkandy, Prajitha and Subrahmanyam, Vinutha and Pandey, Archana and Malagi, Girish and Arya, Pooja and Mahajan, Vibhuti and Bhavikatti, Jayateerth and Pawar, Ketakee and Thorat, Aishwarya and Shah, Priyanki and Goud, Ramakrishna B and Roy, Bishnudeo and Rajukutty, Shon and Immanuel, Sushil and Agarwal, Dhiraj and Saha, Sankhanil and Shivaraj, Akshatha and Panikulam, Patricia and Shome, Rajeshwari and Gulzar, Shah-E-Jahan and Sharma, Anusmrithi U and Naik, Ajinkya and Talashi, Shruti and Belekar, Madhuri and Yadav, Ritu and Khude, Poornima and V, Mamatha and Shivalingaiah, Sudarshan and Deshmukh, Urmila and Bhise, Chinmayee and Joshi, Manjiri and Inbaraj, Leeberk Raja and Chandrasingh, Sindhulina and Ghose, Aurnab and Jamora, Colin and Karumbati, Anandi S and Sundaramurthy, Varadharajan and Johnson, Avita and Ramesh, Naveen and Chetan, Nirutha and Parthiban, Chaitra and Ahmed, Asma and Rakshit, Srabanti and Adiga, Vasista and D{\textquoteright}Souza, George and Rale, Vinay and George, Carolin Elizabeth and John, Jacob and Kawade, Anand and Chaturvedi, Akanksha and Raghunathan, Anu and Dias, Mary and Bhosale, Anand and Raghu, Padinjat and Shashidhara, L S and Vyakarnam, Annapurna and Bal, Vineeta and Kang, Gagandeep and Mayor, Satyajit} } @article {137340, title = {The ubiquitous nanocluster: A molecular scale organizing principle that governs cellular information flow.}, journal = {Curr Opin Cell Biol}, volume = {86}, year = {2024}, month = {2023 Dec 05}, pages = {102285}, abstract = {

The language of biology at the scale of the cell is constituted of alphabets represented by biomolecules. These are stitched together in a variety of ways to create meaning. We argue that the phrases of this language are nanoscale molecular assemblies or nano-hubs for the purpose of information flow. At the cell surface information is sensed and processed via membrane receptors, often configured as multimers. These nano-assemblies serve as receiver nano-hubs, which are flexibly configured with additional nano-hubs that we term modifiers and transducers. This framework serves to process information that is transmitted for execution inside the cell. Here, we explore some examples about how nano-hubs are built and how they may contribute to cellular information flow.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2023.102285}, author = {Garcia-Parajo, Maria F and Mayor, Satyajit} } @article {125204, title = {Cellular compartmentalisation and receptor promiscuity as a strategy for accurate and robust inference of position during morphogenesis. }, journal = {Elife}, year = {2023}, month = {03/2023}, pages = {12:e79257}, abstract = {

Precise spatial patterning of cell fate during morphogenesis requires accurate inference of cellular position. In making such inferences from morphogen profiles, cells must contend with inherent stochasticity in morphogen production, transport, sensing and signalling. Motivated by the multitude of signalling mechanisms in various developmental contexts, we show how cells may utilise multiple tiers of processing (compartmentalisation) and parallel branches (multiple receptor types), together with feedback control, to bring about fidelity in morphogenetic decoding of their positions within a developing tissue. By simultaneously deploying specific and nonspecific receptors, cells achieve a more accurate and robust inference. We explore these ideas in the patterning of Drosophila melanogaster wing imaginal disc by Wingless morphogen signalling, where multiple endocytic pathways participate in decoding the morphogen gradient. The geometry of the inference landscape in the high dimensional space of parameters provides a measure for robustness and delineates stiff and sloppy directions. This distributed information processing at the scale of the cell highlights how local cell autonomous control facilitates global tissue scale design.

}, doi = {10.7554/eLife.79257}, author = {Iyer, Krishnan S and Prabhakara, C and Mayor, Satyajit and Rao, Madan} } @article {129154, title = {A census of actin-associated proteins in humans.}, journal = {Front Cell Dev Biol}, volume = {11}, year = {2023}, month = {2023}, pages = {1168050}, abstract = {

Actin filaments help in maintaining the cell structure and coordinating cellular movements and cargo transport within the cell. Actin participates in the interaction with several proteins and also with itself to form the helical filamentous actin (F-actin). Actin-binding proteins (ABPs) and actin-associated proteins (AAPs) coordinate the actin filament assembly and processing, regulate the flux between globular G-actin and F-actin in the cell, and help maintain the cellular structure and integrity. We have used protein-protein interaction data available through multiple sources (STRING, BioGRID, mentha, and a few others), functional annotation, and classical actin-binding domains to identify actin-binding and actin-associated proteins in the human proteome. Here, we report 2482 AAPs and present an analysis of their structural and sequential domains, functions, evolutionary conservation, cellular localization, abundance, and tissue-specific expression patterns. This analysis provides a base for the characterization of proteins involved in actin dynamics and turnover in the cell.

}, issn = {2296-634X}, doi = {10.3389/fcell.2023.1168050}, author = {Meenakshi S, Iyer and Rao, Madan and Mayor, Satyajit and Sowdhamini, Ramanathan} } @article {131936, title = {An E-cadherin-actin clutch translates the mechanical force of cortical flow for cell-cell contact to inhibit epithelial cell locomotion.}, journal = {Dev Cell}, year = {2023}, month = {2023 Jul 17}, abstract = {

Adherens junctions (AJs) allow cell contact to inhibit epithelial migration yet\ also permit epithelia to move as coherent sheets. How, then, do cells identify which contacts will inhibit locomotion? Here, we show that in human epithelial cells this arises from the orientation of cortical flows at AJs. When the leader cells from different migrating sheets make head-on contact with one another, they assemble AJs that couple together oppositely directed cortical flows. This applies a tensile signal to the actin-binding domain (ABD) of α-catenin, which provides a clutch to promote lateral adhesion growth and inhibit the lamellipodial activity necessary for migration. In contrast, AJs found between leader cells in the same migrating sheet have cortical flows aligned in the same direction, and no such mechanical inhibition takes place. Therefore, α-catenin mechanosensitivity in the clutch between E-cadherin and cortical F-actin allows cells to interpret the direction of motion via cortical flows and signal for contact to inhibit locomotion.

}, issn = {1878-1551}, doi = {10.1016/j.devcel.2023.06.011}, author = {Noordstra, Ivar and Hermoso, Mario D{\'\i}ez and Schimmel, Lilian and Bonfim-Melo, Alexis and Currin-Ross, Denni and Duong, Cao Nguyen and Kalappurakkal, Joseph Mathew and Morris, Richard G and Vestweber, Dietmar and Mayor, Satyajit and Gordon, Emma and Roca-Cusachs, Pere and Yap, Alpha S} } @article {135857, title = {A Survey of Models of Cell Membranes: Toward a New Understanding of Membrane Organization.}, journal = {Cold Spring Harb Perspect Biol}, volume = {15}, year = {2023}, month = {2023 Oct 03}, abstract = {

The cell membrane, the boundary that separates living cells from their environment, has been the subject of study for over a century. The fluid-mosaic model of Singer and Nicolson in 1972 proposed the plasma membrane as a two-dimensional fluid composed of lipids and proteins. Fifty years hence, advances in biophysical and biochemical tools, particularly optical imaging techniques, have allowed for a better understanding of the physical nature, organization, and composition of cell membranes. This has been made possible by visualizing membrane heterogeneities and their dynamics and appreciating the asymmetrical arrangement of lipids in living cell membranes. Despite these advances, mechanisms underlying the local spatiotemporal organization of membrane components remain unclear. This review surveys various models of membrane organization, culminating in a new model that incorporates nonequilibrium processes and forces exerted by interactions with extramembrane elements such as the actin cytoskeleton. The proposed model provides a comprehensive understanding of membrane organization, taking into account the dynamic nature of the cell membrane and its interactions with its immediate environment.

}, keywords = {Actin Cytoskeleton, Cell Membrane, Membrane Lipids, Proteins}, issn = {1943-0264}, doi = {10.1101/cshperspect.a041394}, author = {Mayor, Satyajit and Bhat, Abrar and Kusumi, Akihiro} } @article {131285, title = {Validating saliva as a biological sample for cost-effective, rapid and routine screening for SARS-CoV-2}, journal = {INDIAN JOURNAL OF MEDICAL MICROBIOLOGY}, volume = {45}, year = {2023}, month = {09/2023}, abstract = {

Purpose: Compared to nasopharyngeal/oropharyngeal swabs (N/OPS-VTM), non-invasive saliva samples have enormous potential for scalability and routine population screening of SARS-CoV-2. In this study, we investigate the efficacy of saliva samples relative to N/OPS-VTM for use as a direct source for RT-PCR based SARS-CoV-2 detection. Methods: We collected paired nasopharyngeal/oropharyngeal swabs and saliva samples from suspected positive SARS-CoV-2 patients and tested using RT-PCR. We used generalized linear models to investigate factors that explain result agreement. Further, we used simulations to evaluate the effectiveness of saliva-based screening in restricting the spread of infection in a large campus such as an educational institution. Results: We observed a 75.4\% agreement between saliva and N/OPS-VTM, that increased drastically to 83\% in samples stored for less than three days. Such samples processed within two days of collection showed 74.5\% test sensitivity. Our simulations suggest that a test with 75\% sensitivity, but high daily capacity can be very effective in limiting the size of infection clusters in a workspace. Guided by these results, we successfully implemented a saliva-based screening in the Bangalore Life Sciences Cluster (BLiSC) campus. Conclusion: These results suggest that saliva may be a viable alternate source for SARS-CoV-2 surveillance if samples are processed immediately. Although saliva shows slightly lower sensitivity levels when compared to N/ OPS-VTM, saliva collection is logistically advantageous. We strongly recommend the implementation of salivabased screening strategies for large workplaces and in schools, as well as for population-level screening and routine surveillance as we learn to live with the SARS-CoV-2 virus.

