Some of the projects carried over and continued from the previous year but new collaborations were also initiated with several intramural researchers during this year. We continued our NIDCR (Yamada Lab) collaboration by mapping elastic moduli of gels used as substrates to examine the dependence of cancer cell motility on substrate stiffness. Both uniform gels and gels with stiffness gradients were examined to ensure the quality of gels produced by the various substrate preparation protocols. Moreover, knowledge of the variability at scales relevant to cell movement is also important for the correct interpretation of the cell motility data. A paper is in preparation. Our collaboration with NCI (Dalal Lab) on the various aspects of the structure/stoichiometry of the centromere has recently moved in a different direction. There have been reports that the difference in structure of the cenP-A histone of the centromere compared to the canonical H3 of standard nucleosomes would alter the compactness of the tetramer/octamer. We have been using the AFM to probe whether we can detect a difference in apparent elasticity of reconstituted nucleosomes using recombinant H3 and cenP-A histones. We perform indentations of nucleosomes in appropriate buffers and compare the responses of H3 and cenP-A nucleosomes. We are currently fine-tuning the indentation protocol, primarily using the Quantitative Nanomechanical Mapping (QNM) technique to obtain low noise and very high resolution nanomechanics of the nucleosomes. The effort is ongoing. We completed the AFM imaging part of another NCI project (Adhya Lab) where we examined the binding patterns of a number of variant forms of a small RNA construct (80 bases) expressed in bacteria with DNA and with the abundant HU protein. The project is motivated by observations that led to the hypothesis of a general organizational role of small RNAs in bacterial nucleoids. A paper is under review. We initiated a project with NHLBI (Kruth Lab) to study the effect of cholesterol enrichment on macrophage membrane interactions with the extracellular matrix (ECM). It has been observed that cholesterol in macrophages may be segregated into ordered domains either on the plasma membrane or on detached, extracellular microdomains. These are hypothesized to play an important role in plaque formation on blood vessel walls. The nature of these microdomains is poorly understood but it appears that macrophages, deposit excess cholesterol to the ECM to maintain cholesterol homeostasis. We used the AFM to image and probe such micro-domains outside the macrophages. Consistent with immune-fluorescence, super-resolution observations, we observed micron-sized particles that were obviously pinched-off from the macrophage plasma membranes and which maintained irregular shapes, rather than take the form expected of pure lipid vesicles. In addition, these particles had a narrow distribution of thicknesses that was much bigger than a double bilayer raising further questions about their internal contents. Force indentation experiments point to highly inhomogeneous and surprisingly stiff structures that would be consistent with the ordered cholesterol domains although further work is needed to quantify the mechanics. So far, no other protein or enzyme that may be driving the formation and detachment has been found on these particles leaving open the proposition that they form and detach purely due to the changes in membrane fluidity caused by the presence of ordered cholesterol arrays, namely purely physical/chemical effects. A paper is in preparation and further work is ongoing. A new project with NCI (Roche Lab) seeks to examine the activation of CD4 T cells by antigen presenting cells (APCs) such as dendritic cells (DCs) and B cells via peptide-loaded MHC class II molecules. The question posed was how overexpression or absence of the peptide affects the binding of T cells to APCs. In addition, how does a mutation in the MHC class II molecules affects that binding. For that purpose, we used streptavidin coated tipless, AFM probes to attach biotinylated T cells. We then brought the T cells in contact with APCs (dendritic cells), that were firmly attached to their substrate, applying controlled force for a short time followed by slow retraction. The recorded retraction force curves contain information about the strength and nature of the binding. We could measure a minimum unbinding step of a few tens of pN that may