Dynamic biophysical responses of neuronal cell nuclei and cytoskeletal structure following high impulse loading

Biomechanical cues guide and regulate cellular behavior including nuclear mechanics, gene expression, and tissue homeostasis [1], [2], [3]. Global forces that act upon tissues during normal physical loading or trauma transfer to the cell and organelles through physical links and biochemical signaling. Alterations in nuclear envelope mechanics, chromatin organization, and gene expression have demonstrated the response of the nucleus to changes in the microenvironment [4,5]. How cells integrate cues from the mechanical environment and respond through changes in the cell phenotype is known as mechanoreciprocity. Directed by mechanosensors in the cell membrane, cytoskeleton, and nuclear envelope, mechanoreciprocity is necessary for mechanical stability in the extracellular environment and intracellular compartments [3,[6], [7], [8]]. For example, biochemical signals observed in disease or during prolonged inflammation effect the constituents of the microenvironment over time, which can lead to increases in cell and nuclear stiffness [9,10]. Similarly, microenvironments in which constant mechanical signaling is applied also demonstrate alterations in tissue, cellular, and nuclear stiffness with changes in mechanical differentials over time [11]. However, during short-term mechanical loading, high intensity forces can cause excessive strain (e.g., tensile stretch) and immediate disruption of regulatory pathways in the cell and nucleus, and in turn can lead to cellular and nuclear mechanical instability [12].

One physiological example of immediate mechanical failure within the cell due to impulse loading is traumatic brain injury (TBI), which affects 1.7 million people each year in the United States alone [7,13,14], and depending on the nature of the trauma multiple impacts can be sustained [15]. The complex nature of TBI arises from a combination of mechanical insult (i.e., primary injury) and activation of biochemical cascades (i.e., secondary injury). On a cellular level, the sequelae of TBI is frequently associated with diffuse axonal injury, characterized by axonal blebbing and neurite swelling [16]. These hallmark morphological features represent disruption in the cytoskeletal network of the neuronal axon. Additionally, in vitro studies varying strain and rates of strain during mechanical loading have provided insight into response mechanisms of neuron activity and death [17], [18], [19], [20], including axonal damage [16,18,21,22]. However, the extent that axonal damage extends or transfers to influence biophysical responses of the nucleus has received less attention.

Studies of neuronal axonal injury typically only examine the axon and dendrites before and after mechanical loading without considering the mechanical load experienced by the soma or internal organelles like the nucleus. Earlier work has shown that the soma and axon have different material properties [23] and responses after applied forces [24]. Furthermore, it is well established that alterations in the microenvironment influence axonal length and growth cone extension in neuronal cultures [25,26], demonstrating the mechanoreciprocity of the neuron within its environment. During traumatic loading, it was hypothesized that the soma of the neuron might receive a majority of the dissipating energy during axonal injury [7,24]. However, how this mechanical energy affects nuclear mechanics and directs behavior is still unclear.

In our lab, we have explored mechanoreciprocity and noted that while environmental and extracellular matrix stiffness affected cellular stiffness, changes in cell mechanics did not reciprocally influence matrix stiffness [10]. Additionally, we showed that disruption of the native tissue (extracellular) environment propagates to the plasma membrane and interior nuclear envelope structures of viable cells [9], and that environmental stiffening or disruption of complexes of the linker of nucleoskeleton and cytoskeleton influence nuclear mechanics and cellular behavior [4,[27], [28], [29]]. Several studies have shown that characterization of biophysical perturbations of the cell and nucleus may better depict the underlying collective molecular responses [30], [31], [32]. In our studies, we demonstrated how alterations in the microenvironment have an immediate influence on nuclear mechanics and biophysical features of the cells [4,5,33].

Consequently, we were motivated to explore biophysical responses of the nucleus in neuronal cells to impulse loading in light of the cellular disruption and deformation experienced in traumatic axonal injury. Here we use nuclear movement and temporal tracking of biophysical features to better understand neuronal nuclear biomechanics after high intensity loading. We hypothesized that single and multiple impulse loading, leading to substrate and cellular stretching would result in nuclear remodeling. Temporal analysis of the nuclear behavior revealed the mechanosensitive nature of the neuronal nucleus and provided insight into the damage response of the soma following impulse loading.

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