}, doi = {10.1016/j.ijmmb.2023.100384}, author = {Ansil, B R and George, Carolin Elizabeth and Chandrasingh, Sindhulina and Viswanathan, Ashwin and Thattai, Mukund and Raghu, Padinjat and Devadiga, Santhosha and Harikumar, Arun Geetha and Harsha, Pulleri Kandi and Nair, Indu and Ramakrishnan, Uma and Mayor, Satyajit} } @article {115040, title = {Active emulsions in living cell membranes driven by contractile stresses and transbilayer coupling}, journal = {Proc Natl Acad Sci U S A.}, volume = {119}, year = {2022}, month = {07/2022}, pages = {e2123056119}, abstract = {

The spatiotemporal organization of proteins and lipids on the cell surface has direct functional consequences for signaling, sorting, and endocytosis. Earlier studies have shown that multiple types of membrane proteins, including transmembrane proteins that have cytoplasmic actin binding capacity and lipid-tethered glycosylphosphatidylinositol-anchored proteins (GPI-APs), form nanoscale clusters driven by active contractile flows generated by the actin cortex. To gain insight into the role of lipids in organizing membrane domains in living cells, we study the molecular interactions that promote the actively generated nanoclusters of GPI-APs and transmembrane proteins. This motivates a theoretical description, wherein a combination of active contractile stresses and transbilayer coupling drives the creation of active emulsions, mesoscale liquid order (lo) domains of the GPI-APs and lipids, at temperatures greater than equilibrium lipid phase segregation. To test these ideas, we use spatial imaging of molecular clustering combined with local membrane order, and we demonstrate that mesoscopic domains enriched in nanoclusters of GPI-APs are maintained by cortical actin activity and transbilayer interactions and exhibit significant lipid order, consistent with predictions of the active composite model.

}, doi = {10.1073/pnas.2123056119}, author = {Saha, Suvrajit and Das, A and Patra, C and Anilkumar, AA and Sil, Parijat and Mayor, Satyajit and Rao, Madan} } @article {115047, title = {Reconstitution of Membrane-tethered Minimal Actin Cortices on Supported Lipid Bilayers.}, journal = {J Vis Exp}, year = {2022}, month = {2022 Jul 12}, abstract = {

The surface of a living cell provides a versatile active platform for numerous cellular processes, which arise from the interplay of the plasma membrane with the underlying actin cortex. In the past decades, reconstituted, minimal systems based on supported lipid bilayers in combination with actin filament networks have proven to be very instrumental in unraveling basic mechanisms and consequences of membrane-tethered actin networks, as well as in studying the functions of individual membrane-associated proteins. Here, we describe how to reconstitute such active composite systems in vitro that consist of fluid supported lipid bilayers coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors that can be readily observed via total internal reflection fluorescence microscopy. An open-chamber design allows one to assemble the system in a step-by-step manner and to systematically control many parameters such as linker protein concentration, actin concentration, actin filament length, actin/myosin ratio, as well as ATP levels. Finally, we discuss how to control the quality of the system, how to detect and troubleshoot commonly occurring problems, and some limitations of this system in comparison with the living cell surface.

}, keywords = {Actin Cytoskeleton, Actins, Cell Membrane, Lipid Bilayers, Myosins}, issn = {1940-087X}, doi = {10.3791/63968}, author = {K{\"o}ster, Darius Vasco and Bhat, Abrar and Talluri, Sankarshan and Mayor, Satyajit} } @article {100626, title = {Cargo-specific recruitment in clathrin- and dynamin-independent endocytosis.}, journal = {Nat Cell Biol}, year = {2021}, month = {2021 Oct 06}, abstract = {

Spatially controlled, cargo-specific endocytosis is essential for development, tissue homeostasis and cancer invasion. Unlike cargo-specific clathrin-mediated endocytosis, the clathrin- and dynamin-independent endocytic pathway (CLIC-GEEC, CG pathway) is considered a bulk internalization route for the fluid phase, glycosylated membrane proteins and lipids. While the core molecular players of CG-endocytosis have been recently defined, evidence of cargo-specific adaptors or selective uptake of proteins for the pathway are lacking. Here we identify the actin-binding protein Swiprosin-1 (Swip1, EFHD2) as a cargo-specific adaptor for CG-endocytosis. Swip1 couples active Rab21-associated integrins with key components of the CG-endocytic machinery-Arf1, IRSp53 and actin-and is critical for integrin endocytosis. Through this function, Swip1 supports integrin-dependent cancer-cell migration and invasion, and is a negative prognostic marker in breast cancer. Our results demonstrate a previously unknown cargo selectivity for the CG pathway and a role for specific adaptors in recruitment into this endocytic route.

}, issn = {1476-4679}, doi = {10.1038/s41556-021-00767-x}, author = {Moreno-Layseca, Paulina and J{\"a}ntti, Niklas Z and Godbole, Rashmi and Sommer, Christian and Jacquemet, Guillaume and Al-Akhrass, Hussein and Conway, James R W and Kronqvist, Pauliina and Kallionp{\"a}{\"a}, Roosa E and Oliveira-Ferrer, Leticia and Cervero, Pasquale and Linder, Stefan and Aepfelbacher, Martin and Zauber, Henrik and Rae, James and Parton, Robert G and Disanza, Andrea and Scita, Giorgio and Mayor, Satyajit and Selbach, Matthias and Veltel, Stefan and Ivaska, Johanna} } @article {94677, title = {Ceramide structure dictates glycosphingolipid nanodomain assembly and function.}, journal = {Nat Commun}, volume = {12}, year = {2021}, month = {2021 06 16}, pages = {3675}, abstract = {

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.

}, issn = {2041-1723}, doi = {10.1038/s41467-021-23961-9}, author = {Arumugam, Senthil and Schmieder, Stefanie and Pezeshkian, Weria and Becken, Ulrike and Wunder, Christian and Chinnapen, Dan and Ipsen, John Hjort and Kenworthy, Anne K and Lencer, Wayne and Mayor, Satyajit and Johannes, Ludger} } @article {89987, title = {Distinct actin-dependent nanoscale assemblies underlie the dynamic and hierarchical organization of E-cadherin.}, journal = {Curr Biol}, year = {2021}, month = {2021 Feb 12}, abstract = {

Cadherins are transmembrane adhesion proteins required for the formation of cohesive tissues. Intracellular interactions of E-cadherin with the Catenin family proteins, α- and β-catenin, facilitate connections with the cortical actomyosin network. This is necessary for maintaining the integrity of cell-cell adhesion in epithelial tissues. The supra-molecular architecture of E-cadherin is an important feature of its adhesion function; cis and trans interactions of E-cadherin are deployed to form clusters, both in cis and trans. Studies in Drosophila embryo have also shown that Drosophila E-cadherin (dE-cad) is organized as finite-sized dynamic clusters that localize with actin patches at cell-cell junctions, in continuous exchange with the extra-junctional pool of dE-cad surrounding the clusters. Here, we use the ectopic expression of dE-cad in larval hemocytes, which lack endogenous dE-cad to recapitulate functional cell-cell junctions in a convenient model system. We find that, while dE-cad at cell-cell junctions in hemocytes exhibits a clustered trans-paired organization similar to that reported previously in embryonic epithelial tissue, extra-junctional dE-cad is also organized as relatively immobile nanoclusters as well as more loosely packed diffusive oligomers. Oligomers are promoted by cis interactions of the ectodomain, and their growth is counteracted by the activity of cortical actomyosin. Oligomers in turn promote assembly of dense nanoclusters that require cortical actomyosin activity. Thus, cortical actin activity remodels oligomers and generates nanoclusters. The requirement for dynamic actin in the organization of dE-cad at the nanoscale may provide a mechanism to dynamically tune junctional strength.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2021.01.059}, author = {Chandran, Rumamol and Kale, Girish and Philippe, Jean-Marc and Lecuit, Thomas and Mayor, Satyajit} } @article {91674, title = {Formin nanoclustering-mediated actin assembly during plant flagellin and DSF signaling.}, journal = {Cell Rep}, volume = {34}, year = {2021}, month = {2021 Mar 30}, pages = {108884}, abstract = {

Plants respond to bacterial infection acutely with actin remodeling during plant immune responses. The mechanisms by which bacterial virulence factors (VFs) modulate plant actin polymerization remain enigmatic. Here, we show that plant-type-I formin serves as the molecular sensor for actin remodeling in response to two bacterial VFs: Xanthomonas campestris pv. campestris (Xcc) diffusible signal factor (DSF), and pathogen-associated molecular pattern (PAMP) flagellin in pattern-triggered immunity (PTI). Both VFs regulate actin assembly by tuning the clustering and nucleation activity of formin on the plasma membrane (PM) at the nano-sized scale. By being integrated within the cell-wall-PM-actin cytoskeleton (CW-PM-AC) continuum, the dynamic behavior and function of formins are highly dependent on each scaffold layer{\textquoteright}s composition within the CW-PM-AC continuum during both DSF and PTI signaling. Our results reveal a central mechanism for rapid actin remodeling during plant-bacteria interactions, in which bacterial signaling molecules fine tune plant formin nanoclustering in a host mechanical-structure-dependent manner.

