THE LABORATORY OF ATOMIC FORCE MICROSCOPY

Maria Gaczynska, Ph.D.

CORE DESCRIPTION

The technique of AFM takes advantage of possibility to non-invasively and non-destructively detect topography of microscopic objects using a small, sharp vibrating tip (AFM probe), which interacts with atoms of a sample by the van der Waals forces. The interactions change the resonant frequency or vibrational amplitude of the tip. The changes are monitored by the system and reflect the sample topography and other physical and chemical properties of its surface. The technique possesses unique capabilities to study the dynamics of structure, interactions and surface properties of biological objects from whole cells to single biomacromolecules. Under conditions most closely resembling the native environment, a practical resolution achievable for biological objects reaches 1 nm in lateral direction, 0.1 nm in height, and milliseconds to minutes in a temporal domain.

Objects of the studies can be either dried or immersed in a liquid. In the latter case, ligands can be added or washed out from the sample without ceasing AFM image acquiring. The output data suitable for further mathematical processing are in the form of height, amplitude or surface plot images, or in the form of force plots, where the force of interactions between tip, including modified tip, and sample is measured.

In addition to AFM, the laboratory is fully equipped to perform scanning tunneling microscopy imaging (STM), another method from the scanning probe microscopies (SPM) group, providing an advanced information about topography, charge and electric properties of biological and non-biological objects. The laboratory also functions in a rich network of collaborations with research institutions in USA and abroad. Examples of the research conducted in the laboratory include among others:

Osmulski P.A. and Gaczynska M. (1998) A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast. Cur. Biol. 8: 1023-1026.

Osmulski P.A. and Gaczynska, M. (2000) Atomic force microscopy reveals two conformations of the 20S proteasome from fission yeast. J. Biol. Chem. 275: 13171-13174.

Chen L., Trujillo K., Sung P., and Tomkinson A.E. (2000) Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase. J. Biol. Chem. 275: 26196-26205.

Chen L., Trujillo K., Ramos W., Sung P., and Tomkinson A.E. (2001) Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol. Cell 8: 1105-1115.

Osmulski P.A. and Gaczynska M. (2002) Nanoenzymology of the 20S proteasome: Proteasomal actions are controlled by the allosteric transition. Biochemistry 41: 7047-7053.

Mukherjee S., Brieba L.G., and Sousa R. (2002) Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell 110: 81-91.

Gaczynska, M., Osmulski, P.A., Gao, Y., Post, M., and Simons, M. (2003) Proline and arginine rich peptides constitute a novel class of allosteric inhibitors of proteasome activity. Biochemistry 42: 8663-8670.

Gaczynska, M., Osmulski, P.A., Jiang, Y., Lee, J.K., Bermudez, V. and Hurwitz, J. (2004) Atomic force microscopic analysis of the binding of the Schizosaccharomyces pombe origin recognition complex and the spOrc4 protein with origin DNA. Proc. Natl. Acad. Sci. 101: 17952-17957.

Yasmin, R., Yeung, K.T., Chung, R.H., Gaczynska, M.E., Osmulski, P.A. and Noy, N. (2004) DNA-looping by RXR tetramers permits transcriptional regulation "at a distance". J. Mol. Biol. 343: 327-338.

Osmulski, P.A. and Gaczynska, M. (2005) Atomic force microscopy of the proteasome. In: Ubiquitin nd Protein Degradation, Part A, Methods in Enzymology, R.J. Deshaies, ed., Elsevier, 414-425,

Tan, X., Osmulski, P.A. and Gaczynska, M. (2006) Allosteric regulators of the proteasome: Potential drugs and a novel approach for drug design. Curr. Med. Chem. 13: 155-164.

Gaczynska, M., Rodriguez, K.A., Madabhushi, S, and Osmulski, P.A. (2006) Highbrow proteasome in high-throughput technology. Expert Rev. Proteomics 3: 115-127, 2006.

Gaczynska, M., Osmulski, P.A. (2008) AFM of protein complexes: what can we learn? Current Opinion in Colloid and Interface Science, 13, 351-367.

Gaczynska, M. Osmulski, P.A. (2008) Atomic force microscopy as a tool to study the proteasome assemblies in Methods in Nano Cell Biology (Methods in Cell Biology series, vol. 90), Bhanu Jena, Editor, Elsevier; pp. 39-60.

Rosenzweig, R., Osmulski, P.A., Gaczynska, M., Glickman, M. (2008) The central unit within the 19S regulatory particle of the proteasome. Nature Struct. Mol. Biol. 15: 573-580.

Osmulski, P.A., Hochstrasser, M., Gaczynska, M. (2009) A Tetrahedral Transition State at the Active Sites of the 20S Proteasome is Coupled to Opening of the Alpha- Ring Channel. Structure 17, 1137-1147.

Gaczynska, M. Osmulski, P.A. (2010) Atomic force microscopy of proteasome assemblies. In: Atomic Force Microscopy: Methods and Biomedical Applications. Ed. Braga & Ricci. Methods in Molecular Medicine series, Humana Press. In press.

The facility consists of a NanoScope IIIa Microscope (Digital Instruments) and controller with three vertical engagement scanners, wet-mode and dry-mode sample chambers, a PicoForce scanner, a temperature controlling module and NanoScope optical viewing system (Sony). The data are collected on a Pentium PC computer with NanoScope software v. 5.12, and 6.12, equipped with two text and two graphic monitors and Phaser 450 color printer. For additional image processing and data analysis there is a Pentium PC with SPIP software and an SGI Octane workstation with SPIDER software. The laboratory is furnished with a system isolating the microscope from vibrations, a laminar flow hood (“PCR workstation”) for the dust-free sample preparation, compressed nitrogen tank with a setup for sample drying and antistatic mat.



Objects and processes routinely studied with AFM or STM include: protein-protein and protein - DNA interactions, formation of protein complexes, mechanisms of protein polymerization, interactions of proteins and DNA with drugs, dynamics of structural changes in protein and DNA molecules, and mechanical properties of cells.