Atomic Force Microscopy in Cell Biology_Methods in Cell Biology Volume 68 pptx

[...]... contrast for imaging membrane proteins Due to this newfound motility, the consequences for cell physiology remain to be determined IV Force Spectroscopy The approach to molecular structures in cells and to their interaction in molecular biology is traditionally a chemical one Therefore, molecular interactions are characterized by binding constants, on- and off-rates, and corresponding binding energies... feasibility and to gain hands-on experience prior to investing on equipment and personnel Bhanu P Jena, Ph.D Heinrich H¨ rber, Ph.D o CHAPTER 1 Local Probe Techniques J K Heinrich Horber ¨ EMBL Meyerhofstrasse 1 69117 Heidelberg, Germany I Introduction II Scanning Tunneling Microscopy III Atomic Force Microscopy A Combination with Optical Microscopy B Combination with Patch-Clamp Technique IV Force Spectroscopy... affinities of proteins to surfaces In addition, in vitro cell cultures require cell- surface adhesion, which is mediated by a sublayer of adsorbed proteins Protein adsorption is a net result of various complex interactions between and within all components including the solid surface, the protein, the solvent, and any other solutes present These interaction forces include dipole and induced dipole moments,... Folded proteins can be stretched to more than 10 times their length, reaching almost their total contour length The force extension curves show a characteristic sawtooth-like pattern The reaction coordinate of unfolding is imposed by the direction of pulling, and in this way unfolding events occurring in a single protein can be examined Each peak is attributed to a breakage of a main stabilizing connection... the unfolding of spectrin repeats can occur in a stepwise fashion during stretching The force extention patterns exhibit features that are compatible with the existence of at least one intermediate state These new details regarding the unfolding of single domains revealed by precise AFM measurements show that force spectroscopy can be used not only to determine forces that stabilize protein structures... measuring various material properties at or close to surfaces This can be done in vacuum, in gas, or in liquids in a broad temperature range with a resolution down to either the atomic or the molecular level In this way, it is the only type of microscopy that can complement optical microscopy in biology on a smaller scale Additionally, these instruments allow manipulations at either the single -atomic. .. been the driving force for major scientific developments Local probe techniques extend our sense of touching into the micro- and nanoworld and in this way provide complementary new insight into these worlds with microscopic techniques Furthermore, touching things is an essential prerequisite to manipulating things, and the ability to feel and to manipulate single molecules and atoms certainly marks another... abruptly disappear, leaving behind small scars on the cell surface Considering the timing and their size, we believe these protrusions are progeny viruses exiting the cell Assuming that approximately 20–100 viruses exit the living cell and that roughly 1/40 of the cell surface is accessible to our SFM, one should be able to observe one or two of these events for each infected cell We actually observed... become irreversible The adhesion forces established by single proteins, e.g., protein A and tubulin molecules, within contact times from milliseconds to seconds, can be measured by the AFM Protein A and tubulin, both globular proteins, can be seen as examples for different types of protein binding The molecules can be attached to the cantilever tip and then brought into contact with different surfaces,... interaction between tip and plasma membrane structures, which can be in uenced quite strongly by the socalled extracellular matrix of cells containing a broad variety of sugars and other polymer structures As with the integrated tip of the cantilever, forces in the range of some 10 to 100 pN are applied to the investigated cell membrane, and the mechanical properties of cell surface structures dominate . Heinrich H ¨ orber I. Introduction 1 II. Scanning Tunneling Microscopy 4 III. Atomic Force Microscopy 7 IV. Force Spectroscopy 13 V. Photonic Force Microscopy 21 References 30 2. The Atomic Force.