}, issn = {2211-1247}, doi = {10.1016/j.celrep.2021.108884}, author = {Ma, Zhiming and Liu, Xiaolin and Nath, Sangeeta and Sun, He and Tran, Tuan Minh and Yang, Liang and Mayor, Satyajit and Miao, Yansong} } @article {91469, title = {Plk4 triggers autonomous de novo centriole biogenesis and maturation.}, journal = {J Cell Biol}, volume = {220}, year = {2021}, month = {2021 May 03}, abstract = {

Centrioles form centrosomes and cilia. In most proliferating cells, centrioles assemble through canonical duplication, which is spatially, temporally, and numerically regulated by the cell cycle and the presence of mature centrioles. However, in certain cell types, centrioles assemble de novo, yet by poorly understood mechanisms. Herein, we established a controlled system to investigate de novo centriole biogenesis, using Drosophila melanogaster egg explants overexpressing Polo-like kinase 4 (Plk4), a trigger for centriole biogenesis. We show that at a high Plk4 concentration, centrioles form de novo, mature, and duplicate, independently of cell cycle progression and of the presence of other centrioles. Plk4 concentration determines the temporal onset of centriole assembly. Moreover, our results suggest that distinct biochemical kinetics regulate de novo and canonical biogenesis. Finally, we investigated which other factors modulate de novo centriole assembly and found that proteins of the pericentriolar material (PCM), and in particular γ-tubulin, promote biogenesis, likely by locally concentrating critical components.

}, issn = {1540-8140}, doi = {10.1083/jcb.202008090}, author = {Nabais, Catarina and Pessoa, Delphine and de-Carvalho, Jorge and van Zanten, Thomas and Duarte, Paulo and Mayor, Satyajit and Carneiro, Jorge and Telley, Ivo A and Bettencourt-Dias, M{\'o}nica} } @article {95906, title = {Strategies to target SARS-CoV-2 entry and infection using dual mechanisms of inhibition by acidification inhibitors.}, journal = {PLoS Pathog}, volume = {17}, year = {2021}, month = {2021 Jul 12}, pages = {e1009706}, abstract = {

Many viruses utilize the host endo-lysosomal network for infection. Tracing the endocytic itinerary of SARS-CoV-2 can provide insights into viral trafficking and aid in designing new therapeutic strategies. Here, we demonstrate that the receptor binding domain (RBD) of SARS-CoV-2 spike protein is internalized via the pH-dependent CLIC/GEEC (CG) endocytic pathway in human gastric-adenocarcinoma (AGS) cells expressing undetectable levels of ACE2. Ectopic expression of ACE2 (AGS-ACE2) results in RBD traffic via both CG and clathrin-mediated endocytosis. Endosomal acidification inhibitors like BafilomycinA1 and NH4Cl, which inhibit the CG pathway, reduce the uptake of RBD and impede Spike-pseudoviral infection in both AGS and AGS-ACE2 cells. The inhibition by BafilomycinA1 was found to be distinct from Chloroquine which neither affects RBD uptake nor alters endosomal pH, yet attenuates Spike-pseudovirus entry. By screening a subset of FDA-approved inhibitors for functionality similar to BafilomycinA1, we identified Niclosamide as a SARS-CoV-2 entry inhibitor. Further validation using a clinical isolate of SARS-CoV-2 in AGS-ACE2 and Vero cells confirmed its antiviral effect. We propose that Niclosamide, and other drugs which neutralize endosomal pH as well as inhibit the endocytic uptake, could provide broader applicability in subverting infection of viruses entering host cells via a pH-dependent endocytic pathway.

}, issn = {1553-7374}, doi = {10.1371/journal.ppat.1009706}, author = {Prabhakara, Chaitra and Godbole, Rashmi and Sil, Parijat and Jahnavi, Sowmya and Gulzar, Shah-E-Jahan and van Zanten, Thomas S and Sheth, Dhruv and Subhash, Neeraja and Chandra, Anchal and Shivaraj, Akshatha and Panikulam, Patricia and U, Ibrahim and Nuthakki, Vijay Kumar and Puthiyapurayil, Theja Parassini and Ahmed, Riyaz and Najar, Ashaq Hussain and Lingamallu, Sai Manoz and Das, Snigdhadev and Mahajan, Bhagyashri and Vemula, Praveen and Bharate, Sandip B and Singh, Parvinder Pal and Vishwakarma, Ram and Guha, Arjun and Sundaramurthy, Varadharajan and Mayor, Satyajit} } @article {99106, title = {"Viscotaxis"- Directed Migration of Mesenchymal Stem Cells in Response to Loss Modulus Gradient.}, journal = {Acta Biomater}, year = {2021}, month = {2021 Aug 29}, abstract = {

Directed cell migration plays a crucial role in physiological and pathological conditions. One important mechanical cue, known to influence cell migration, is the gradient of substrate elastic modulus (E). However, the cellular microenvironment is viscoelastic and hence the elastic property alone is not sufficient to define its material characteristics. To bridge this gap, in this study, we investigated the influence of the gradient of viscous property of the substrate, as defined by loss modulus (G) on cell migration. We cultured human mesenchymal stem cells (hMSCs) on a collagen-coated polyacrylamide gel with constant storage modulus (G) but with a gradient in the loss modulus (G). We found hMSCs to migrate from high to low loss modulus. We have termed this form of directional cellular migration as "Viscotaxis". We hypothesize that the high loss modulus regime deforms more due to creep in the long timescale when subjected to cellular traction. Such differential deformation drives the observed Viscotaxis. To verify our hypothesis, we disrupted the actomyosin contractility with myosin inhibitor blebbistatin and ROCK inhibitor Y27632, and found the directional migration to disappear. Further, such time-dependent creep of the high loss material should lead to lower traction, shorter lifetime of the focal adhesions, and dynamic cell morphology, which was indeed found to be the case. Together, findings in this paper highlight the importance of considering the viscous modulus while preparing stiffness-based substrates for the field of tissue engineering. STATEMENT OF SIGNIFICANCE: While the effect of substrate elastic modulus has been investigated extensively in the context of cell biology, the role of substrate viscoelasticity is poorly understood. This omission is surprising as our body is not elastic, but viscoelastic. Hence, the role of viscoelasticity needs to be investigated at depth in various cellular contexts. One such important context is cell migration. Cell migration is important in morphogenesis, immune response, wound healing, and cancer, to name a few. While it is known that cells migrate when presented with a substrate with a rigidity gradient, cellular behavior in response to viscoelastic gradient has never been investigated. The findings of this paper not only reveal a completely novel cellular taxis or directed migration, it also improves our understanding of cell mechanics significantly.

}, issn = {1878-7568}, doi = {10.1016/j.actbio.2021.08.039}, author = {Shirke, Pallavi Uday and Goswami, Hiya and Kumar, Vardhman and Shah, Darshan and Beri, Sarayu and Das, Siddhartha and Bellare, Jayesh and Mayor, Satyajit and Venkatesh, K V and Seth, Jyoti R and Majumder, Abhijit} } @article {80781, title = {The bacterial quorum sensing signal DSF hijacks sterol biosynthesis to suppress plant innate immunity.}, journal = {Life Sci Alliance}, volume = {3}, year = {2020}, month = {2020 Oct}, abstract = {

Quorum sensing (QS) is a recognized phenomenon that is crucial for regulating population-related behaviors in bacteria. However, the direct specific effect of QS molecules on host biology is largely understudied. In this work, we show that the QS molecule DSF (-11-methyl-dodecenoic acid) produced by pv. can suppress pathogen-associated molecular pattern-triggered immunity (PTI) in , mediated by flagellin-induced activation of flagellin receptor FLS2. The DSF-mediated attenuation of innate immunity results from the alteration of FLS2 nanoclusters and endocytic internalization of plasma membrane FLS2. DSF altered the lipid profile of , with a particular increase in the phytosterol species, which impairs the general endocytosis pathway mediated by clathrin and FLS2 nano-clustering on the plasma membrane. The DSF effect on receptor dynamics and host immune responses could be entirely reversed by sterol removal. Together, our results highlighted the importance of sterol homeostasis to plasma membrane organization and demonstrate a novel mechanism by which pathogenic bacteria use their communicating molecule to manipulate pathogen-associated molecular pattern-triggered host immunity.

