Main Projects

The Human Lung Cell Atlas

Source: Herbert Schiller, Helmholtz Zentrum München, modified from Human Cell Atlas

Single cell genomics is revolutionizing biology and medicine, combining the advantages of bulk sequencing techniques and microscopic analyses of single cells. Rapid technological advances now allow the profiling of genomes, transcriptomes and epigenomes at an unprecedented level of resolution (see https://www.singlecell.de/). We employ the recently developed Drop-seq method, which uses microfluidics to capture single cells along with sets of uniquely barcoded primer beads into nanoliter-sized aqueous droplets. The smart barcoding approach in Drop-seq allows the massively parallel, and thus cost-effective, analysis of mRNA transcripts from thousands of individual cells simultaneously while remembering transcripts’ cell of origin (Macosko et al 2015).

As collaborative member of the Human Cell Atlas (HCA) Initiative we use single cell RNA-seq to unravel the cellular composition of both mouse and human lungs. See our contribution to the HCA white paper online: https://www.humancellatlas.org/files/HCA_WhitePaper_18Oct2017.pdf 


Chronic lung diseases are a leading cause of death worldwide and are on the rise in a rapidly aging society. The Lung Cell Atlas will reveal the true complexity of cellular composition and spatial organization of the various multicellular units that constitute human lungs. Together with our HCA and DZL (German Center for Lung Research) partners we are currently starting to develop methodologies and an infrastructure for standardized and validated characterization and integration of single-cell transcriptomic and proteomic data into the spatial context of lung tissue architecture. Already the first draft of the Lung Cell Atlas will include well defined cohorts of chronic lung disease samples (including COPD and IPF) in order to proof feasibility and power of delineating the healthy condition from a continuum of possible disease states in individual humans. A Human Lung Cell Atlas will tremendously accelerate both basic and translational research on lung development and disease.

Links:

https://www.humancellatlas.org/

https://www.singlecell.de/

https://www.humancellatlas.org/files/HCA_WhitePaper_18Oct2017.pdf

The Extracellular Matrix in Lung Regeneration and Fibrosis

Despite the tremendous interest in ECM biology in the context of regenerative medicine and cancer research, the systematic characterization of extracellular matrix niches is a much underexplored area of research. Lung regeneration depends on the reactivation of developmental programs, where the crosstalk between mesenchyme and epithelium via secreted proteins is essential. In a process called fibrogenesis, several mesenchymal cell populations secrete and assemble a specialized provisional ECM, which acts as a scaffold and master regulator of developmental programs in concert with extracellular morphogens, such as growth factors, cytokines, or chemokines. We recently characterized the murine lung proteome, including its extracellular matrix content and organization, to unprecedented depth, which enabled a protein-centric systems biology view on tissue injury, fibrosis and repair (Schiller et al, 2015).

Mobilization of tissue-resident progenitor- and stem-cell populations needed for regeneration of the correct alveolar epithelial organization after injury is initiated in the early inflammatory and fibrogenic phase after lung injury. For instance, the perivascular niche plays a key role by contributing paracrine factors and the basement membrane (BM) between epithelial cell layer and capillary endothelial cells of the alveoli, in particular, is strategically located to guide stem cell behavior. Nevertheless, the heterogeneity of basement membrane composition and architecture, its specific functions as part of a stem cell niche, and its plasticity after injury has not been fully characterized so far.

We plan to use a variety of state of the art methods including immunofluorescence imaging, mass spectrometry driven proteomics and single cell expression analysis to pinpoint spatio-temporal changes of ECM composition in lung development, injury repair and metastatic colonization. The functional implications of selected proteins are assessed using transgenic mouse lines or CRISPR/Cas9 constructs.

References

Schiller HB, Fernandez IE, Burgstaller G, Schaab C, Scheltema RA, Schwarzmayr T, Strom TM, Eickelberg O, Mann M (2015) Time- and compartment-resolved proteome profiling of the extracellular niche in lung injury and repair. Mol Syst Biol 11: 819

Unraveling the Molecular Architecture of Extracellular Matrix Niches

Source: David S. Doodsell

Metazoans evolved ~300 large multidomain extracellular matrix (ECM) proteins, which interact with each other and cells to form elaborate composite biomaterials that shape both the form and function of tissues. To determine the topology of this highly complex and insoluble network at molecular resolution in its tissue context has not been possible due to technical challenges. The recent successful combination of chemical crosslinking with mass spectrometry (CXMS), promises to revolutionize structural biology and protein interaction studies, and opens the way for ECM interactomics in situ. In collaboration with Dr. Richard Scheltema (Utrecht University) we are currently developing several mass spectrometry (MS) methods and software (including CXMS) for structural proteomics of the ECM. Using collision induced dissociation (CID) of labile chemical crosslinkers we recently developed a novel CXMS analysis workflow, utilizing the Q-exactive quadrupole-orbitrap type mass spectrometer. As a proof of concept, we successfully used our new method in combination with protein crystallography and cryo‐EM to obtain the first complete pseudoatomic model of a type‐III CRISPR complex (Benda et al, 2014).

