Scanning Probe Microscopy for the Intramural Research Community

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications & trials

Abstract

Most of the collaborative projects carried over and continued from the previous year. A new collaboration was initiated with NIDCR (Robey lab). Our long-term collaboration with NCI (Dalal Lab) continued with the examination of the effect of cenP-A or H1.5 linker histone overexpression on the elasticity of the nucleus and possibly of the cytoskeleton mechanics. We are studying both fibroblasts and epithelial cells comparing normal to histone up-regulating ones. CenP-A and H1.5 histone over-expression occurs in a range of cancers such as gliomas, prostate cancer and others. The hypothesis is that excess cenP-A or H1.5 would alter the genome organization and possibly the cytoskeleton in ways reflected in the mechanics of the cell and of the nucleus. It is known that cell mechanical properties influence tumor cell migration and cell cycle regulation. In parallel, we engaged in a pilot project to investigate the biophysical characteristics of glioblastoma multiforme (GBM) tumors grown in a novel 3D organoid along with other relevant cell types simulating the in-vivo environment. The goal is to compare with patient derived GBM tumors to validate the 3D organoid biophysical characteristics and to investigate whether individual cells change their mechanics when cultured in the 3D organoid. A paper was previously (end of 2019-2020 year) submitted to Nucleic Acid Research (NAR) presenting the results from our collaboration with NIDDK (Hinton Lab) on MotB interactions with T4 bacteriophage and host DNA. Reviewers asked additional data that required many new experiments and data re-analysis. The work was completed, and the revised manuscript was recently accepted for publication. Following this we turned our attention to investigating possible MotB role in DNA packaging and release in the T4 bacteriophage. In particular, we are studying possible regulation of the function of the gp17 protein by MotB. A gp17 protein oligomer forms the motor assembly at the bottom of the T4 heads and its role is to package DNA inside the T4 head. Since we showed that MotB strongly condenses DNA, the hypothesis is that it may regulate the gp17 function. We use the AFM to visualize the internalization of DNA into empty T4 heads in the presence and absence of varying concentrations of MotB. By arresting the DNA internalization at mid-points using ATPgS to inhibit ATP hydrolysis needed by the motor, we quantify the kinetics of the process and the novel role of MotB. The project is ongoing. We continued our collaboration with NEI (Sergeev lab) helping with their effort to produce melanin in-vitro using recombinant human tyrosinase (Tyr) catalytic domain bound to magnetic microbeads (MB). Oxidation of the substrate L-DOPA by Tyr produces dopachrome and melanin polymers. The Tyr-MB platform facilitates the partition of the enzymatic products and will potentially enable large scale production of melanin. With the AFM, we examined the morphology and surface properties of the magnetic beads before and after Tyr immobilization. The observed differences confirmed the presence of the Tyr domain on the MB surface. A paper describing the methodology of melanin production was recently published in the Int. J. Molec. Sci.. A new project was initiated the second half of the year with NIDCR (Robey Lab). The lab is investigating the healing process of bone after a fracture. Upon a bone fracture, skeletal stem cells in the periosteum and in the bone marrow are recruited to the fracture site where they differentiate and repair the periosteum matrix and subsequently the bone. The localization and the matrix microenvironment that the stem cells reside in is not well understood and it is known that stem cell differentiation can be directed by its microenvironment elasticity. We undertook the task of quantifying the mechanics of the matrix at different locations of interest. We used the AFM to show that the periosteum matrix near a fracture is significantly softer than before the fracture. The hypothesis, at this point is that periosteum matrix swelling alters the elasticity of the tissue and that leads to stem cell activation and recruitment to the repair pathway. We subsequently use the AFM to perform high resolution imaging of the matrix fiber architecture focusing on regions of interest guided by fluorescence staining of the various cell types. Tissue swelling should increase the matrix mesh size and alter matrix fiber organization as a result of increased fluid influx.