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Integrin nanoclusters can bridge thin matrix fibres to form cell-matrix adhesions

NATURE MATERIALS 2019年第12期18卷 1366-+页

作者： Changede, Rishita Cai, Haogang Wind, Shalom J. Sheetz, Michael P. Natl Univ Singapore Mechanobiol Inst Singapore Singapore Columbia Univ Dept Mech Engn New York NY 10027 USA Argonne Natl Lab Nanosci & Technol Div Ctr Nanoscale Mat Lemont IL USA Columbia Univ Dept Appl Phys & Appl Math New York NY USA Univ Texas Med Branch Biochem & Mol Biol Dept Mol Mechanomed Program Galveston TX 77555 USA

Integrin-mediated cell-matrix adhesions are key to sensing the geometry and rigidity of extracellular environments and influence vital cellular processes. In vivo, the extracellular matrix is composed of fibrous arrays. To understand the fibre geometries that are required for adhesion formation, we patterned nanolines of various line widths and arrangements in single, crossing or paired arrays with the integrin-binding peptide Arg-Gly-Asp. Single thin lines (width <= 30 nm) did not support cell spreading or formation of focal adhesions, despite the presence of a high density of Arg-Gly-Asp, but wide lines (>40 nm) did. Using super-resolution microscopy, we observed stable, dense integrin clusters formed on parallel (within 110 nm) or crossing thin lines (mimicking a matrix mesh) similar to those on continuous substrates. These dense clusters bridged the line pairs by recruiting activated but unliganded integrins, as verified by integrin mutants unable to bind ligands that coclustered with ligand-bound integrins when present in an active extended conformation. Thus, in a fibrous extracellular matrix mesh, stable integrin nano-clusters bridge between thin (<= 30 nm) matrix fibres and bring about downstream consequences of cell motility and growth.

关键词： Ligands

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Fungi stabilize connectivity in the lung and skin microbial ecosystems

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MICROBIOME 2018年第1期6卷 1-14页

作者： Tipton, Laura Mueller, Christian L. Kurtz, Zachary D. Huang, Laurence Kleerup, Eric Morris, Alison Bonneau, Richard Ghedin, Elodie Univ Pittsburgh Sch Med Dept Computat & Syst Biol Pittsburgh PA 15260 USA NYU Ctr Genom & Syst Biol New York NY 10003 USA Simons Fdn Ctr Computat Biol Flatiron Inst New York NY 10010 USA NYU Sch Med Dept Microbiol New York NY 10016 USA Univ Calif San Francisco Div Pulm & Crit Care Med San Francisco CA 94110 USA Univ Calif San Francisco HIV AIDS Div San Francisco CA 94110 USA Univ Calif Los Angeles David Geffen Sch Med Dept Med Div Pulm & Crit Care Los Angeles CA 90095 USA Univ Pittsburgh Sch Med Div Pulm Allergy & Crit Care Med Pittsburgh PA USA NYU Coll Global Publ Hlth Dept Epidemiol New York NY USA

Background: No microbe exists in isolation, and few live in environments with only members of their own kingdom or domain. As microbiome studies become increasingly more interested in the interactions between microbes than in cataloging which microbes are present, the variety of microbes in the community should be considered. However, the majority of ecological interaction networks for microbiomes built to date have included only bacteria. Joint association inference across multiple domains of life, e.g., fungal communities (the mycobiome) and bacterial communities, has remained largely elusive. Results: Here, we present a novel extension of the SParse InversE Covariance estimation for Ecological ASsociation Inference (SPIEC-EASI) framework that allows statistical inference of cross-domain associations from targeted amplicon sequencing data. For human lung and skin micro- and mycobiomes, we show that cross-domain networks exhibit higher connectivity, increased network stability, and similar topological re-organization patterns compared to single-domain networks. We also validate in vitro a small number of cross-domain interactions predicted by the skin association network. Conclusions: For the human lung and skin micro- and mycobiomes, our findings suggest that fungi play a stabilizing role in ecological network organization. Our study suggests that computational efforts to infer association networks that include all forms of microbial life, paired with large-scale culture-based association validation experiments, will help formulate concrete hypotheses about the underlying biological mechanisms of species interactions and, ultimately, help understand microbial communities as a whole.

