Scanning Probe Microscopy for the Intramural Research Community

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Several of the collaborative projects carried over and continued from the previous year. New collaborations were initiated with NIDCR (Ten Hagen lab) and NCI (Rudloff lab). Our NCI collaborations with the Dalal Lab continued with further efforts to investigate the source of chromosome fragility upon overexpression of the cenP-A histone. Such over-expression leads to formation of ectopic centromeres resulting in widespread deregulation of gene expression. This is observed in several cancers. With the AFM we perform high resolution mapping of elastic properties in search of regions of high elasticity gradients. Focusing on chromosome 8, initial results failed to locate such sites, but we continue with a modified protocol that should greatly improve data quality and acquisition time (a major hitherto problem). The other project in the collaboration continued investigating the 3-D printed, glioblastoma tumors. By mapping elastic properties of different regions of the tissue, we ascertain that its mechanical properties are similar to in-vivo tumors thus supporting the 3-D printing platform. Further, changes in those mechanical properties caused by certain cancer drugs are evaluated and correlated with parallel biochemical and genetic analyses of the tissues. Another collaboration was with NEI (Sergeev lab) has been examining the structures formed by different types of melanins. Melanins are the natural pigments found in most organisms. They are produced by specialized cells, the melanocytes, and in humans, they determine the skin and hair color, but are also present elsewhere in the body. Melanins are small molecules produced by oxidation of amino-acids, notably tyrosine and L-cysteine. These small molecules aggregate forming different structures depending on the pattern of polymerizing bonds. Absence of one type of melanin, the eumelanin, is thought to be the cause of albinism. Our collaborators have developed a melanin production protocol and have provided several different melanin types. We undertook to image and to characterize the aggregates formed by those melanins. With the AFM, we observed different melanins forming aggregates of different sizes and morphologies and with different surface nano-topography. We also observed variations in fragility, after applying forces with the AFM probes to the aggregates and subjecting them to shear stresses with micropipettes. The collaboration with a team from the NHLBI (Lee Lab) on the role of the detailed amino acid sequence of a-synuclein in driving fiber formation, continued. a-synuclein is an intrinsically disordered protein strongly associated with Parkinsons and other neurodegenerative diseases, although possible causative role has not been rigorously established. There are several potential binding sites on the disordered a-synuclein monomer that can drive aggregation, oligomerization and fiber formation, but the binding sites critical for the latter are not known. The wildtype protein forms characteristic twisted fibers exhibiting well defined periodicity. Several protein mutants also result in fiber formation, but generally, with different periods, different cross-sections and morphologies. Salt bridges between Lys and Glu residues, have been suggested as the dominant mechanism. But the large number of these residues in the full-length protein, led our collaborators to fist examine a shorter, 44-140aa peptide, that includes the proteolytic-resistant core, to limit the number of possible salt-bridges. Using the AFM, we have completed imaging and characterization of fibers formed by this shorter protein after a systematically chosen array of one- and two-amino-acid substitutions. We are currently evaluating the results to detail the hierarchy of binding pair relative significance. We took part in a project with NICHD (Fields Lab) on the development of a sulfated nanocellulose material to prevent viral transmission. Our collaborators discovered that a novel cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC), obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This capacity can also be further enhanced by chemical sulfation. They showed that low concentration ENC suspensions efficiently encapsulated spike (S) protein and SARS-CoV-2 pseudoviruses and prevented infection of 293T-ACE2 cells. We used high-resolution AFM imaging to visualize the nanocellulose fiber matrix, and confirmed the cross-linking between the S-protein and the pseudoviruses with the nano-fiber network by demonstrating specific fiber network changes upon the introduction of the viral objects. A project with NHLBI (Ten Hagen lab) examined the mechanics of mucin granules from the salivary glands of the Drosophila. Mucins are large, heavily O-glycosylated proteins that line and protect epithelial surfaces by forming a dense mucus layer. Upon synthesis, mucins are tightly packaged within small vesicles to be transported to the plasma membrane before exocytosis. The packaging mechanism is poorly understood. Our collaborators discovered a small cysteine-rich, non-glycosylated protein, Sgs7, that is essential to packaging Sgs3, an abundant member of the mucin family. Upon Sgs7 knockout, the granules become larger and attain a spherical morphology and are more fragile upon osmotic challenge. Electron microscopy shows structured packaging of mucins within wildtype granules, while no such structures are seen in Sgs7-knockout granules. We used the AFM to measure membrane tension and elastic modulus of both types of granules. We showed that the elastic modulus of the wildtype granules is significantly higher as is their membrane tension. The existence of structured arrangement of Sgs3 within wildtype granules, most likely affected by cysteine intra-protein bonds, indicate that the mucin contents are gel-like, while the Sgs7-knockout contents are more like a mucin solution. This is consistent with our finding of higher elastic modulus and higher effective membrane tension for the wildtype granules. A very new project with NCI (Rudloff lab) is investigating the effects of a novel cancer drug to the structure for nucleoli. Nucleoli are regions in the cell nuclei where ribosomal biogenesis takes place. It is a tightly packed assembly of DNA, RNA and a few proteins. Computational modeling and imaging studies with fluorescence and electron microscopy indicate that upon introduction of the drug, certain interactions within the nucleolus are abolished leading to an altered phase separation of its different components. At that point, different components needed in the transcription pathway are separated, transcription of the different proteins for ribosome production are deregulated, ribosomes cease to be produced and apoptosis follows. The phase separation after treatment is seen as producing a looser structure which should be reflected in the elastic properties of the nucleolus. We are using the AFM to image and measure the elastic properties of purified nucleoli. Our preliminary data of untreated nucleoli are consistent with previous measurements and continue examining populations of treated and untreated nucleoli