}, issn = {2575-1077}, doi = {10.26508/lsa.202000720}, author = {Tran, Tuan Minh and Ma, Zhiming and Triebl, Alexander and Nath, Sangeeta and Cheng, Yingying and Gong, Ben-Qiang and Han, Xiao and Wang, Junqi and Li, Jian-Feng and Wenk, Markus R and Torta, Federico and Mayor, Satyajit and Yang, Liang and Miao, Yansong} } @article {66088, title = {Dynamic actin-mediated nano-scale clustering of CD44 regulates its meso-scale organization at the plasma membrane.}, journal = {Mol Biol Cell}, volume = {31}, year = {2020}, month = {03/2020}, pages = {561-579}, abstract = {

Transmembrane adhesion receptors at the cell surface, such as CD44, are often equipped with modules to interact with the extracellular-matrix(ECM) and the intra-cellular cytoskeletal machinery. CD44 has been recently shown to compartmentalize the membrane into domains by acting as membrane pickets, facilitating the function of signaling receptors. While spatial organization and diffusion studies of membrane proteins are usually conducted separately, here we combine observations of organization and diffusion by using high spatio-temporal resolution imaging on living cells to reveal a hierarchical organization of CD44. CD44 is present in a meso-scale meshwork pattern where it exhibits enhanced confinement and is enriched in nano-clusters of CD44 along its boundaries. This nanoclustering is orchestrated by the underlying cortical actin dynamics. Interaction with actin is mediated by specific segments of the intracellular-domain(ICD). This influences the organization of the protein at the nano-scale, generating a selective requirement for formin over Arp2/3-based actin-nucleation machinery. The extracellular-domain(ECD) and its interaction with elements of ECM do not influence the meso-scale organization, but may serve to reposition the meshwork with respect to the ECM. Taken together, our results capture the hierarchical nature of CD44 organization at the cell surface, with active cytoskeleton-templated nano-clusters localized to a meso-scale meshwork pattern. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

}, issn = {1939-4586}, doi = {10.1091/mbc.E18-11-0715}, author = {Sil, Parijat and Mateos, Nicolas and Nath, Sangeeta and Buschow, Sonja and Manzo, Carlo and Suzuki, Kenichi G N and Fujiwara, Takahiro and Kusumi, Akihiro and Garcia-Parajo, Maria F and Mayor, Satyajit} } @article {77384, title = {Myosin II Filament Dynamics in Actin Networks Revealed with Interferometric Scattering Microscopy.}, journal = {Biophys J}, volume = {118}, year = {2020}, month = {2020 Apr 21}, pages = {1946-1957}, abstract = {

The plasma membrane and the underlying cytoskeletal cortex constitute active platforms for a variety of cellular processes. Recent work has shown that the remodeling acto-myosin network modifies local membrane organization, but the molecular details are only partly understood because of difficulties with experimentally accessing the relevant time and length scales. Here, we use interferometric scattering microscopy to investigate a minimal acto-myosin network linked to a supported lipid bilayer membrane. Using the magnitude of the interferometric contrast, which is proportional to molecular mass, and fast acquisition rates, we detect and image individual membrane-attached actin filaments diffusing within the acto-myosin network and follow individual myosin II filament dynamics. We quantify myosin II filament dwell times and processivity as functions of ATP concentration, providing experimental evidence for the predicted ensemble behavior of myosin head domains. Our results show how decreasing ATP concentrations lead to both increasing dwell times of individual myosin II filaments and a global change from a remodeling to a contractile state of the acto-myosin network.

}, issn = {1542-0086}, doi = {10.1016/j.bpj.2020.02.025}, author = {Mosby, Lewis S and Hundt, Nikolas and Young, Gavin and Fineberg, Adam and Polin, Marco and Mayor, Satyajit and Kukura, Philipp and K{\"o}ster, Darius V} } @article {77439, title = {Toward a new picture of the living plasma membrane.}, journal = {Protein Sci}, volume = {29}, year = {2020}, month = {2020 Jun}, pages = {1355-1365}, abstract = {

Our understanding of the plasma membrane structure has undergone a major change since the proposal of the fluid mosaic model of Singer and Nicholson in the 1970s. In this model, the membrane, composed of over thousand lipid and protein species, is organized as a well-equilibrated two-dimensional fluid. Here, the distribution of lipids is largely expected to reflect a multicomponent system, and proteins are expected to be surrounded by an annulus of specialized lipid species. With the recognition that a multicomponent lipid membrane is capable of phase segregation, the membrane is expected to appear as patchwork quilt pattern of membrane domains. However, the constituents of a living membrane are far from being well equilibrated. The living cell membrane actively maintains a trans-bilayer asymmetry of composition, and its constituents are subject to a number of dynamic processes due to synthesis, lipid transfer as well as membrane traffic and turnover. Moreover, membrane constituents engage with the dynamic cytoskeleton of a living cell, and are both passively as well as actively manipulated by this engagement. The extracellular matrix and associated elements also interact with membrane proteins contributing to another layer of interaction. At the nano- and mesoscale, the organization of lipids and proteins emerge from these encounters, as well as from protein-protein, protein-lipid, and lipid-lipid interactions in the membrane. New methods to study the organization of membrane components at these scales have also been developed, and provide an opportunity to synthesize a new picture of the living cell surface as an active membrane composite.

}, issn = {1469-896X}, doi = {10.1002/pro.3874}, author = {Kalappurakkal, Joseph Mathew and Sil, Parijat and Mayor, Satyajit} } @article {62769, title = {A composition-dependent molecular clutch between T cell signaling condensates and actin.}, journal = {Elife}, volume = {8}, year = {2019}, month = {2019 Jul 03}, abstract = {

During T cell activation, biomolecular condensates form at the immunological synapse (IS) through multivalency-driven phase separation of LAT, Grb2, Sos1, SLP-76, Nck, and WASP. These condensates move radially at the IS, traversing successive radially-oriented and concentric actin networks. To understand this movement, we biochemically reconstituted LAT condensates with actomyosin filaments. We found that basic regions of Nck and N-WASP/WASP promote association and co-movement of LAT condensates with actin, indicating conversion of weak individual affinities to high collective affinity upon phase separation. Condensates lacking these components were propelled differently, without strong actin adhesion. In cells, LAT condensates lost Nck as radial actin transitioned to the concentric network, and engineered condensates constitutively binding actin moved aberrantly. Our data show that Nck and WASP form a clutch between LAT condensates and actin in vitro and suggest that compositional changes may enable condensate movement by distinct actin networks in different regions of the IS.

}, issn = {2050-084X}, doi = {10.7554/eLife.42695}, author = {Ditlev, Jonathon A and Vega, Anthony R and K{\"o}ster, Darius Vasco and Su, Xiaolei and Tani, Tomomi and Lakoduk, Ashley M and Vale, Ronald D and Mayor, Satyajit and Jaqaman, Khuloud and Rosen, Michael K} } @article {59720, title = {Integrin Mechano-chemical Signaling Generates Plasma Membrane Nanodomains that Promote Cell Spreading.}, journal = {Cell}, volume = {177}, year = {2019}, month = {2019 Jun 13}, pages = {1738-1756.e23}, abstract = {

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a major class of lipid-anchored plasma membrane proteins. GPI-APs form nanoclusters generated by cortical acto-myosin activity. While our understanding of the physical principles governing this process is emerging, the molecular machinery and functional relevance of GPI-AP nanoclustering are unknown. Here, we first show that a membrane receptor signaling pathway directs nanocluster formation. Arg-Gly-Asp motif-containing ligands bound to the β1-integrin receptor activate src and focal adhesion kinases, resulting in RhoA signaling. This cascade triggers actin-nucleation via specific formins, which, along with myosin activity, drive the nanoclustering of membrane proteins with actin-binding domains. Concurrently, talin-mediated activation of the mechano-transducer vinculin is required for the coupling of the acto-myosin machinery to inner-leaflet lipids, thereby generating GPI-AP nanoclusters. Second, we show that these nanoclusters are functional; disruption of their formation either in GPI-anchor remodeling mutants or in vinculin mutants impairs cell spreading and migration, hallmarks of integrin function.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2019.04.037}, author = {Kalappurakkal, Joseph Mathew and Anilkumar, Anupama Ambika and Patra, Chandrima and van Zanten, Thomas S and Sheetz, Michael P and Mayor, Satyajit} } @article {66089, title = {Phosphorylation of Nephrin induces phase separated domains that move through actomyosin contraction.}, journal = {Mol Biol Cell}, year = {2019}, month = {2019 Oct 10}, pages = {mbcE18120823}, abstract = {

The plasma membrane of eukaryotic cells is organized into lipid and protein microdomains, whose assembly mechanisms and functions are incompletely understood. We demonstrate that proteins in the Nephrin/Nck/N-WASP actin-regulatory pathway cluster into micron-scale domains at the basal plasma membrane upon triggered phosphorylation of transmembrane Nephrin. The domains are persistent but readily exchange components with their surroundings, and their formation is dependent on the number of Nck SH3 domains, suggesting they are phase separated polymers assembled through multivalent interactions among the three proteins. The domains form independent of the actin cytoskeleton, but acto-myosin contractility induces their rapid lateral movement. Nephrin phosphorylation induces larger clusters at the cell periphery, which are associated with extensive actin assembly and dense filopodia. Our studies illustrate how multivalent interactions between proteins at the plasma membrane can produce micron-scale organization of signaling molecules, and how the resulting clusters can both respond to and control the actin cytoskeleton. [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text] [Media: see text].