关键词： Cross-domain Associations Single Network Domain Mycobiome Sparse Inverse Covariance Estimation Operational Taxonomic Units (OTUs)

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Full control of ligand positioning reveals spatial thresholds for T cell receptor triggering

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NATURE NANOTECHNOLOGY 2018年第7期13卷 610-+页

作者： Cai, Haogang Muller, James Depoil, David Mayya, Viveka Sheetz, Michael P. Dustin, Michael L. Wind, Shalom J. Columbia Univ Dept Mech Engn New York NY 10027 USA NYU Sch Med Skirball Inst Biomol Med New York NY 10003 USA Univ Oxford Nuffield Dept Orthoped Rheumatol & Musculoskeleta Kennedy Inst Rheumatol Oxford England Natl Univ Singapore Mechanobiol Inst Singapore Singapore Singapore Columbia Univ Dept Biol Sci New York NY 10027 USA Columbia Univ Dept Appl Phys & Appl Math New York NY 10027 USA Argonne Natl Lab Ctr Nanoscale Mat 9700 S Cass Ave Argonne IL 60439 USA

Elucidating the rules for receptor triggering in cell-cell and cell-matrix contacts requires precise control of ligand positioning in three dimensions. Here, we use the T cell receptor (TCR) as a model and subject T cells to different geometric arrangements of ligands, using a nanofabricated single-molecule array platform. This comprises monovalent TCR ligands anchored to lithographically patterned nanoparticle clusters surrounded by mobile adhesion molecules on a supported lipid bilayer. The TCR ligand could be co-planar with the supported lipid bilayer (2D), excluding the CD45 transmembrane tyrosine phosphatase, or elevated by 10 nm on solid nanopedestals (3D), allowing closer access of CD45 to engaged TCR. The two configurations resulted in different T cell responses, depending on the lateral spacing between the ligands. These results identify the important contributions of lateral and axial components of ligand positioning and create a more complete foundation for receptor engineering for immunotherapy.

关键词： Ligands

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Durable Interactions of T Cells with T Cell Receptor Stimuli in the Absence of a Stable Immunological Synapse

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CELL REPORTS 2018年第2期22卷 340-349页

作者： Mayya, Viveka Judokusumo, Edward Abu Shah, Enas Peel, Christopher G. Neiswanger, Willie Depoil, David Blair, David A. Wiggins, Chris H. Kam, Lance C. Dustin, Michael L. Univ Oxford Kennedy Inst Rheumatol Oxford OX3 7FY England NYU Sch Med Skirball Inst Biomol Med New York NY 10016 USA Columbia Univ Dept Biol Engn New York NY 10027 USA Carnegie Mellon Univ Machine Learning Dept Pittsburgh PA 15213 USA Columbia Univ Dept Appl Phys & Appl Math New York NY 10027 USA Immunocore Ltd Abingdon OX14 4RY Oxon England Boehringer Ingelheim Corp Ridgefield CT 06877 USA

T cells engage in two modes of interaction with antigen-presenting surfaces: stable synapses and motile kinapses. Although it is surmised that durable interactions of T cells with antigen-presenting cells involve synapses, in situ 3D imaging cannot resolve the mode of interaction. We have established in vitro 2D platforms and quantitative metrics to determine cell-intrinsic modes of interaction when T cells are faced with spatially continuous or restricted stimulation. All major resting human T cell subsets, except memory CD8 T cells, spend more time in the kinapse mode on continuous stimulatory surfaces. Surprisingly, we did not observe any concordant relationship between the mode and durability of interaction on cell-sized stimulatory spots. Naive CD8 T cells maintain kinapses for more than 3 hr before leaving stimulatory spots, whereas their memory counterparts maintain synapses for only an hour before leaving. Thus, durable interactions do not require stable synapses.

