Nano-indentation and Nano-scale imaging using atomic force microscopy: from single cell to single molecule
Creators: name: Al-Hammood, Orooba
Publisher: name: Universität Bielefeld
Year: 2021
Subjects: subject: 530

description: Abstract 1 TMEM43 is a protein embedded inside the inner nuclear envelope and cytoplasm, which is related to the LINC (linker of nucleoskeleton and cytoskeleton) complex. Studies indicates that the Ser358Leu mutation in the TMEM43 gene lead to arrhythmogenic right ventricular cardiomyopathy type V (ARVC-5) is a distinct form of ARVC, but information about the molecular mechanisms of evolution of these diseases are still limited. Understanding how the Ser358Leu mutation affect the mechanical properties of the nucleus and as a whole in a transfect HeLa cells model, may give new insights into the potential molecular mechanisms of these diseases. By using the AFM force spectroscopy mode the elasticity of the nuclear region and cytoplasm region under native conditions were measured. By means of the Hertz model the force curve data were analyzed. We found the HeLa cell carrying GFP-TMEM43-pS358L showed a noticeable increase Young modulus in the nucleus and cytoplasm compared to GFP-TMEM43-Wt. Here, our results strongly suggest that the impact of the TMEM43 mutation is not limited to nuclear mechanics but extends to the entire cell. The endoplasmic reticulum (ER) plays exceptional role of folding and maturation of newly synthesized secretory and transmembrane proteins, and maintaining calcium homeostasis. The ER stress is accumulation of misfolded proteins at the ER, resulting in unfolded protein response (UPR). In this study, we were focused in two-way. (1) To characterize whether disorder cellular homeostasis that causes ERS is affects the mechanical properties of the nucleus. (2) To create a new control to understand the pathological mechanisms of ARVC5 disease. To accomplish the first goal, the treated HEK293 cell line with two different volumes 0.1% and 0.25% of with pharmacological agents the thapsigargin and tunicamycin, which is standardized model to induce ER stress were used. After the data analysis, our results revealed a significant increase Young modulus in the nucleus compared with control (DMSO) volumes a significant increase Young modulus in the nucleus compared with control (DMSO) volumes. Here, these results proved that the disorder cellular homeostasis produces the dynamic feedback between intracellular and extra cellular environments, resulting in a change the biophysical activity of cells. As for the second aim, the transfected HEK293 GFP-TMEM43-S358L, GFP-TMEM43-WT, and GFP-without TMEM43 were used. The GFP-TMEM43-S358L cells exhibited significantly different stiffness values than the nuclear zone of the transfected HEK293 cell (GFP-TMEM43-WT) and (GFP-WT-without TMEM43) cells. In briefly, the pS358L mutation in TMEM43 gene affects the structure of physical connections in cell and stimulates a cellular stress response, apoptosis and thus cell death. Abstract 2 The xanthan gum is an exopolysaccharide which is secreted by the gram- negative phytopathogen the Xanthomonas campestrisis (Xcc). Given its distinctive rheological properties such as the capability to form a highly viscous solution at low shear forces, xanthan was used in various applications such as the foods industry, cosmetics, pharmaceuticals and technical applications. With the growing industrial importance, many biotechnological approaches used to improve the xanthan gum production. Targeted metabolic engineering of Xanthomonas campestris (Xcc) could be an effective approach in which xanthan production efficiency and optimize shear- thickening potency is improved. By means of atomic force microscopy (AFM), the secondary structure of the single molecules of the wild type xanthan strain B100, the mutant xanthan strain Xcc H21012 (wxcB), the commercial acetate, pyruvate-free xanthan, and JBL007 were analyzed. The topological images of the single molecules of xanthan revealed characteristic differences between these strains. The structures ranged from branched xanthan double-strands of both the B100 and the strain Xcc H21012 (wxcB), while other products showed a single-stranded coiled polymer. Here, these results proved that the secondary structure of xanthan strongly correlates with its viscosity properties. Overall, the results of this study provide a better understanding of the polymerization and secretion-machinery related to the biosynthesis of xanthan gum which will benefit future applications.