}, issn = {1939-4586}, doi = {10.1091/mbc.E18-12-0823}, author = {Kim, Soyeon and Kalappurakkal, Joseph M and Mayor, Satyajit and Rosen, Michael K} } @article {53189, title = {Recent advances in clathrin-independent endocytosis.}, journal = {F1000Res}, volume = {8}, year = {2019}, month = {2019}, abstract = {

Endocytic pathways are broadly classified into clathrin dependent and independent on the basis of the requirement for the coat protein, clathrin. The molecular pathways and mechanisms underlying the formation of clathrin-independent pathways are still being explored, and this review summarizes recent advances and emerging functional roles of these diverse pathways. In particular, this review will discuss the growing consensus on the role of BAR domain proteins and the actin machinery in different clathrin-independent pathways and its significance to the functions fulfilled by these endocytic pathways.

}, issn = {2046-1402}, doi = {10.12688/f1000research.16549.1}, author = {Hemalatha, Anupama and Mayor, Satyajit} } @article {66848, title = {Spoiled for Choice: Diverse Endocytic Pathways Function at the Cell Surface.}, journal = {Annu Rev Cell Dev Biol}, volume = {35}, year = {2019}, month = {2019 Oct 06}, pages = {55-84}, abstract = {

Endocytosis has long been identified as a key cellular process involved in bringing in nutrients, in clearing cellular debris in tissue, in the regulation of signaling, and in maintaining cell membrane compositional homeostasis. While clathrin-mediated endocytosis has been most extensively studied, a number of clathrin-independent endocytic pathways are continuing to be delineated. Here we provide a current survey of the different types of endocytic pathways available at the cell surface and discuss a new classification and plausible molecular mechanisms for some of the less characterized pathways. Along with an evolutionary perspective of the origins of some of these pathways, we provide an appreciation of the distinct roles that these pathways play in various aspects of cellular physiology, including the control of signaling and membrane tension.

}, issn = {1530-8995}, doi = {10.1146/annurev-cellbio-100617-062710}, author = {Thottacherry, Joseph Jose and Sathe, Mugdha and Prabhakara, Chaitra and Mayor, Satyajit} } @article {45437, title = {Editorial Overview: Membranes and organelles: rethinking membrane structure, function and compartments.}, journal = {Curr Opin Cell Biol}, volume = {53}, year = {2018}, month = {2018 Aug}, pages = {A1-A3}, issn = {1879-0410}, doi = {10.1016/j.ceb.2018.07.005}, author = {Spang, Anne and Mayor, Satyajit} } @article {45305, title = {Mechanochemical feedback control of dynamin independent endocytosis modulates membrane tension in adherent cells.}, journal = {Nat Commun}, volume = {9}, year = {2018}, month = {2018 Oct 11}, pages = {4217}, abstract = {

Plasma membrane tension regulates many key cellular processes. It is modulated by, and can modulate, membrane trafficking. However, the cellular pathway(s) involved in this interplay is poorly understood. Here we find that, among a number of endocytic processes operating simultaneously at the cell surface, a dynamin independent pathway, the CLIC/GEEC (CG) pathway, is rapidly and specifically upregulated upon a sudden reduction of tension. Moreover, inhibition (activation) of the CG pathway results in lower (higher) membrane tension. However, alteration in membrane tension does not directly modulate CG endocytosis. This requires vinculin, a mechano-transducer recruited to focal adhesion in adherent cells. Vinculin acts by controlling the levels of a key regulator of the CG pathway, GBF1, at the plasma membrane. Thus, the CG pathway directly regulates membrane tension and is in turn controlled via a mechano-chemical feedback inhibition, potentially leading to homeostatic regulation of membrane tension in adherent cells.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-06738-5}, author = {Thottacherry, Joseph Jose and Kosmalska, Anita Joanna and Kumar, Amit and Vishen, Amit Singh and Elosegui-Artola, Alberto and Pradhan, Susav and Sharma, Sumit and Singh, Parvinder P and Guadamillas, Marta C and Chaudhary, Natasha and Vishwakarma, Ram and Trepat, Xavier and Del Pozo, Miguel A and Parton, Robert G and Rao, Madan and Pullarkat, Pramod and Roca-Cusachs, Pere and Mayor, Satyajit} } @article {34070, title = {Quantitative Control of GPCR Organization and Signaling by Endocytosis in Epithelial Morphogenesis.}, journal = {Curr Biol}, year = {2018}, month = {2018 Apr 25}, abstract = {

Tissue morphogenesis arises from controlled cell deformations in response to cellular contractility. During Drosophila gastrulation, apical activation of the actomyosin networks drives apical constriction in the invaginating mesoderm and cell-cell intercalation in the extending ectoderm. Myosin II (MyoII) is activated by cell-surface G protein-coupled receptors (GPCRs), such as Smog and Mist, that activate G proteins, the small GTPase Rho1, and the kinase Rok. Quantitative control over GPCR and Rho1 activation underlies differences in deformation of mesoderm and ectoderm cells. We show that GPCR Smog activity is concentrated on two different apical plasma membrane compartments, i.e., the surface and plasma membrane invaginations. Using fluorescence correlation spectroscopy, we probe the surface of the plasma membrane, and we show that Smog homo-clusters in response to its activating ligand Fog. Endocytosis of Smog is regulated by the kinase Gprk2 and β-arrestin-2 that clears active Smog from the plasma membrane. When Fog concentration is high or endocytosis is low, Smog rearranges in homo-clusters and accumulates in plasma membrane invaginations that are hubs for Rho1 activation. Lastly, we find higher Smog homo-cluster concentration and numerous apical plasma membrane invaginations in the mesoderm compared to the ectoderm, indicative of reduced endocytosis. We identify that dynamic partitioning of active Smog at the surface of the plasma membrane or plasma membrane invaginations has a direct impact on Rho1 signaling. Plasma membrane invaginations accumulate high Rho1-guanosine triphosphate (GTP) suggesting they form signaling centers. Thus, Fog concentration and Smog endocytosis form coupled regulatory processes that regulate differential Rho1 and MyoII activation in the Drosophila embryo.

}, issn = {1879-0445}, doi = {10.1016/j.cub.2018.03.068}, author = {Jha, Ankita and van Zanten, Thomas S and Philippe, Jean-Marc and Mayor, Satyajit and Lecuit, Thomas} } @article {34255, title = {Small GTPases and BAR domain proteins regulate branched actin polymerisation for clathrin and dynamin-independent endocytosis.}, journal = {Nat Commun}, volume = {9}, year = {2018}, month = {2018 May 09}, pages = {1835}, abstract = {

Using real-time TIRF microscopy imaging, we identify sites of clathrin and dynamin-independent CLIC/GEEC (CG) endocytic vesicle formation. This allows spatio-temporal localisation of known molecules affecting CG endocytosis; GBF1 (a GEF for ARF1), ARF1 and CDC42\ which appear sequentially over 60 s, preceding scission. In an RNAi screen for BAR domain proteins affecting CG endocytosis, IRSp53 and PICK1, known interactors of CDC42 and ARF1, respectively, were selected. Removal of IRSp53, a negative curvature sensing protein, abolishes CG endocytosis. Furthermore, the identification of ARP2/3 complex at CG endocytic sites, maintained in an inactive state reveals a function for PICK1, an ARP2/3 inhibitor. The spatio-temporal sequence of the arrival and disappearance of the molecules suggest a mechanism for a clathrin and dynamin-independent endocytic process. Coincident with the loss of PICK1 by GBF1-activated ARF1, CDC42 recruitment leads to the activation of IRSp53 and the ARP2/3 complex, resulting in a burst of F-actin polymerisation potentially powering scission.

}, issn = {2041-1723}, doi = {10.1038/s41467-018-03955-w}, author = {Sathe, Mugdha and Muthukrishnan, Gayatri and Rae, James and Disanza, Andrea and Thattai, Mukund and Scita, Giorgio and Parton, Robert G and Mayor, Satyajit} } @article {29827, title = {Transmembrane Pickets Connect Cyto- and Pericellular Skeletons Forming Barriers to Receptor Engagement.}, journal = {Cell}, volume = {172}, year = {2018}, month = {2018 Jan 11}, pages = {305-317.e10}, abstract = {

Phagocytic receptors must diffuse laterally to become activated upon clustering by multivalent targets. Receptor diffusion, however, can be obstructed by transmembrane proteins ("pickets") that are immobilized by interacting with the cortical cytoskeleton. The molecular identity of these pickets and their role in\ phagocytosis have not been defined. We used single-molecule tracking to study the interaction between Fcγ receptors and CD44, an abundant transmembrane protein capable of indirect association with F-actin, hence likely to serve as a picket. CD44 tethers reversibly to formin-induced actin filaments, curtailing receptor diffusion. Such linear filaments predominate in the trailing end of polarized macrophages, where receptor mobility was minimal. Conversely, receptors were most mobile at the leading edge, where Arp2/3-driven actin branching predominates. CD44 binds hyaluronan, anchoring a pericellular coat that also limits receptor displacement and obstructs access to phagocytic targets. Force must be applied to traverse the pericellular barrier, enabling receptors to engage their targets.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2017.12.023}, author = {Freeman, Spencer A and Vega, Anthony and Riedl, Magdalena and Collins, Richard F and Ostrowski, Phillip P and Woods, Elliot C and Bertozzi, Carolyn R and Tammi, Markku I and Lidke, Diane S and Johnson, Pauline and Mayor, Satyajit and Jaqaman, Khuloud and Grinstein, Sergio} } @article {29629, title = {Wnt and Hedgehog: Secretion of Lipid-Modified Morphogens.}, journal = {Trends Cell Biol}, volume = {28}, year = {2018}, month = {2018 Feb}, pages = {157-170}, abstract = {

Morphogens are signaling molecules produced by a localized source, specifying cell fate in a graded manner. The source secretes morphogens into the extracellular milieu to activate various target genes in an autocrine or paracrine manner. Here we describe various secreted forms of two canonical morphogens, the lipid-anchored Hedgehog (Hh) and Wnts, indicating the involvement of multiple carriers in the transport of these morphogens. These different extracellular secreted forms are likely to have distinct functions. Here we evaluate newly identified mechanisms that morphogens use to traverse the required distance to activate discrete paracrine signaling.

