לוח שנה משולב

Much of our understanding of the biological mechanisms that underlie cellular functions, such as migration, differentiation and force sensing has been garnered from studying cells cultured on two-dimensional (2D) substrates. In the recent years there has been intense interest and effort to understand cell mechanics in three-dimensional (3D) cultures, which more closely resemble the in vivo microenvironment. However, a major challenge unique to 3D settings is the dynamic feedback between cells and their surroundings. In many 3D matrices, cells remodel and reorient local extracellular microenvironment, which in turn alters the active mechanics and in many cases, the cell phenotype. Most models for matrices to date do not account for such positive feedback. Such models, validated by experiments, can provide a quantitative framework to study how injury related factors (in pathological conditions such as fibrosis and cancer metastasis) alter extracellular matrix (ECM) mechanics. They can also be used to analyze tissue morphology in complex 3D environments such as during morphogenesis and organogenesis, and guide such processes in engineered 3D tissues. In this talk, I will present discrete network simulations to study how cells remodel matrices and how this remodeling can lead to force transmission over large distances in cells. I will also discuss an active tissue model to quantitatively study the influence of mechanical constraints and matrix stiffness on contractility and stability of micropatterned tissues.

Site-specific installation of paramagnetic lanthanide ions in proteins is a powerful method in delineating the structures, dynamics and interactions of proteins by NMR and EPR. Since most proteins do not have a paramagnetic center, efforts towards site-specific labeling of proteins with paramagnetic ions have thus been made via thiol chemistry, click chemistry, and molecular biology. The formation of disulfide bond between a protein and the paramagnetic tag is mostly applied in protein modifications, whereas the disulfide bond tether succumbs to low stability in reducing conditions or high pH. We have been focusing on development of paramagnetic tagging proteins in formation of a stable thioether bond for analysis of proteins in vitro and in cells using NMR and EPR. A number of stable paramagnetic tags have been designed and the performance of the respective protein conjugates has been evaluated in vitro and in cells by high resolution NMR spectroscopy. Using these high-performance paramagnetic tags, we were able to determine the 3D structure of a protein in live cells and 3D structure of unstable and short-lived thioester intermediate of Sortase A with pseudocontact shifts (PCSs) as structural restraints.

: Intracellular Ca2+ concentration ([Ca2+]i) is tightly regulated in neurons. Ca2+ plays important roles in signal transduction pathways, synaptic plasticity, energy metabolism and apoptosis. In dendritic spines, [Ca2+]i is controlled by voltage and ligand-gated channels that allow Ca2+ entry from the extracellular space and by ryanodine receptors (RyR) and inositol 1,4,5-trisphosphate receptors (IP3R) that release Ca2+ from intracellular stores. Disruption in Ca2+ homeostasis is linked to several pathologies and is suggested to play a pivotal role in the cascade of events leading to Alzheimer disease (AD). In line with this, I found that low concentrations of caffeine, known to release Ca2+ from stores, is more effective in facilitating long-term potentiation (LTP) induction in hippocampal slices of a triple-transgenic (3xTg) mouse model of AD than controls. Synaptopodin (SP) is a protein residing in the dendritic spines. SP is an essential component in the formation of the spine apparatus (SA), which is a specialized form of smooth endoplasmic reticulum (ER) found in dendritic spines. Spines lacking SP were shown to release less Ca2+ from stores. The present study is aimed to explore the involvement of Ca2+ stores in 3xTg mouse model of AD. By crossing 3xTg and SPKO mice lines, I studied the effect of SP deficiency on AD markers in the 3xTg mouse. I found that the 3xTg/SPKO mice show normal learning in a spatial memory task by comparison to the deficiency found in the 3xTg mouse, and express normal LTP in hippocampal slices, which is deficient in 3xTg mice. Furthermore, low concentration of ryanodine has a facilitating effect on LTP induction only in the 3xTg mice group. In addition, these brains do not express amyloid plaques, activated microglia, p-tau overexpression and high RyR expression seen in age matched 3xTg mice, These results suggest that SP deficiency restores [Ca2+]i homeostasis in the 3xTg so as to suppress the progression of AD symptoms.

Membranes compartmentalize living matter into cells and subcellular structures. Many life processes involve membrane topological changes and remodelling: the uptake of materials via endocytosis and secretion by exocytosis, the generation of intra or extra-cellular vesicles as well as various membrane fusion processes. In order to get to the bottom of these fundamental physiological processes, it is vital to study membrane mechanical properties and membrane deformation. In this talk I will present the results of our research on several aspects of vesicle generation and membrane fusion using single molecule techniques. By means of an AFM force spectroscopy study we characterized the mechanical properties of small natural vesicles, called extracellular vesicles (EVs). Investigating the mechanical properties of these vesicles and their lipid and protein content provided new insights into the still poorly understood processes underlying vesicle generation. Acoustic Force Spectroscopy (AFS) was the choice for our novel methodology to measure cell mechanical properties. It enabled our finding that uptake of EVs by cells changes cellular deformability, a process that may have implications in several disease states where EV levels are significantly elevated, such as malaria and breast cancer. Combining optical tweezers with confocal fluorescence microscopy was the perfect tool for the investigation of membrane remodelling by calcium sensor proteins which are crucial in neuronal communication. We discovered surprising differences between the action mechanisms of two structurally similar proteins, Doc2b and Synaptotagmin-1 (Syt1), as determined by quantifying the strength and probabilities of protein-induced membrane-membrane interactions. Overall these fundamentally new insights into central biological processes were possible by our biophysical characterization of membranes using a powerful combination of single molecule techniques: Optical tweezers combined with confocal fluorescent microscopy, AFS and AFM.