关键词： adhesion chemokines immunological synapse kinapse live imaging lymphocytes micro-contact printing migration

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Improving T Cell Expansion with a Soft Touch

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NANO LETTERS 2017年第2期17卷 821-826页

作者： Lambert, Lester H. Goebrecht, Geraldine K. E. De Leo, Sarah E. O'Connor, Roddy S. Nunez-Cruz, Selene Li, Tai-De Yuan, Jinglun Milone, Michael C. Kam, Lance C. Columbia Univ Dept Biomed Engn New York NY 10027 USA Univ Penn Dept Pathol & Lab Med Perelman Sch Med Philadelphia PA 19104 USA Univ Penn Ctr Cellular Immunotherapies Perelman Sch Med Philadelphia PA 19104 USA CUNY Adv Sci Res Ctr New York NY 10031 USA

Protein-coated microbeads provide a consistent approach for activating and expanding populations of T cells for immunotherapy but do not fully capture the properties of antigen presenting cells. In this report, we enhance T cell expansion by replacing the conventional, rigid bead with a mechanically soft elastomer. Polydimethylsiloxane (PDMS) was prepared in a microbead format and modified with activating antibodies to CD3 and CD28. A total of three different formulations of PDMS provided an extended proliferative phase in both CD4(+)-only and mixed CD4(+)- CD8(+) T cell preparations. CD8(+) T cells retained cytotoxic function, as measured by a set of biomarkers (perforin production, LAMP2 mobilization, and IFN-gamma secretion) and an in vivo assay of targeted cell killing. Notably, PDMS beads presented a nanoscale polymer structure and higher rigidity than that associated with conventional bulk material. These data suggest T cells respond to this higher rigidity, indicating an unexpected effect of curing conditions. Together, these studies demonstrate that adopting mechanobiology ideas into the bead platform can provide new tools for T cell based immunotherapy.

关键词： T cell mechanobiology immunotherapy nanoscale rigidity

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EGFR and HER2 activate rigidity sensing only on rigid matrices

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NATURE MATERIALS 2017年第7期16卷 775-+页

作者： Saxena, Mayur Liu, Shuaimin Yang, Bo Hajal, Cynthia Changede, Rishita Hu, Junqiang Wolfenson, Haguy Hone, James Sheetz, Michael P. Columbia Univ Dept Biomed Engn New York NY 10027 USA Columbia Univ Dept Mech Engn New York NY 10027 USA Natl Univ Singapore Mechanobiol Inst Singapore 117411 Singapore Columbia Univ Dept Biol Sci New York NY 10027 USA Technion Israel Inst Technol Ruth & Bruce Rappaport Fac Med Dept Genet & Dev Biol IL-31096 Haifa Israel

Epidermal growth factor receptor (EGFR) interacts with integrins during cell spreading and motility, but little is known about the role of EGFR in these mechanosensing processes. Here we show, using two different cell lines, that in serum-and EGF-free conditions, EGFR or HER2 activity increase spreading and rigidity-sensing contractions on rigid, but not soft, substrates. Contractions peak after 15-20 min, but diminish by tenfold after 4 h. Addition of EGF at that point increases spreading and contractions, but this can be blocked by myosin-II inhibition. We further show that EGFR and HER2 are activated through phosphorylation by Src family kinases (SFK). On soft surfaces, neither EGFR inhibition nor EGF stimulation have any effect on cell motility. Thus, EGFR or HER2 can catalyse rigidity sensing after associating with nascent adhesions under rigidity-dependent tension downstream of SFK activity. This has broad implications for the roles of EGFR and HER2 in the absence of EGF both for normal and cancerous growth.