We currently work on ECM protein complex retrieval methods from tissues as well as CXMS approaches to develop and apply `discovery mode´ analysis tools for protein interactions from complex tissue environments in situ.

References

Benda C, Ebert J, Scheltema RA, Schiller HB, Baumgartner M, Bonneau F, Mann M, Conti E (2014) Structural Model of a CRISPR RNA-Silencing Complex Reveals the RNA-Target Cleavage Activity in Cmr4. Mol Cell 56: 43-54

Functional proteomics of cellular rigidity sensing mechanisms

Source: Herbert Schiller, Max Iglesias, modified after EMBO Reports Cover art

In this project, we aim at an understanding of fundamental molecular principles of cellular mechanosensing by using a combination of advanced cell biology techniques with mass spectrometry driven (phospho-)proteomics and CRISPR/Cas9 mediated genome editing.

In the last 10 years the field of mechanobiology rapidly evolved and a variety of data strengthens the hypothesis that most if not all cells process mechanical inputs from their environment. The molecular details of these mechanosensitive signaling cascades are currently not very well characterized. On encounter of their extracellular substrate cells reciprocate the stiffness of that substrate. This mechanoreciprocity is generated by feedback connections between cell-matrix adhesions and the cytoskeleton, which tune the strength of myosin-II mediated contractile forces to an equilibrium of applied force and tensile strength of the ECM substrate (Schiller & Fassler, 2013). The precise molecular nature of many elements in these feedback connections is currently unknown and it is also unclear to which extend an equilibrium of mechanosensing to mechanoresponse differs between cell types and how such differences can be programmed. It is conceivable that regulation of the cellular mechanosensing properties is likely to happen on proteins with functions in cell-matrix adhesions and the cytoskeleton. Both integrin- and cadherin-mediated adhesions connect to the filamentous (F-) actin cytoskeleton by using a variety of adaptor and signalling proteins. These proteins assemble into a dense and highly dynamic network visible as a protein plaque at the plasma membrane, which we refer to as the adhesome.

We and others pioneered the use of mass spectrometry driven proteomics to systematically analyze the molecular composition of the adhesome and its dynamic changes under the influence of mechanical tension. Our own findings revealed that β1-family integrins and associated focal adhesion proteins feed into signaling pathways that produce myosin-II mediated force when bound to fibronectin, while the αv-family of integrins and specific associated focal adhesion proteins respond to myosin-II and matrix stiffness dependent tension at focal adhesions to reinforce the adhesion site (Schiller et al, 2011; Schiller et al, 2013).

Recent developments in phosphoproteomic workflows substantially increase sensitivity of detection and sample throughput, which enables us to follow the dynamic changes of the phosphorylation landscape (serine, threonine, tyrosine phosphorylation on proteins) of cells during cell spreading, polarization and contraction at a depth of more than 10,000 quantified phosphosites. Using this technology we identified interesting subsets of integrin dependent phosphorylation sites that are controlled by ECM substrate stiffness. We will analyze the functional implications of selected sites with the aim of uncovering novel pathways that are important in the control of cellular activities during tissue fibrosis.

References

Schiller HB, Fassler R (2013) Mechanosensitivity and compositional dynamics of cell-matrix adhesions. EMBO Rep 14: 509-519

Schiller HB, Friedel CC, Boulegue C, Fassler R (2011) Quantitative proteomics of the integrin adhesome show a myosin II-dependent recruitment of LIM domain proteins. EMBO Rep 12: 259-266

Schiller HB, Hermann MR, Polleux J, Vignaud T, Zanivan S, Friedel CC, Sun Z, Raducanu A, Gottschalk KE, Thery M, Mann M, Fassler R (2013) beta1- and alphav-class integrins cooperate to regulate myosin II during rigidity sensing of fibronectin-based microenvironments. Nat Cell Biol 15: 625-636