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.

USE AND PUBLICATIONS

The laboratory is used extensively (on average about 20-30 hrs per week, excluding analysis) not only by the researchers from Dr. Gaczynska’s and neighboring laboratories, but also through collaborative efforts with researchers from other Departments of the University. The laboratory also functions in a rich network of collaborations with other Universities in the USA. 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. and Osmulski, P.A. (2008) AFM of biological complexes: what can we learn? Current Opinion in Colloid & Interface Science, in press.

Rina Rosenzweig, Pawel A. Osmulski, Maria Gaczynska, and Michael H. Glickman: The central unit within the 19S regulatory particle of the proteasome. (Nature, in revision).

Chen Y., Polci R., Litchfield P., Gaczynska M. and Riley D.J. Nek1 regulates renal epithelial cell apoptosis through phosphorylation of the mitochondrial transition pore channel VDAC. (submitted to Molecular Cell).


FUNDING

This resource is funded by the Cancer Therapy and Research Center (P30 CA54174), David Curiel, M.D., Principal Investigator. All users of this core must cite the support of the CTRC Support Grant (P30CA54174) in all publications and presentations involving work performed by this Shared Resource.
Department of Molecular Medicine provides additional funding to the Core.


EQUIPMENT

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.



SERVICES

Objects and processes routinely studied with AFM include: protein-protein and protein - DNA interactions, formation of protein complexes, interactions of proteins and DNA with drugs, dynamics of structural changes in protein and DNA molecules.