关键词： Rigidity

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Improved Glass Surface Passivation for Single-Molecule Nanoarrays

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LANGMUIR 2016年第39期32卷 10034-10041页

作者： Cai, Haogang Wind, Shalom J. Columbia Univ Dept Mech Engn New York NY 10027 USA Columbia Univ Dept Appl Phys & Appl Math New York NY 10027 USA

Single-molecule fluorescence techniques provide a critical tool for probing biomolecular and cellular interactions with unprecedented resolution and, precision. Unfortunately, many of these techniques are hindered by a common problem, namely, the nonspecific adsorption of target biomolecules. This issue is mostly addressed by passivating the glass surfaces with a poly(ethylene glycol) (PEG) brush. This is effective only at low concentrations of the probe molecule because there are defects inherent to polymer brushes formed on glass coverslips due to the presence of surface impurities. Tween-20, a detergent, is a promising alternative that can improve surface passivation, but it is incompatible with living cells, and it also possesses limited selectivity for glass background over metallic nanoparticles, which are frequently used as anchors for the probe molecules. To address these issues, we have developed a more versatile method to improve the PEG passivation. A thin film of hydrogen silsesquioxane (HSQ) is spin-coated and thermally cured on glass coverslips in order to cover the surface impurities. This minimizes the formation of PEG defects and reduces nonspecific adsorption, resulting in an improvement comparable to Tween-20 treatment. This approach was applied to single-molecule nanoarrays of streptavidin bound to AuPd nanodots patterned by e-beam lithography (EBL). The fluorescence signal to background ratio (SBR) on HSQ:coated glass was improved by similar to 4-fold as compared to PEG directly on glass. This improvement enables direct imaging of ordered arrays of single molecules anchored to lithographically patterned arrays of metallic nanodots.

关键词： Adsorption Nanodots Amorphous materials Imaging probes Nanoarchitectures

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Robust classification of protein variation using structural modelling and large-scale data integration

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NUCLEIC ACIDS RESEARCH 2016年第6期44卷 2501-2513页

作者： Baugh, Evan H. Simmons-Edler, Riley Mueller, Christian L. Alford, Rebecca F. Volfovsky, Natalia Lash, Alex E. Bonneau, Richard NYU Dept Biol New York NY 10003 USA NYU Ctr Genom & Syst Biol New York NY 10003 USA NYU Dept Comp Sci New York NY 10003 USA Simons Fdn Simons Ctr Data Anal New York NY 10010 USA Carnegie Mellon Univ Dept Chem 5000 Forbes Ave Pittsburgh PA 15289 USA Commack High Sch Commack NY 11725 USA Simons Fdn New York NY 10010 USA

Existing methods for interpreting protein variation focus on annotating mutation pathogenicity rather than detailed interpretation of variant deleteriousness and frequently use only sequence-based or structure-based information. We present VIPUR, a computational framework that seamlessly integrates sequence analysis and structural modelling (using the Rosetta protein modelling suite) to identify and interpret deleterious protein variants. To train VIPUR, we collected 9477 protein variants with known effects on protein function from multiple organisms and curated structural models for each variant from crystal structures and homology models. VIPUR can be applied to mutations in any organism's proteome with improved generalized accuracy (AUROC .83) and interpretability (AUPR .87) compared to other methods. We demonstrate that VIPUR's predictions of deleteriousness match the biological phenotypes in ClinVar and provide a clear ranking of prediction confidence. We use VIPUR to interpret known mutations associated with inflammation and diabetes, demonstrating the structural diversity of disrupted functional sites and improved interpretation of mutations associated with human diseases. Lastly, we demonstrate VIPUR's ability to highlight candidate variants associated with human diseases by applying VIPUR to de novo variants associated with autism spectrum disorders.

