Scanning Probe Microscopy for the Intramural Research Community

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

Abstract

Several of the collaborative projects from the previous year carried over, continued and some expanded and are ongoing. Our collaboration with the NEI (Sergeev lab) on melanins continued from last year. Melanins are the natural pigments found in most organisms. They are generally produced by specialized cells, the melanocytes, and in humans, they determine the skin and hair color, but are also present elsewhere in the body. Albinism is caused by deficiencies in eumelanin, a form of melanin. Melanins are small molecules (a few 100s Da) produced by oxidation of amino-acids, notably tyrosine and L-cysteine. The structures of these small molecules make them prone to polymerization in diverse ways leading to a range of large aggregates whose form depends on the pattern of polymerizing bonds, the proportion of the different monomers and other factors. Our collaborators have developed an in-vitro melanin production protocol and can generate several different melanin types. We previously imaged and characterized the aggregates formed by those melanins and compare them with corresponding structures extracted from melanocytes. Recently, we examined in detail the various intermediate products in the melanin production pathways. Those reaction products included tyrosinase, L-DOPA, and other reactants encountered along the pathways leading to the formation of different types of melanin. We now have a more complete description of those intermediates and their striking variability. Some of those products show well organized aggregates, pointing to dominant polymerization binding sites while others form amorphous structures pointing to multiple, competing such binding sites. Comparisons with some of the products with ones extracted from melanocytes further supports the in-vitro melanin production protocol. The collaboration with NHLBI (Lee Lab) continued examining a-synuclein (-Syn) constructs. We imaged full length a-Syn and confirmed the well-known morphology of fibers resulting from -Syn polymerization. In addition, we examined -Syn fibers extracted from Parkinson’s patients and fibers formed by various seeding strategies of the same samples. Seeding is the process by which short, preformed fibers are added to a solution of polymerizing macromolecules, and they are known to bias the polymerization process guiding the morphology of the fibers. The next step was to examine the interactions of a lysosomal enzyme, GCase (Glucocerebrosidase) with -Syn. Mutations in its encoding gene are strong risk factors for Parkinson’s disease. It is hypothesized that GCase interacts with -Syn and it has been shown that mutant GCase promotes -Syn accumulation in the endoplasmic reticulum. We used the AFM to visualize possible modes of enzyme binding onto a-Syn fibers. Under the experimental conditions for fiber formation, we observe the -Syn fibers strongly bind to each other forming large structures resembling balls of yarn. That would probably prevent most -Syn from binding GCase. Indeed, we did not observe GCase bound to -Syn fibers. Besides, it is not clear whether GCase may interact with -Syn within fibers or with its monomeric form. We are currently modifying experimental methods and trying different strategies to investigate further. During the year we collaborated with NHGRI (Hanchard lab) investigating the role of hemicentin, a large extracellular protein critical for the maintenance of the architectural integrity of various tissues. The roles of hemicentin in the extracellular space and in the cytoplasm have not been clarified. Our collaborators identified hemicentin variants as potential markers for childhood-onset essential hypertension, a significant risk factor for heart disease later in life. It is known that hemicentin is highly expressed in vascular tissue. We used the AFM to compare the elastic properties of wildtype and hemicentin-knockdown (KD) human aortic vascular smooth muscle cells (VSMC). So far, we have confirmed that hemicentin KD cells have reduced elastic modulus (by a factor of 2). Such reduction in elastic modulus would certainly affect the response of the vascular tissue to blood circulation. We continue examining different hemicentin variant KD cells to further clarify the role of aberrant hemicentin. We are also collaborating with NCI-CCR (Hunter lab) on an effort to elucidate the role of a poorly understood nuclear protein, the retroelement silencing factor 1 (Resf1). Resf1 has been identified as a critical tumor suppressor since reduced Resf1 activity results in faster tumor growth and increased metastases in estrogen receptor (ER)-negative breast cancer and even worse, in triple-negative cases. The way tumor suppression is achieved by Resf1 is not understood at all and our collaborators hypothesized that lack of Resf1 might affect the elasticity of the nucleus. We undertook to measure the elasticity of 4T1 murine cell nuclei derived from breast cancer tissue. We compared wildtype (WT) and Resf1-knockout (KO) cells by extracting elastic moduli from the region directly above the cell nuclei, and found that the KO cell nuclei are more than twice as stiff as the WT. This result was consistent with independent deformable cytometry measurements and consistent across two different KO-clones. We are currently examining a third clone to make sure that the results are not clone-dependent. The surprising finding is that cell lines appear to exhibit the opposite behavior compared to what is observed in actual breast tissue. Our collaborators are currently isolating breast cancer cells from cancer tissue and trying to culture them avoiding any phenotypic changes. Another collaboration was with NIA (Sung lab) on A-42 protein, a well-known factor in Alzheimer’s and other neurodegenerative diseases. The objective was to visualize and characterize the oligomeric state of the A-42 protein samples at concentrations in the physiological range (~50nM). We used the AFM for high-resolution imaging of A-42 solutions at different concentrations, from 2mM on 50nM. At room temperature, we showed that the peptide forms small oligomers, mostly 1-8 monomers, while larger oligomers were in lesser proportions. Although the peptide is an intrinsically disordered, AFM could image monomers and small oligomers and can quantify the effective aggregation binding strength. When the solutions were incubated at 37oC for 24 hrs, the peptide formed larger oligomers with a distribution centered around the 20-mer but larger oligomers of more than 100 monomers were also present. Another collaboration was with NIDCR (Robey lab). The lab is producing fibrin beads (a few hundred mM in size) with the goal to use then as scaffolds for regenerative medicine. Since fibrin is a natural agent in cartilage and bone healing, it is very suitable for cell incorporation and proliferation. At this stage, the project is refining the bead production method and needed to evaluate the elastic properties and surface morphology of the produced beads. We used AFM indentation to extract elastic moduli and performed high resolution imaging at scales relevant to cell sizes. The elastic moduli are in the range of a several hundreds of kPa which is similar to that of natural cartilage. We were also involved in several other pilot projects. For example, we attempted imaging microtubules (MTs) (NINDS) under physiological buffer conditions with the goal of examining the binding of the tau protein along the MTs. The AFM environment proved too noisy making imaging challenging and causing damage to the MTs. We are investigating improvements to the MT attachment to our substrates. Another pilot project involved imaging RNA molecules (NIAMS) and visualizing the binding of a synthetic peptide that is expected to bind the RNA. The RNA molecules formed unexpected structures, and the project went back to the bench to re-evaluate the RNA production protocol.