Regulation of Cellular Responses on the Nanometer Scale

Transkript

1 Regulation of Cellular Responses on the Nanometer Scale Joachim P. Spatz Max-Planck-Institute for Metals Research Department of New Materials and Biosystems University of Heidelberg Department of Biophysical Chemistry

2 Life Science Materials Science Biophysics

3 Life Science Materials Science Biophysics

4 The Extracellular Matrix Cell 67 nm Materials Science & Life Science 10 µm C. Ploetz et al. J. Struct. Biol., 106 (1991) P. Fratzl: Current Opinion in Colloid and Interface Science 8 (2003) D.J. Müller et al. J. Struc. Biol. 2004, 148, 268 K. Meller et al. Cell Tissue Res (1997) 288:

5 Nanolithography by Self-Assembly unloaded micelle loaded micelle loaded micelle

6 Nanolithography by Self-Assembly 20 nm Au-dot pattern

7 Nanolithography by Self-Assembly 10nm 28nm 58nm silicon oxide silicon 73nm 85nm

8 Nanolithography by Self-Assembly Dr. Max-Planck-Institut Roman Glass, für Metallforschung, MPI-MF Stuttgart / Uni-HD

9 Nanolithography by Self-Assembly 500 nm 100 nm 500 nm 100 nm

10 Nanolithography by Self-Assembly Dr. Max-Planck-Institut Roman Glass, für Metallforschung, MPI-MF Stuttgart / Uni-HD

11 Nanolithography by Self-Assembly Dr. F.-M. Kamm, Ulm University

12 Biomimetic Antireflective Interfaces The Moth Eye 5 µm 1 µm h Light R [%] n 0 n nm T [%] n 1 n 1 Collaboration Max-Planck-Institut für Metallforschung, with Stuttgart ZEISS & BOSCH

13 Biomimetic Antireflective Interfaces Optical Lense Nanostructured Optical Lense 20 nm 200 nm 200 nm

14 Biomimetic Antireflective Interfaces 100 nm Collaboration with Zeiss 22.4 mm [1010] 10 A

15 Biomimetic Antireflective Interfaces f) unstructured glass 0 unstructured glass 45 c) Collaboration with ZEISS & BOSCH

16 Nanoporous Electrochemical Sensors Enzyme label Detection-Ab Antigen Capture-Ab e - Ox Redox-inactive Redox-active Red e - Incubation with antigen (sample) wash incubation with enzyme labeled detection antibody wash add substrate measurement Enzyme converts redox-inactive molecule to redox active mediator Repeated oxidation / reduction will increase signal: redox - cycling Martin Stelzle, NMI Reutlingen

17 Nanoporous Electrochemical Sensors Anode Cathode Ox d Red Requires small anode / cathode spacing <1µm State of the art: amplification factor of 10 Difficult & expensive fabrication limits applicability in diagnostics Martin Stelzle, NMI Reutlingen

18 Nanoporous Electrochemical Sensors Red Ox d Multiple micro- or nanopores, laterally connected Electrode spacing defined by insulator thickness Micro-structure technology based on self-assembly process Martin Stelzle, NMI Reutlingen

19 Nanoporous Electrochemical Sensors top electrode Red Ox insulator bottom electrode Martin Stelzle, NMI Reutlingen [1010] 10 A

20 Nanoporous Electrochemical Sensors -500mV 450mV CE -500mV Ox Red d 4 Inch 300 µm Electrode pad 500 nm Martin Stelzle, NMI Reutlingen [1010] current / na only bottom-electrode redoxcycling y = 0,0095x - 0,0526 R 2 = 0,9958 y = 0,0015x + 0,0244 R 2 = 0, c /nm Amplification by redox-cycling! Para-Aminophenol

21 Cell Adhesion to Nanostructured Interfaces Life Science Materials Science Biophysics

22 Focal Adhesions

23 The Extracellular Matrix nm Alberts et al. Molecular Biology of the Cell

24 Cell Adhesion to Nanoscopic Interfaces Integrin Cell membrane PEG silicon oxide 10nm silicon ligand for cell surface receptor d metal nanoparticles

25 Biofunctionalisation of Nanostructures Site-directed protein immobilization via his-tag leads to enhanced activity Applicable for a wide variety of proteins Indirect immobilization of Fc-tagged proteins or antibodies

26 Cell Adhesion to Nanoscopic Interfaces 58 nm presentation of RGD ligands 73 nm presentation of RGD ligands

27 Cell Adhesion to Nanoscopic Interfaces 100 nm

28 Cell Adhesion to Nanoscopic Interfaces ON / OFF Selection of mrna from Fibronectin

29 Cell Adhesion to Nanostructured Interfaces

30 Cell Adhesion to Nanostructured Interfaces Homogeneous 60 nm pattern 15 nm/mm; including 60 nm 20 µm 20 µm Cells sense peptide spacing of ~1 nm between opposed cell sides

31 Ordered and Random Nanopattern ordered random 53 R1 High density 82 R8 Low density

32 Ordered and Random Nanopattern Pattern Au nanoparticle density (μm -2 ) Average Order Parameter ±Standard Deviation Average Interdot Spacing±Standard Deviation (nm) Cell Adhesion R1: Random 408 ± ± ± 14 YES High density (1) R8: Random 151 ± ± ± 33 YES Low density (2) 53: Ordered 416 ± ± ± 8 YES High density (3) 82: Ordered 171 ± ± ± 12 NO Low density (4)

33 Cell Adhesion to Nanostructured Interfaces MOLECULARLY DEFINED DIGITAL SURFACES 250 nm 2 µm Dr. Max-Planck-Institut Marco Arnold, für Metallforschung,,Dr. Ada Stuttgart Cavalcanti-Adam, MPI-MF / UNI HD