}, issn = {1879-3088}, doi = {10.1016/j.tcb.2017.10.003}, author = {Parchure, Anup and Vyas, Neha and Mayor, Satyajit} } @article {25251, title = {Actin retrograde flow actively aligns and orients ligand-engaged integrins in focal adhesions.}, journal = {Proc Natl Acad Sci U S A}, volume = {114}, year = {2017}, month = {2017 Oct 03}, pages = {10648-10653}, abstract = {

Integrins are transmembrane receptors that, upon activation, bind extracellular ligands and link them to the actin filament (F-actin) cytoskeleton to mediate cell adhesion and migration. Cytoskeletal forces in migrating cells generated by polymerization- or contractility-driven "retrograde flow" of F-actin from the cell leading edge have been hypothesized to mediate integrin activation for ligand binding. This predicts that these forces should align and orient activated, ligand-bound integrins at the leading edge. Here, polarization-sensitive fluorescence microscopy of GFP-αVβ3 integrins in fibroblasts shows that integrins are coaligned in a specific orientation within focal adhesions (FAs) in a manner dependent on binding immobilized ligand and a talin-mediated linkage to the F-actin cytoskeleton. These findings, together with Rosetta modeling, suggest that integrins in FA are coaligned and may be highly tilted by cytoskeletal forces. Thus, the F-actin cytoskeleton sculpts an anisotropic molecular scaffold in FAs, and this feature may underlie the ability of migrating cells to sense directional extracellular cues.

}, issn = {1091-6490}, doi = {10.1073/pnas.1701136114}, author = {Swaminathan, Vinay and Kalappurakkal, Joseph Mathew and Mehta, Shalin B and Nordenfelt, Pontus and Moore, Travis I and Koga, Nobuyasu and Baker, David A and Oldenbourg, Rudolf and Tani, Tomomi and Mayor, Satyajit and Springer, Timothy A and Waterman, Clare M} } @article {28246, title = {Direction of actin flow dictates integrin LFA-1 orientation during leukocyte migration.}, journal = {Nat Commun}, volume = {8}, year = {2017}, month = {2017 Dec 11}, pages = {2047}, abstract = {

Integrin αβ heterodimer cell surface receptors mediate adhesive interactions that provide traction for cell migration. Here, we test whether the integrin, when engaged to an extracellular ligand and the cytoskeleton, adopts a specific orientation dictated by the direction of actin flow on the surface of migrating cells. We insert GFP into the rigid, ligand-binding head of the integrin, model with Rosetta the orientation of GFP and its transition dipole relative to the integrin head, and measure orientation with fluorescence polarization microscopy. Cytoskeleton and ligand-bound integrins orient in the same direction as retrograde actin flow with their cytoskeleton-binding β-subunits tilted by applied force. The measurements demonstrate that intracellular forces can orient cell surface integrins and support a molecular model of integrin activation by cytoskeletal force. Our results place atomic, {\r A}-scale structures of cell surface receptors in the context of functional and cellular, μm-scale measurements.

}, issn = {2041-1723}, doi = {10.1038/s41467-017-01848-y}, author = {Nordenfelt, Pontus and Moore, Travis I and Mehta, Shalin B and Kalappurakkal, Joseph Mathew and Swaminathan, Vinay and Koga, Nobuyasu and Lambert, Talley J and Baker, David and Waters, Jennifer C and Oldenbourg, Rudolf and Tani, Tomomi and Mayor, Satyajit and Waterman, Clare M and Springer, Timothy A} } @article {14978, title = {A DNA-Based T Cell Receptor Reveals a Role for Receptor Clustering in Ligand Discrimination.}, journal = {Cell}, volume = {169}, year = {2017}, month = {2017 Mar 23}, pages = {108-119.e20}, abstract = {

A T\ cell mounts an immune response by measuring the binding strength of its T\ cell receptor (TCR) for peptide-loaded MHCs (pMHC) on an antigen-presenting cell. How T\ cells convert the lifetime of the extracellular TCR-pMHC interaction into an intracellular signal remains unknown. Here, we developed a synthetic signaling system in which the extracellular domains of the TCR and pMHC were replaced with short hybridizing strands of DNA. Remarkably, T\ cells can discriminate between DNA ligands differing by a single base pair. Single-molecule imaging reveals that signaling is initiated when single ligand-bound receptors are converted into clusters, a time-dependent process requiring ligands with longer bound times. A computation model reveals that receptor clustering serves a kinetic proofreading function, enabling ligands with longer bound times to have disproportionally greater signaling outputs. These results suggest that spatial reorganization of receptors plays an important role in ligand discrimination in T\ cell signaling.

}, issn = {1097-4172}, doi = {10.1016/j.cell.2017.03.006}, author = {Taylor, Marcus J and Husain, Kabir and Gartner, Zev J and Mayor, Satyajit and Vale, Ronald D} } @article {15479, title = {The mystery of membrane organization: composition, regulation and roles of lipid rafts.}, journal = {Nat Rev Mol Cell Biol}, year = {2017}, month = {2017 Mar 30}, abstract = {

Cellular plasma membranes are laterally heterogeneous, featuring a variety of distinct subcompartments that differ in their biophysical properties and composition. A large number of studies have focused on understanding the basis for this heterogeneity and its physiological relevance. The membrane raft hypothesis formalized a physicochemical principle for a subtype of such lateral membrane heterogeneity, in which the preferential associations between cholesterol and saturated lipids drive the formation of relatively packed (or ordered) membrane domains that selectively recruit certain lipids and proteins. Recent studies have yielded new insights into this mechanism and its relevance in vivo, owing primarily to the development of improved biochemical and biophysical technologies.

}, issn = {1471-0080}, doi = {10.1038/nrm.2017.16}, author = {Sezgin, Erdinc and Levental, Ilya and Mayor, Satyajit and Eggeling, Christian} } @article {3546, title = {Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer.}, journal = {Proc Natl Acad Sci U S A}, volume = {113}, year = {2016}, month = {2016 Mar 22}, pages = {E1645-54}, abstract = {

The surface of a living cell provides a platform for receptor signaling, protein sorting, transport, and endocytosis, whose regulation requires the local control of membrane organization. Previous work has revealed a role for dynamic actomyosin in membrane protein and lipid organization, suggesting that the cell surface behaves as an active composite composed of a fluid bilayer and a thin film of active actomyosin. We reconstitute an analogous system in vitro that consists of a fluid lipid bilayer coupled via membrane-associated actin-binding proteins to dynamic actin filaments and myosin motors. Upon complete consumption of ATP, this system settles into distinct phases of actin organization, namely bundled filaments, linked apolar asters, and a lattice of polar asters. These depend on actin concentration, filament length, and actin/myosin ratio. During formation of the polar aster phase, advection of the self-organizing actomyosin network drives transient clustering of actin-associated membrane components. Regeneration of ATP supports a constitutively remodeling actomyosin state, which in turn drives active fluctuations of coupled membrane components, resembling those observed at the cell surface. In a multicomponent membrane bilayer, this remodeling actomyosin layer contributes to changes in the extent and dynamics of phase-segregating domains. These results show how local membrane composition can be driven by active processes arising from actomyosin, highlighting the fundamental basis of the active composite model of the cell surface, and indicate its relevance to the study of membrane organization.

}, issn = {1091-6490}, doi = {10.1073/pnas.1514030113}, author = {K{\"o}ster, Darius Vasco and Husain, Kabir and Iljazi, Elda and Bhat, Abrar and Bieling, Peter and Mullins, R Dyche and Rao, Madan and Mayor, Satyajit} } @article {3544, title = {Cortical actin and the plasma membrane: inextricably intertwined.}, journal = {Curr Opin Cell Biol}, volume = {38}, year = {2016}, month = {2016 Feb}, pages = {81-9}, abstract = {

The plasma membrane serves as a barrier, separating the cell from its external environment. Simultaneously it acts as a site for information transduction, entry of nutrients, receptor signaling, and adapts to the shape of the cell. This requires local control of organization at multiple scales in this heterogeneous fluid lipid bilayer with a plethora of proteins and a closely juxtaposed dynamic cortical cytoskeleton. New membrane models highlight the influence of the underlying cortical actin on the diffusion of membrane components. Myosin motors as well as proteins that remodel actin filaments have additionally been implicated in defining the organization of many membrane constituents. Here we provide a perspective of the intimate relationship of the membrane lipid matrix and the underlying cytoskeleton.

