THE LABORATORY OF ATOMIC FORCE MICROSCOPY
Maria Gaczynska, Ph.D.
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:
Gaczynska M and Osmulski PA: (2011) Atomic force microscopy of proteasome assemblies. In: Atomic Force Microscopy in Biomedical Research: Methods and Protocols, Ed. Braga & Ricci., ISBN: 978-1-61779-104-8, Humana Press. DOI: 10.1007/978-1-61779-105-5_9 Series: Methods in Molecular Biology, 736: 117-132.
Osmulski PA, Hochstrasser M, and 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(8): 1137-47.
Rosenzweig R, Osmulski PA, Gaczynska M, and Glickman MH: (2008) The central unit within the 19S regulatory particle of the proteasome. Nat Struct Mol Biol. 15(6): 573-80.
Gaczynska M and Osmulski PA: (2008) Chapter 3 Atomic force microscopy as a tool to study the proteasome assemblies In: Methods in Nano Cell Biology (Methods in Cell Biology series, Vol. 90), Edited by Bhanu P. Jena. Elsevier Science; p. 39-60.
Gaczynska M and Osmulski PA: (2008) AFM of biological complexes: what can we learn? Curr Opin Colloid Interface Sci. 13(5): 351-67.
Gaczynska M, Rodriguez K, Madabhushi S, and Osmulski PA: (2006) Highbrow proteasome in high-throughput technology. Expert Rev Proteomics. 3(1): 115-27.
Tan X, Osmulski PA, and Gaczynska M: (2006) Allosteric regulators of the proteasome: potential drugs and a novel approach for drug design. Curr Med Chem. 13(2): 155-65.
Osmulski PA and Gaczynska M: (2005) Atomic force microscopy of the proteasome. In: Ubiquitin and Protein Degradation, Part A Methods Enzymol. 398: 414-25. RJ Deshaies, ed., Elsevier.
Yasmin R, Yeung KT, Chung RH, Gaczynska ME, Osmulski PA, and Noy N: (2004) DNA-looping by RXR tetramers permits transcriptional regulation "at a distance." J Mol Biol. 343(2): 327-38.
Gaczynska M, Osmulski PA, Jiang Y, Lee JK, 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 USA. 101(52): 17952-7.
Gaczynska M, Osmulski PA, Gao Y, Post MJ, and Simons M: (2003) Proline- and arginine-rich peptides constitute a novel class of allosteric inhibitors of proteasome activity. Biochemistry. 42(29): 8663-70.
Mukherjee S, Brieba LG, and Sousa R: (2002) Structural transitions mediating transcription initiation by T7 RNA polymerase. Cell. 110(1): 81-91.
Osmulski PA and Gaczynska M: (2002) Nanoenzymology of the 20S proteasome: proteasomal actions are controlled by the allosteric transition. Biochemistry. 41(22): 7047-53.
Chen L, Trujillo K, Ramos W, Sung P, and Tomkinson AE: (2001) Promotion of Dnl4-catalyzed DNA end-joining by the Rad50/Mre11/Xrs2 and Hdf1/Hdf2 complexes. Mol Cell. 8(5): 1105-15.
Chen L, Trujillo K, Sung P, and Tomkinson AE: (2000) Interactions of the DNA ligase IV-XRCC4 complex with DNA ends and the DNA-dependent protein kinase. J Biol Chem. 275(34): 26196-205.
Osmulski PA and Gaczynska M: (2000) Atomic force microscopy reveals two conformations of the 20S proteasome from fission yeast. J Biol Chem. 275(18): 13171-4.
Osmulski PA and Gaczynska M: (1998) A new large proteolytic complex distinct from the proteasome is present in the cytosol of fission yeast. Curr Biol. 8(18): 1023-6.
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.