Cytoskeletal Dynamics of Neurons Measured by Combined Fluorescence and Atomic Force Microscopy
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MRS Advances © 2018 Materials Research Society DOI: 10.1557/adv.2018.101
Cytoskeletal Dynamics of Neurons Measured by Combined Fluorescence and Atomic Force Microscopy Peter Moore1 and Cristian Staii1 1
Department of Physics and Astronomy, Tufts University, 574 Boston Avenue, Medford, MA 02155
ABSTRACT
Mechanical properties of neurons represent a key factor that determines the functionality of neuronal cells and the formation of neural networks. The main source of mechanical stability for the cell is a biopolymer network of microtubules and actin filaments that form the main components of the cellular cytoskeleton. This biopolymer network is responsible for the growth of neuronal cells as they extend neurites to connect with other neurons, forming the nervous system. Here we present experimental results that combine atomic force microscopy (AFM) and fluorescence microscopy to produce systematic, high-resolution elasticity and fluorescence maps of cortical neurons. This approach allows us to apply external forces to neurons, and to monitor the dynamics of the cell cytoskeleton. We measure how the elastic modulus of neurons changes upon changing the ambient temperature, and identify the cytoskeletal components responsible for these changes. These results demonstrate the importance of taking into account the effect of ambient temperature when measuring the mechanical properties of cells.
INTRODUCTION Detailed knowledge of the structure and dynamics of the neuronal cytoskeleton is essential for understanding the mechanisms that control neuronal growth, development and repair. Mechanical properties of the cell are determined by the two main components of the cytoskeleton: actin filaments and microtubules [1-3]. The dynamics of microtubules is powered by two families of motor proteins: kinesins and dyneins. Actin filaments, generally smaller and more densely spaced than microtubules, are linked together by binding proteins such filamin A or the molecular motor myosin. Rather than forming a static network, the molecular motors constantly bind and unbind to microtubules and actin filaments, giving the network a dynamic nature. In general, cells use the mechanical motors to generate internal forces for processes such as growth and cytokinesis [1-3]. Many in vitro studies of biopolymer networks measure their elastic properties while varying the concentration molecular motors in the substrate [2]. Recent studies also show that temperature also has a significant effect on the cytoskeletal dynamics in living cells [4-6]. Much of the existing study of cellular networks has been performed at room temperature (25ºC), while living cells in vivo normally exist at physiological temperatures (close to 37ºC). In previous work [4] we have shown that neurons display a significant increase in the average elastic modulus upon a decrease in
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