34 Cell Adhesion to Nanostructured Interfaces Length of PAX Pattern [µm] 3 µm 1 µm 500 nm 250 nm 100 nm extended 60 nm # particles per patch Patch Size [µm] 1 µm 250 nm 6 Integrin proteins per patch are required to initiate stable adhesion

35 Receptor Cross Talk

36 Binary Nanoarrays - Receptor Cross-talk INPUT matrix composition growth factors Julien Polleux

37 {TiO 2 - Au} binary nanoarrays {TiO 2 35nm} {TiO 2 35nm - Au 110nm} Julien Polleux 300 Ti for 45 Au 6.7 times more RGD than EGF

38 Au silicon oxide Julien Polleux

39 {TiO 2 35nm@RGD - Au 110nm@EGF} binary nanoarrays Julien Polleux

40 Immune Response is triggered by encounters

41 Role of Force and Adhesion in Recognition Alon & Dustin 2007 Cross-talk Induced from outside

42 Separation Forces in T cell B Cell Conjugates PNAS 2009

43 Role of Force and Adhesion in Recognition Alon & Dustin 2007 Cross-talk Induced from outside

44 Mechano-Sensing of Interfaces Y- Wang, UPEN

45 Substrates BCMN quasi hexagonal ordered gold nanopattern: extended or micro-nano structured Interparticle spacing: 20 nm < L< 200 nm Aydin et al. SMALL 2009

46 Substrates Transfer Lithography Poly(ethylene glycol) Diacrylate (PEG-DA) Biologically Inert & non-toxic Water soluble MW g/mol 100 nm 500 nm Aydin D. & Louban I. In prep.

47 Substrates PEG-DA Hydrogels 0.6 kpa < E Y (M W, C PEG )< 6 MPa

48 Readout Cell spreading area: A SP (E Y, ΔL) [µm 2 ] Adhesion force density: F ADH (E Y, ΔL) [nn/µm 2 ]

49 Readout Adhesion Force Density

50 Results Detachment Forces F ADH (E Y ) F ADH (ΔL)

51 Readout Cell Spreading Area

52 Results Cell Spreading spreading area µm

53 Results Cell Spreading A (E Y ) A (ΔL)

54 Results Cell Spreading

55 Conclusions Cell spreading area change

56 Migration of Human Tumour Cell's - Regulation of Metastases Formation - 5 µm 10 µm Prof. Dr. Thomas Seufferlein Innere Medizin I, Universitätsklinikum Ulm Nature Cell Biology 2003 Nature Cell Biology 2003

57 H 3 C CH 3 H 3 C N primary tumor O O P O O sphingosylphosphorylcholine (SPC) bioactive lipid high concentrations found in patients with metastatic invasions metastases H HO H NH 2 Prof. Dr. Thomas Seufferlein Innere Medizin I, Universitätsklinikum Ulm CH 3

58 0 min 15 min 30 min 45 min adding SPC keratin Fibre architecture - elasticity?

59 Uniaxial deformation of a single cell

60 Nature Cell Biology 2003

61 Design and fabrication Schematic view of the migration chip : Reservoir Channels Reservoir Microscope with incubation chamber allows live cell imaging - bright field or fluorescence.

62 Design and fabrication Reservoir Channel s Reservoir Reservoir Channel s Reservoir Channel properties: LxWxH 150x20x11 µm 150x7x11 µm

63 Panc-1 Cell Migration Cytoplasm can penetrate the channels. Cells are not able to migrate through the channels. scale bar 50 µm 150x7x11 µm

64 Panc-1 Cell Migration Addition of 10µM SPC: Cells can migrate through channels. Cytoplasm can penetrate the channels. scale bar 50 µm 150x7x11 µm 8h

65 Panc-1 Cell Migration penetration invasion permeation channel dimensions 150x7x11 µm 7 µm N=115 N=100 Rolli et al. PLoS ONE submitted

66 High-Throughput Screens Identifying Cell Specific Material Parameters Substrate Stiffness 1 Substrate Stiffness 2

67 High-Throughput Screens

68 Sensing the Environment Chemistry Mechanics Nano- Presentation

69 Acknowledgment Benjamin Geiger, Weizmann Institute of Science Horst Kessler, TU München Reinhard Fässler, MPI Biochemistry Viola Vogel, ETH Zürich Mike Sheetz, Columbia University Financial Support Alfried Krupp von Bohlen und Halbach-Stiftung Boehringer Ingelheim Fonds German-Israeli Foundation (GIF) Deutsch-Israel Projekt (DIP) Fonds der Chemischen Industrie VolkswagenStiftung Deutsche Krebshilfe e. V. BMBF, Humboldt-Stiftung, DFG EU-STREP, EU-IP, Marie Curie BASF, Qiagen, Zeiss, Evotec, National Institutes of Health (NIH) Max-Planck-Gesellschaft

70

71 Particle Synthesis: Au Nanoparticles on Silica Beads 200 µm 20 µm 200 nm

72 Spatial Arrangement of Receptors & Forces - Cell Signaling vary d ligand for cell surface receptor Signal Molecule d metal nanoparticles nanometer scale Transmembrane Receptors

73 Role of FORCES in Cell Signaling

74 Biomimetic Cell-Cell Contacts

75 Biomimetic Cell-Cell Contacts 71 nm Ecad spacing 53 nm Ecad spacing

76 Biomimetic Cell-Cell Contacts

77 Biomimetic Cell-Cell Contacts

78 Biomimetic Cell-Cell Contacts

79 Biomimetic Cell-Cell Contacts

80 Cell-Cell Contacts