Scanning Probe Microscopy for the Intramural Research Community

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

Abstract

We continued some of the projects that were started during the previous year but also initiated new collaborations with several intramural researchers. We continued our NIDCR collaboration on the factors that induce invadopodia formation and concomitant local ECM remodeling. Invadopodia are actin-rich protrusions from cells (particularly cancer cells) that secrete proteases to locally degrade the ECM and invade adjacent tissues. The question addressed was whether stiffness alone could be the primary driver for their formation. We showed that, in addition to stiffness, both composition and structure of the matrix regulate invadopodia formation. A novel, cross-linked, high-density, fibrillar collagen matrix closely imitating the stroma layer adjacent to tumors induced very high degree of invadopodia formation while other substrates of similar stiffness but different composition had a much weaker effect. Our collaboration with NCI on the structure/stoichiometry of the centromere focused on a number of mutations of the centromeric cenP-A histone. Recently, a lab expressed and purified mutant cenP-A mimicking acetylated mutants. We have been examining reconstituted chromatin using wildtype and mutant cenP-A to examine the role of acetylation in determining the centromere structure. In parallel we are developing recognition imaging methodologies for molecular identification of cenPA and other histones as well as components of the kinetochore in an effort to gain insights into the topological arrangement of the components of the inner plate. We have a long-standing collaboration with NICHD for the study of cartilage. The goal is to correlate composition with mechanics of cartilage tissues. Composition is obtained using Fourier Transform Infrared Spectroscopy (FTIR) and MRI methods with the goal of developing MRI techniques for early arthritis diagnosis. We have developed a sophisticated methodology for data acquisition and analysis overcoming a number of issues relating to the nature of the experimental samples. We have obtained the most detailed map of mouse cartilage properties to date. These data should be of general interest due to the mouse being the most common animal for model diseases, including arthritis. The data are being expanded to include osteoarthritic cartilage and cartilage from old age mice. These data, in addition to providing a link between mechanics and age or disease, will also be used in the development of MRI diagnostic techniques. Another NCI project focused on a novel, small RNA (80-100 bases) expressed in bacteria that appears to interact both with DNA and with the abundant HU protein. This suggested another, hitherto unknown, pathway for nucleoid organization. We have been examining the complexes formed between DNA and HU in the presence of the small RNA and the images point to definite cooperative action involving small RNA. The initial findings were recently published but more work is ongoing to further understand the role of the sRNA. We completed the project with NCI on the possible chromatin de-compacting effected by HMGN5 protein overexpression. We completed additional control experiments to verify that just GFP co-expression with HMGN5 does not alter nuclear stiffness and also that the cell properties away from the cell remain unaffected. This meant that the observed stiffness change in HMGN5 over-expressing cells over the nucleus was actually most likely due to changes within the nucleus itself. This points to chromatin de-compaction. A recent project with NEI looks at visualization and characterization of tyrosinase and several of its mutants relating to albinism. Tyrosinase is the primary enzyme regulating production of melanin. The protein and its mechanism of action are rather poorly understood. We perform imaging of individual enzyme molecules to examine their oligomeric state in the purified solutions under different buffers and aim at providing a quasi-3-D shape of the protein in its wildtype and mutant forms. The aim is to see if mutations cause significant shape changes, indicating severe misfolding. We have already established the conditions under which the protein remains monomeric and are proceeding with the description of its 3-D shape on our substrates. Homology-based models for the folding of the wildtype protein can also be fitted to the volumes obtained from the AFM images in an effort to discern differences between wildtype and mutants.