}, issn = {1879-0410}, doi = {10.1016/j.ceb.2016.02.021}, author = {K{\"o}ster, Darius V and Mayor, Satyajit} } @article {11611, title = {Endocytosis of Wingless via a dynamin-independent pathway is necessary for signaling in Drosophila wing discs.}, journal = {Proc Natl Acad Sci U S A}, year = {2016}, month = {2016 Oct 25}, abstract = {

Endocytosis of ligand-receptor complexes regulates signal transduction during development. In particular, clathrin and dynamin-dependent endocytosis has been well studied in the context of patterning of the Drosophila wing disc, wherein apically secreted Wingless (Wg) encounters its receptor, DFrizzled2 (DFz2), resulting in a distinctive dorso-ventral pattern of signaling outputs. Here, we directly track the endocytosis of Wg and DFz2 in the wing disc and demonstrate that Wg is endocytosed from the apical surface devoid of DFz2 via a dynamin-independent CLIC/GEEC pathway, regulated by Arf1, Garz, and class I PI3K. Subsequently, Wg containing CLIC/GEEC endosomes fuse with DFz2-containing vesicles derived from the clathrin and dynamin-dependent endocytic pathway, which results in a low pH-dependent transfer of Wg to DFz2 within the merged and acidified endosome to initiate Wg signaling. The employment of two distinct endocytic pathways exemplifies a mechanism wherein cells in tissues leverage multiple endocytic pathways to spatially regulate signaling.

}, issn = {1091-6490}, doi = {10.1073/pnas.1610565113}, author = {Hemalatha, Anupama and Prabhakara, Chaitra and Mayor, Satyajit} } @article {3560, title = {GPI-anchored protein organization and dynamics at the cell surface.}, journal = {J Lipid Res}, volume = {57}, year = {2016}, month = {2016 Feb}, pages = {159-75}, abstract = {

The surface of eukaryotic cells is a multi-component fluid bilayer in which glycosylphosphatidylinositol (GPI)-anchored proteins are an abundant constituent. In this review, we discuss the complex nature of the organization and dynamics of GPI-anchored proteins at multiple spatial and temporal scales. Different biophysical techniques have been utilized for understanding this organization, including fluorescence correlation spectroscopy, fluorescence recovery after photobleaching, single particle tracking, and a number of super resolution methods. Major insights into the organization and dynamics have also come from exploring the short-range interactions of GPI-anchored proteins by fluorescence (or F{\"o}rster) resonance energy transfer microscopy. Based on the nanometer to micron scale organization, at the microsecond to the second time scale dynamics, a picture of the membrane bilayer emerges where the lipid bilayer appears inextricably intertwined with the underlying dynamic cytoskeleton. These observations have prompted a revision of the current models of plasma membrane organization, and suggest an active actin-membrane composite.

}, issn = {1539-7262}, doi = {10.1194/jlr.R062885}, author = {Saha, Suvrajit and Anilkumar, Anupama Ambika and Mayor, Satyajit} } @article {866, title = {Building endocytic pits without clathrin.}, journal = {Nat Rev Mol Cell Biol}, volume = {16}, year = {2015}, month = {2015 May}, pages = {311-21}, abstract = {

How endocytic pits are built in clathrin- and caveolin-independent endocytosis still remains poorly understood. Recent insight suggests that different forms of clathrin-independent endocytosis might involve the actin-driven focusing of membrane constituents, the lectin-glycosphingolipid-dependent construction of endocytic nanoenvironments, and Bin-Amphiphysin-Rvs (BAR) domain proteins serving as scaffolding modules. We discuss the need for different types of internalization processes in the context of diverse cellular functions, the existence of clathrin-independent mechanisms of cargo recruitment and membrane bending from a biological and physical perspective, and finally propose a generic scheme for the formation of clathrin-independent endocytic pits.

}, keywords = {Actins, Animals, Cell Membrane, Clathrin, Endocytosis, Humans, Lectins, Metabolic Networks and Pathways}, issn = {1471-0080}, doi = {10.1038/nrm3968}, author = {Johannes, Ludger and Parton, Robert G and Bassereau, Patricia and Mayor, Satyajit} } @article {1019, title = {Diffusion of GPI-anchored proteins is influenced by the activity of dynamic cortical actin.}, journal = {Mol Biol Cell}, volume = {26}, year = {2015}, month = {2015 Nov 5}, pages = {4033-45}, abstract = {

Molecular diffusion at the surface of living cells is believed to be predominantly driven by thermal kicks. However, there is growing evidence that certain cell surface molecules are driven by the fluctuating dynamics of cortical cytoskeleton. Using fluorescence correlation spectroscopy, we measure the diffusion coefficient of a variety of cell surface molecules over a temperature range of 24-37{\textdegree}C. Exogenously incorporated fluorescent lipids with short acyl chains exhibit the expected increase of diffusion coefficient over this temperature range. In contrast, we find that GPI-anchored proteins exhibit temperature-independent diffusion over this range and revert to temperature-dependent diffusion on cell membrane blebs, in cells depleted of cholesterol, and upon acute perturbation of actin dynamics and myosin activity. A model transmembrane protein with a cytosolic actin-binding domain also exhibits the temperature-independent behavior, directly implicating the role of cortical actin. We show that diffusion of GPI-anchored proteins also becomes temperature dependent when the filamentous dynamic actin nucleator formin is inhibited. However, changes in cortical actin mesh size or perturbation of branched actin nucleator Arp2/3 do not affect this behavior. Thus cell surface diffusion of GPI-anchored proteins and transmembrane proteins that associate with actin is driven by active fluctuations of dynamic cortical actin filaments in addition to thermal fluctuations, consistent with expectations from an "active actin-membrane composite" cell surface.

}, issn = {1939-4586}, doi = {10.1091/mbc.E15-06-0397}, author = {Saha, Suvrajit and Lee, Il-Hyung and Polley, Anirban and Groves, Jay T and Rao, Madan and Mayor, Satyajit} } @article {863, title = {Homo-FRET imaging highlights the nanoscale organization of cell surface molecules.}, journal = {Methods Mol Biol}, volume = {1251}, year = {2015}, month = {2015}, pages = {151-73}, abstract = {

Several models have been proposed to understand the structure and organization of the plasma membrane in living cells. Predicated on equilibrium thermodynamic principles, the fluid-mosaic model of Singer and Nicholson and the model of lipid domains (or membrane rafts) are dominant models, which account for a fluid bilayer and functional lateral heterogeneity of membrane components, respectively. However, the constituents of the membrane and its composition are not maintained by equilibrium mechanisms. Indeed, the living cell membrane is a steady state of a number of active processes, namely, exocytosis, lipid synthesis and transbilayer flip-flop, and endocytosis. In this active milieu, many lipid constituents of the cell membrane exhibit a nanoscale organization that is also at odds with passive models based on chemical equilibrium. Here we provide a detailed description of microscopy and cell biological methods that have served to provide valuable information regarding the nature of nanoscale organization of lipid components in a living cell.

}, keywords = {Animals, Cell Membrane, CHO Cells, Cricetinae, Cricetulus, Fluorescence Polarization, Fluorescence Resonance Energy Transfer, Lipid Bilayers, Microscopy, Fluorescence, Models, Biological, Nanotechnology}, issn = {1940-6029}, doi = {10.1007/978-1-4939-2080-8_9}, author = {Saha, Suvrajit and Raghupathy, Riya and Mayor, Satyajit} } @article {864, title = {Live cell plasma membranes do not exhibit a miscibility phase transition over a wide range of temperatures.}, journal = {J Phys Chem B}, volume = {119}, year = {2015}, month = {2015 Mar 26}, pages = {4450-9}, abstract = {

Lipid/cholesterol mixtures derived from cell membranes as well as their synthetic reconstitutions exhibit well-defined miscibility phase transitions and critical phenomena near physiological temperatures. This suggests that lipid/cholesterol-mediated phase separation plays a role in the organization of live cell membranes. However, macroscopic lipid-phase separation is not generally observed in cell membranes, and the degree to which properties of isolated lipid mixtures are preserved in the cell membrane remain unknown. A fundamental property of phase transitions is that the variation of tagged particle diffusion with temperature exhibits an abrupt change as the system passes through the transition, even when the two phases are distributed in a nanometer-scale emulsion. We support this using a variety of Monte Carlo and atomistic simulations on model lipid membrane systems. However, temperature-dependent fluorescence correlation spectroscopy of labeled lipids and membrane-anchored proteins in live cell membranes shows a consistently smooth increase in the diffusion coefficient as a function of temperature. We find no evidence of a discrete miscibility phase transition throughout a wide range of temperatures: 14-37 {\textdegree}C. This contrasts the behavior of giant plasma membrane vesicles (GPMVs) blebbed from the same cells, which do exhibit phase transitions and macroscopic phase separation. Fluorescence lifetime analysis of a DiI probe in both cases reveals a significant environmental difference between the live cell and the GPMV. Taken together, these data suggest the live cell membrane may avoid the miscibility phase transition inherent to its lipid constituents by actively regulating physical parameters, such as tension, in the membrane.