represent the unbinding of a single MHC molecule from a single antigen on the T cell. Most of the unbinding events were those of a small population of bound pairs. There were clear differences between different cell pairs. In the absence of peptide we only observed non-specific adhesion between the cells and no unbinding between MHC molecules and antigens. In the presence of mutated MHC molecules we observe severely compromised ability to bind. Peptide enrichment greatly enhanced the unbinding forces. These findings explain observed behavior of T cell activation under the given conditions and reinforces proposed models of this important function of immune system cells. Our longstanding collaboration on cartilage and gels with NICHD (Basser Lab) continued and a first paper was just submitted detailing our methodology and our results mapping the mouse knee cartilage mechanics. In the next stage, we are examining more closely the effects of GAG depletion and tissue fixation and have embarked into a finite element modeling effort to that would aims at modeling the fundamental factors defining the mechanical response of joints under physiological loads. An older NIDDK collaboration (Craigie lab) has been re-initiated focused on the detailed mechanics of the function of HIV-integrase (HIV-IN). In the past, our collaborators succeeded in creating a stable synaptic complex (SSC) in-vitro consisting of two viral DNA molecules and a HIV-IN tetramer bringing the two DNA stand ends together. This complex is the necessary step prior to the integration of viral into host DNA. It proved impossible, however, to form this complex using short DNA (<200bp) even though only about 12bp at the ends of DNA strands form the DNA binding domain for HIV-IN. This excess DNA precluded crystallization of the SSC. The function of the excess DNA has remained a mystery and no HIV-IN has been observed to bind at any other location along the DNA except at the end binding sequence. It was recently proposed that the recombinant addition of a DNA binding peptide (Sso7d, a known, DNA binding archeal protein) at the N-terminal of HIV-IN may allow the formation of the SSC with short DNA. We used the AFM to image and analyze the complexes formed by wt-IN and by the Sso7d-IN with such short DNA. The goal is to establish the conditions under which Sso7d-IN forms SSCs that will not aggregate non-specifically as happens with wt-IN and long DNA. We have shown the formation of regular SSC arrays under high salt conditions and hope to fine tune conditions that will lead to ever larger arrays that would be crystal precursors. The effort is ongoing.
| Hategan, Alina; Bianchet, Mario A; Steiner, Joseph et al. (2017) HIV Tat protein and amyloid-? peptide form multifibrillar structures that cause neurotoxicity. Nat Struct Mol Biol 24:379-386 |
| Masuda, Takaaki; Xu, Xiaoling; Dimitriadis, Emilios K et al. (2016) ""DNA Binding Region"" of BRCA1 Affects Genetic Stability through modulating the Intra-S-Phase Checkpoint. Int J Biol Sci 12:133-43 |
| Gao, Yuan; Nieuwendaal, Ryan; Dimitriadis, Emilios K et al. (2016) Supramolecular Self-assembly of a Model Hydrogelator: Characterization of Fiber Formation and Morphology. Gels 2: |
| Furusawa, Takashi; Rochman, Mark; Taher, Leila et al. (2015) Chromatin decompaction by the nucleosomal binding protein HMGN5 impairs nuclear sturdiness. Nat Commun 6:6138 |
| Artym, Vira V; Swatkoski, Stephen; Matsumoto, Kazue et al. (2015) Dense fibrillar collagen is a potent inducer of invadopodia via a specific signaling network. J Cell Biol 208:331-50 |
| Qian, Zhong; Macvanin, Mirjana; Dimitriadis, Emilios K et al. (2015) A New Noncoding RNA Arranges Bacterial Chromosome Organization. MBio 6: |
| Walkiewicz, Marcin P; Dimitriadis, Emilios K; Dalal, Yamini (2014) CENP-A octamers do not confer a reduction in nucleosome height by AFM. Nat Struct Mol Biol 21:2-3 |
| Chandran, Preethi L; Dimitriadis, Emilios K; Lisziewicz, Julianna et al. (2014) DNA nanoparticles with core-shell morphology. Soft Matter 10:7653-60 |
| Li, Lina; Dimitriadis, Emilios K; Yang, Yu et al. (2013) Production and characterization of novel recombinant adeno-associated virus replicative-form genomes: a eukaryotic source of DNA for gene transfer. PLoS One 8:e69879 |
| Bui, Minh; Walkiewicz, Marcin P; Dimitriadis, Emilios K et al. (2013) The CENP-A nucleosome: a battle between Dr Jekyll and Mr Hyde. Nucleus 4:37-42 |
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