Pascal D. Odermatt

Triggered cargo release by encapsulated enzymatic catalysis in capsosomes.

We report the coencapsulation of glutathione reductase and disulfide-linked polymer-oligopeptide conjugates into capsosomes, polymer carrier capsules containing liposomal subcompartments. The architecture of the capsosomes enables a temperature-triggered conversion of oxidized glutathione to its reduced sulfhydryl form by the encapsulated glutathione reductase. The reduced glutathione subsequently induces the release of the encapsulated oligopeptides from the capsosomes by reducing the disulfide linkages of the conjugates. This study highlights the potential of capsosomes to continuously generate a potent antioxidant while simultaneously releasing small molecule therapeutics.

Noncovalent Liposome Linkage and Miniaturization of Capsosomes for Drug Delivery

We report the synthesis of poly(methacrylic acid)-co-(oleyl methacrylate) with three different amounts of oleyl methacrylate and compare the ability of these polymers with that of poly(methacrylic acid)-co-(cholesteryl methacrylate) (PMA(c)) to noncovalently anchor liposomes to polymer layers. We subsequently assembled ∼1 μm diameter PMA(c)-based capsosomes, polymer hydrogel capsules that contain up to ∼2000 liposomal subcompartments, and investigate the potential of these carriers to deliver water-insoluble drugs by encapsulating two different antitumor compounds, thiocoraline or paclitaxel, into the liposomes. The viability of lung cancer cells is used to substantiate the cargo concentration-dependent activity of the capsosomes. These findings cover several crucial aspects for the application of capsosomes as potential drug delivery vehicles.

Chronic inflammation imposes aberrant cell fate in regenerating epithelia through mechanotransduction

Chronic inflammation is associated with a variety of pathological conditions in epithelial tissues, including cancer, metaplasia and aberrant wound healing. In relation to this, a significant body of evidence suggests that aberration of epithelial stem and progenitor cell function is a contributing factor in inflammation-related disease, although the underlying cellular and molecular mechanisms remain to be fully elucidated. In this study, we have delineated the effect of chronic inflammation on epithelial stem/progenitor cells using the corneal epithelium as a model tissue. Using a combination of mouse genetics, pharmacological approaches and in vitro assays, we demonstrate that chronic inflammation elicits aberrant mechanotransduction in the regenerating corneal epithelium. As a consequence, a YAP–TAZ/β-catenin cascade is triggered, resulting in the induction of epidermal differentiation on the ocular surface. Collectively, the results of this study demonstrate that chronic inflammation and mechanotransduction are linked and act to elicit pathological responses in regenerating epithelia.

High-Resolution Correlative Microscopy: Bridging the Gap between Single Molecule Localization Microscopy and Atomic Force Microscopy

Nanoscale characterization of living samples has become essential for modern biology. Atomic force microscopy (AFM) creates topological images of fragile biological structures from biomolecules to living cells in aqueous environments. However, correlating nanoscale structure to biological function of specific proteins can be challenging. To this end we have built and characterized a correlated single molecule localization microscope (SMLM)/AFM that allows localizing specific, labeled proteins within high-resolution AFM images in a biologically relevant context. Using direct stochastic optical reconstruction microscopy (dSTORM)/AFM, we directly correlate and quantify the density of localizations with the 3D topography using both imaging modalities along (F-)actin cytoskeletal filaments. In addition, using photo activated light microscopy (PALM)/AFM, we provide correlative images of bacterial cells in aqueous conditions. Moreover, we report the first correlated AFM/PALM imaging of live mammalian cells. The complementary information provided by the two techniques opens a new dimension for structural and functional nanoscale biology.

High-frequency multimodal atomic force microscopy

Summary Multifrequency atomic force microscopy imaging has been recently demonstrated as a powerful technique for quickly obtaining information about the mechanical properties of a sample. Combining this development with recent gains in imaging speed through small cantilevers holds the promise of a convenient, high-speed method for obtaining nanoscale topography as well as mechanical properties. Nevertheless, instrument bandwidth limitations on cantilever excitation and readout have restricted the ability of multifrequency techniques to fully benefit from small cantilevers. We present an approach for cantilever excitation and deflection readout with a bandwidth of 20 MHz, enabling multifrequency techniques extended beyond 2 MHz for obtaining materials contrast in liquid and air, as well as soft imaging of delicate biological samples.

Division site selection linked to inherited cell surface wave troughs in mycobacteria

Cell division is tightly controlled in space and time to maintain cell size and ploidy within narrow bounds. In bacteria, the canonical Minicell (Min) and nucleoid occlusion (Noc) systems together ensure that division is restricted to midcell after completion of chromosome segregation1. It is unknown how division site selection is controlled in bacteria that lack homologues of the Min and Noc proteins, including mycobacteria responsible for tuberculosis and other chronic infections2. Here, we use correlated optical and atomic-force microscopy3,4 to demonstrate that morphological landmarks (waveform troughs) on the undulating surface of mycobacterial cells correspond to future sites of cell division. Newborn cells inherit wave troughs from the (grand)mother cell and ultimately divide at the centre-most wave trough, making these morphological features the earliest known landmark of future division sites. In cells lacking the chromosome partitioning (Par) system, missegregation of chromosomes is accompanied by asymmetric cell division at off-centre wave troughs, resulting in the formation of anucleate cells. These results demonstrate that inherited morphological landmarks and chromosome positioning together restrict mycobacterial division to the midcell position.

Single-molecule kinetic analysis of HP1-chromatin binding reveals a dynamic network of histone modification and DNA interactions

Abstract Chromatin recruitment of effector proteins involved in gene regulation depends on multivalent interaction with histone post-translational modifications (PTMs) and structural features of the chromatin fiber. Due to the complex interactions involved, it is currently not understood how effectors dynamically sample the chromatin landscape. Here, we dissect the dynamic chromatin interactions of a family of multivalent effectors, heterochromatin protein 1 (HP1) proteins, using single-molecule fluorescence imaging and computational modeling. We show that the three human HP1 isoforms are recruited and retained on chromatin by a dynamic exchange between histone PTM and DNA bound states. These interactions depend on local chromatin structure, the HP1 isoforms as well as on PTMs on HP1 itself. Of the HP1 isoforms, HP1α exhibits the longest residence times and fastest binding rates due to DNA interactions in addition to PTM binding. HP1α phosphorylation further increases chromatin retention through strengthening of multivalency while reducing DNA binding. As DNA binding in combination with specific PTM recognition is found in many chromatin effectors, we propose a general dynamic capture mechanism for effector recruitment. Multiple weak protein and DNA interactions result in a multivalent interaction network that targets effectors to a specific chromatin modification state, where their activity is required.

Applications of MEMS to Cell Biology

Relatively simple microscale devices such as cantilevers already have found distinct applications in the study of cells and subcellular structures. The ability to measure small forces, small features, and small masses has led to unique and elegant measurement solutions. In this chapter, we give a brief background of some relevant fundamental biology and provide examples of research in cell biology, ranging from mechanobiology, physiology, microbiology to cancer biology, where MEMS- and NEMS-based methods have had a significant and, most likely, lasting impact.

Correlated Atomic Force Microscopy and Single Molecule Localization Microscopy

Laboratory for Bioand Nano-Instrumentation, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland Laboratory of Nanoscale Biology, Institute of Bioengineering, School of Engineering, EPFL, 1015 Lausanne, Switzerland National High Magnetic Field Laboratory, Florida State University, Tallahassee, Florida, USA. Department of Biological Science, Florida State University, Tallahassee, Florida, USA.