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Molecular Occupancy of Nanodot Arrays

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ACS NANO 2016年第4期10卷 4173-4183页

作者： Cai, Haogang Wolfenson, Haguy Depoil, David Dustin, Michael L. Sheetz, Michael P. Wind, Shalom J. Columbia Univ Dept Mech Engn New York NY 10027 USA Columbia Univ Dept Biol Sci New York NY 10027 USA Columbia Univ Dept Appl Phys & Appl Math New York NY 10027 USA Univ Oxford Kennedy Inst Rheumatol Oxford OX3 7FY England

Single-molecule nanodot arrays, in which a biomolecule of choice (protein, nucleic acid, etc.) is bound to a metallic nanoparticle on a solid substrate, are becoming an increasingly important tool in the study of biomolecular and cellular interactions. We have developed an on-chip measurement protocol to monitor and control the molecular occupancy of nanodots. Arrays of widely spaced nanodots and nanodot clusters were fabricated on glass surfaces by nanolithography and functionalized with fluorescently labeled proteins. The molecular occupancy was determined by monitoring individual fluorophore bleaching events, while accounting for fluorescence quenching effects. We found that the occupancy can be interpreted as a packing problem, and depends on nanodot size and binding ligand concentration, where the latter is easily adjusted to compensate the flexibility of dimension control in nanofabrication. The results are scalable with nanodot cluster size, extending to large area close packed arrays. As an example, the nanoarray platform was used to probe the geometric requirement of T-cell activation at the single-molecule level.

关键词： molecular occupancy stoichiometry single-molecule assays photobleaching fluorescence quenching electron beam lithography

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4C-ker: A Method to Reproducibly Identify Genome-Wide Interactions Captured by 4C-Seq Experiments

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PLOS COMPUTATIONAL BIOLOGY 2016年第3期12卷 e1004780页

作者： Raviram, Ramya Rocha, Pedro P. Mueller, Christian L. Miraldi, Emily R. Badri, Sana Fu, Yi Swanzey, Emily Proudhon, Charlotte Snetkova, Valentina Bonneau, Richard Skok, Jane A. NYU Sch Med Dept Pathol New York NY USA NYU Dept Biol New York NY 10003 USA NYU Courant Inst Math Sci Dept Comp Sci New York NY 10012 USA Simons Ctr Data Anal New York NY USA NYU Sch Med Skirball Inst New York NY USA

4C-Seq has proven to be a powerful technique to identify genome-wide interactions with a single locus of interest (or "bait") that can be important for gene regulation. However, analysis of 4C-Seq data is complicated by the many biases inherent to the technique. An important consideration when dealing with 4C-Seq data is the differences in resolution of signal across the genome that result from differences in 3D distance separation from the bait. This leads to the highest signal in the region immediately surrounding the bait and increasingly lower signals in far-cis and trans. Another important aspect of 4C-Seq experiments is the resolution, which is greatly influenced by the choice of restriction enzyme and the frequency at which it can cut the genome. Thus, it is important that a 4C-Seq analysis method is flexible enough to analyze data generated using different enzymes and to identify interactions across the entire genome. Current methods for 4C-Seq analysis only identify interactions in regions near the bait or in regions located in far-cis and trans, but no method comprehensively analyzes 4C signals of different length scales. In addition, some methods also fail in experiments where chromatin fragments are generated using frequent cutter restriction enzymes. Here, we describe 4C-ker, a Hidden-Markov Model based pipeline that identifies regions throughout the genome that interact with the 4C bait locus. In addition, we incorporate methods for the identification of differential interactions in multiple 4C-seq datasets collected from different genotypes or experimental conditions. Adaptive window sizes are used to correct for differences in signal coverage in near-bait regions, far-cis and trans chromosomes. Using several datasets, we demonstrate that 4C-ker outperforms all existing 4C-Seq pipelines in its ability to reproducibly identify interaction domains at all genomic ranges with different resolution enzymes.

关键词： Genomics B cells Hidden Markov models Genomic signal processing T cells Chromatin Genome analysis Genetic loci

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