}, issn = {1520-5207}, doi = {10.1021/jp512839q}, author = {Lee, Il-Hyung and Saha, Suvrajit and Polley, Anirban and Huang, Hector and Mayor, Satyajit and Rao, Madan and Groves, Jay T} } @article {3536, title = {Oligomerization and endocytosis of Hedgehog is necessary for its efficient exovesicular secretion.}, journal = {Mol Biol Cell}, volume = {26}, year = {2015}, month = {2015 Dec 15}, pages = {4700-17}, abstract = {

Hedgehog (Hh) is a secreted morphogen involved in both short- and long-range signaling necessary for tissue patterning during development. It is unclear how this dually lipidated protein is transported over a long range in the aqueous milieu of interstitial spaces. We previously showed that the long-range signaling of Hh requires its oligomerization. Here we show that Hh is secreted in the form of exovesicles. These are derived by the endocytic delivery of cell surface Hh to multivesicular bodies (MVBs) via an endosomal sorting complex required for transport (ECSRT)-dependent process. Perturbations of ESCRT proteins have a selective effect on long-range Hh signaling in Drosophila wing imaginal discs. Of importance, oligomerization-defective Hh is inefficiently incorporated into exovesicles due to its poor endocytic delivery to MVBs. These results provide evidence that nanoscale organization of Hh regulates the secretion of Hh on ESCRT-derived exovesicles, which in turn act as a vehicle for long-range signaling.

}, issn = {1939-4586}, doi = {10.1091/mbc.E15-09-0671}, author = {Parchure, Anup and Vyas, Neha and Ferguson, Charles and Parton, Robert G and Mayor, Satyajit} } @article {1007, title = {Physical principles of membrane remodelling during cell mechanoadaptation.}, journal = {Nat Commun}, volume = {6}, year = {2015}, month = {2015}, pages = {7292}, abstract = {

Biological processes in any physiological environment involve changes in cell shape, which must be accommodated by their physical envelope--the bilayer membrane. However, the fundamental biophysical principles by which the cell membrane allows for and responds to shape changes remain unclear. Here we show that the 3D remodelling of the membrane in response to a broad diversity of physiological perturbations can be explained by a purely mechanical process. This process is passive, local, almost instantaneous, before any active remodelling and generates different types of membrane invaginations that can repeatedly store and release large fractions of the cell membrane. We further demonstrate that the shape of those invaginations is determined by the minimum elastic and adhesive energy required to store both membrane area and liquid volume at the cell-substrate interface. Once formed, cells reabsorb the invaginations through an active process with duration of the order of minutes.

}, issn = {2041-1723}, doi = {10.1038/ncomms8292}, author = {Kosmalska, Anita Joanna and Casares, Laura and Elosegui-Artola, Alberto and Thottacherry, Joseph Jose and Moreno-Vicente, Roberto and Gonz{\'a}lez-Tarrag{\'o}, V{\'\i}ctor and del Pozo, Miguel {\'A}ngel and Mayor, Satyajit and Arroyo, Marino and Navajas, Daniel and Trepat, Xavier and Gauthier, Nils C and Roca-Cusachs, Pere} } @article {882, title = {Salt-induced remodeling of spatially restricted clathrin-independent endocytic pathways in Arabidopsis root.}, journal = {Plant Cell}, volume = {27}, year = {2015}, month = {2015 Apr}, pages = {1297-315}, abstract = {

Endocytosis is a ubiquitous cellular process that is characterized well in animal cells in culture but poorly across intact, functioning tissue. Here, we analyze endocytosis throughout the Arabidopsis thaliana root using three classes of probes: a lipophilic dye, tagged transmembrane proteins, and a lipid-anchored protein. We observe a stratified distribution of endocytic processes. A clathrin-dependent endocytic pathway that internalizes transmembrane proteins functions in all cell layers, while a sterol-dependent, clathrin-independent pathway that takes up lipid and lipid-anchored proteins but not transmembrane proteins is restricted to the epidermal layer. Saline stress induces a third pathway that is clathrin-independent, nondiscriminatory in its choice of cargo, and operates across all layers of the root. Concomitantly, small acidic compartments in inner cell layers expand to form larger vacuole-like structures. Plants lacking function of the Rab-GEF (guanine nucleotide exchange factor) VPS9a (vacuolar protein sorting 9A) neither induce the third endocytic pathway nor expand the vacuolar system in response to salt stress. The plants are also hypersensitive to salt. Thus, saline stress reconfigures clathrin-independent endocytosis and remodels endomembrane systems, forming large vacuoles in the inner cell layers, both processes correlated by the requirement of VPS9a activity.

}, keywords = {Arabidopsis, Biological Transport, Clathrin, Endocytosis, Plant Roots, Signal Transduction, Sodium Chloride}, issn = {1532-298X}, doi = {10.1105/tpc.15.00154}, author = {Baral, Anirban and Irani, Niloufer G and Fujimoto, Masaru and Nakano, Akihiko and Mayor, Satyajit and Mathew, M K} } @article {867, title = {Tailor-made ezrin actin binding domain to probe its interaction with actin in-vitro.}, journal = {PLoS One}, volume = {10}, year = {2015}, month = {2015}, pages = {e0123428}, abstract = {

Ezrin, a member of the ERM (Ezrin/Radixin/Moesin) protein family, is an Actin-plasma membrane linker protein mediating cellular integrity and function. In-vivo study of such interactions is a complex task due to the presence of a large number of endogenous binding partners for both Ezrin and Actin. Further, C-terminal actin binding capacity of the full length Ezrin is naturally shielded by its N-terminal, and only rendered active in the presence of Phosphatidylinositol bisphosphate (PIP2) or phosphorylation at the C-terminal threonine. Here, we demonstrate a strategy for the design, expression and purification of constructs, combining the Ezrin C-terminal actin binding domain, with functional elements such as fusion tags and fluorescence tags to facilitate purification and fluorescence microscopy based studies. For the first time, internal His tag was employed for purification of Ezrin actin binding domain based on in-silico modeling. The functionality (Ezrin-actin interaction) of these constructs was successfully demonstrated by using Total Internal Reflection Fluorescence Microscopy. This design can be extended to other members of the ERM family as well.

}, issn = {1932-6203}, doi = {10.1371/journal.pone.0123428}, author = {Shrivastava, Rohini and K{\"o}ster, Darius and Kalme, Sheetal and Mayor, Satyajit and Neerathilingam, Muniasamy} } @article {881, title = {Transbilayer lipid interactions mediate nanoclustering of lipid-anchored proteins.}, journal = {Cell}, volume = {161}, year = {2015}, month = {2015 Apr 23}, pages = {581-94}, abstract = {

Understanding how functional lipid domains in live cell membranes are generated has posed a challenge. Here, we show that transbilayer interactions are necessary for the generation of cholesterol-dependent nanoclusters of GPI-anchored proteins mediated by membrane-adjacent dynamic actin filaments. We find that long saturated acyl-chains are required for forming GPI-anchor nanoclusters. Simultaneously, at the inner leaflet, long acyl-chain-containing phosphatidylserine (PS) is necessary for transbilayer coupling. All-atom molecular dynamics simulations of asymmetric multicomponent-membrane bilayers in a mixed phase provide evidence that immobilization of long saturated acyl-chain lipids at either leaflet stabilizes cholesterol-dependent transbilayer interactions forming local domains with characteristics similar to a liquid-ordered (lo) phase. This is verified by experiments wherein immobilization of long acyl-chain lipids at one leaflet effects transbilayer interactions of corresponding lipids at the opposite leaflet. This suggests a general mechanism for the generation and stabilization of nanoscale cholesterol-dependent and actin-mediated lipid clusters in live cell membranes.

}, keywords = {Actins, Animals, Cell Membrane, CHO Cells, Cricetulus, Glycosylphosphatidylinositols, Lipid-Linked Proteins, Molecular Dynamics Simulation, Phosphatidylserines}, issn = {1097-4172}, doi = {10.1016/j.cell.2015.03.048}, author = {Raghupathy, Riya and Anilkumar, Anupama Ambika and Polley, Anirban and Singh, Parvinder Pal and Yadav, Mahipal and Johnson, Charles and Suryawanshi, Sharad and Saikam, Varma and Sawant, Sanghapal D and Panda, Aniruddha and Guo, Zhongwu and Vishwakarma, Ram A and Rao, Madan and Mayor, Satyajit} } @article {80913, title = {Pathways of clathrin-independent endocytosis.}, journal = {Nat Rev Mol Cell Biol}, volume = {8}, year = {2007}, month = {2007 Aug}, pages = {603-12}, abstract = {

There are numerous ways that endocytic cargo molecules may be internalized from the surface of eukaryotic cells. In addition to the classical clathrin-dependent mechanism of endocytosis, several pathways that do not use a clathrin coat are emerging. These pathways transport a diverse array of cargoes and are sometimes hijacked by bacteria and viruses to gain access to the host cell. Here, we review our current understanding of various clathrin-independent mechanisms of endocytosis and propose a classification scheme to help organize the data in this complex and evolving field.

}, keywords = {Animals, Bacteria, Biological Transport, Active, Clathrin, Endocytosis, Eukaryotic Cells, Humans, Virus Internalization, Viruses}, issn = {1471-0072}, doi = {10.1038/nrm2216}, author = {Mayor, Satyajit and Pagano, Richard E} }