Scanning Probe Microscopy for the Intramural Research Community

National Institute Of Biomedical Imaging And Bioengineering, Bethesda

Investigators

Linked publications, trials & patents

Abstract

Most of the collaborative projects carried over and continued from the previous year. A new collaboration was initiated with NHLBI (Lee lab). Our NCI collaborations with the Dalal Lab continued on two fronts: It is known that the replacement of the H3 histone with the cenP-A variant, results in the formation of non-canonical nucleosomes, called centromeres, at tightly regulated chromosome locations. Centromeres are indispensable to cell mitosis. In several cancers, it has been observed that cenP-A is overexpressed, and centromeres localize at non-standard chromosome sites. These chromosomes tend to physically fragment resulting in widespread deregulation of gene expression. The reasons and mechanisms for this fragmentation are not understood, and we undertook to examine the mechanical properties of such chromosomes, using chromosome #8, a target for colon cancer. High resolution elasticity maps across chromosome spreads were obtained using AFM nanoindentation and compared with corresponding maps of similar spreads from normal cells. The spreads are then stained for the ectopic cenP-A, to identify chromosome #8 and the location of the mis-localized centromeres. The hypothesis is that elasticity variations in certain regions will indicate weaker regions that may be prone to fragmentation. The project is ongoing focusing on optimization of the experimental protocol that will allow reliable identification of the same spreads in different labs. Another project in the collaboration focuses on the examination of 3-D printed, glioblastoma tumors that include several cell types, including the stromal cells that define the tumor microenvironment. This is a more realistic system than 2-D cell cultures, for the study of the development of the tumor and for drug screening. One measure of a realistic tumor is its stiffness. Tumor stiffness is modulated by several factors, many connected with chromatic re-organization and deregulation caused by histone mutants and by histone over-expression, observed in many cancers. We use the AFM to measure the elastic modulus of the tumor and its micro-environment and the effect of a few drug candidates. In parallel, we examine elasticity alterations caused by overexpression of histones, such as cenP-A and H1.5, in a 2-D culture of normal cells, such as fibroblasts. We previously established that centromeres are softer than canonical nucleosomes and we are finding that this is reflected in the nuclear stiffness when cenP-A is overexpressed. We know that cenP-A overexpression increases heterochromatin, a looser organization of chromatin. We continued our work on the NIDCR (Robey Lab) project on the bone healing process following fracture. It is known that upon a bone fracture, skeletal stem cells in the periosteum and in the bone-marrow are recruited to the fracture site where they differentiate into several cell types and rebuild the periosteum matrix and subsequently the bone. The matrix microenvironment and its dynamic transformation post-fracture that guides stem cell recruitment and differentiations is not well understood. It is generally known that stem cell differentiation can be directed by its microenvironment elasticity. We used the AFM to measure elasticity and showed that the periosteum matrix in the vicinity of a fracture becomes significantly softer than before the fracture. The obvious hypothesis then is that the altered periosteum matrix elasticity leads to stem cell activation and recruitment to the repair pathway. High resolution imaging of the periosteum post-fracture shows altered structures. We are working on quantifying those differences. A new collaboration with a team from the NHLBI (Lee Lab) is an effort to better understand the role of the detailed amino acid sequence of alpha-synuclein that drives fiber formation. The binding sites critical to fiber formation are not known. alpha-synuclein fiber aggregates (together with a few other proteins), called Lewy bodies, are found in the brain cells of Parkinsons and of some dementia patients, although possible causative role of these fibers has not been rigorously established. The wildtype protein forms characteristic twisted fibers exhibiting well defined periodicity. Several protein mutants also result in fiber formation, but generally, with different periods. Our collaborators produced several alpha-synuclein variants with one or two amino-acid substitutions and investigated their fiber forming potential. We are using the AFM to perform high-resolution imaging of the formed structures. Most of the amino-acid substitutions did not abolish the fiber formation capacity but we observed fibers with a wide range of periodicities as well as smooth fibers, with no twisting at all. Moreover, several variants exhibit weak fiber formation with varying periodicities, mixed twisted and non-twisted fibers and high proportions of proteins that do not form complete fiber structures. The work is continuing with the examination of further constructs, to better understand fiber formation mechanics of the protein. Putting together the information on how fibers formation is altered by specific amino-acid substitutions will facilitate resolve the question of the detailed structure of these fibers.