Isolated Nucleus Stiffens in Response to Low Intensity Vibration

The nucleus, central to all cellular activity, relies on both direct mechanical input and its molecular transducers to sense and respond to external mechanical stimuli. This response occurs by regulating intra-nuclear organization that ultimately determines gene expression to control cell function and fate. It has long been known that signals propagate from an extracellular environment to the cytoskeleton and into nucleus (outside-in signaling) to regulate cell behavior. Emerging evidence, however, shows that both the cytoskeleton and the nucleus have inherent abilities to sense and adapt to mechanical force, independent of each other. While it has been shown that isolated nuclei can adapt to force directly ex vivo, the role of nuclear mechanoadaptation in response to physiologic forces in vivo remains unclear. To gain more knowledge regarding nuclear mechanoadaptation in cells, we have developed an atomic force microscopy based experimental procedure to isolate live nuclei and specifically test whether nuclear stiffness increases in mesenchymal stem cells (MSCs) following the application of low intensity vibration (LIV). Results indicated that isolated nuclei were on average 36% softer than nuclei of intact MSCs. In intact MSCs, depletion of nuclear structural proteins LaminA/C and Sun-1&2 led to both decreases in nuclear elastic moduli and decreased chromatin condensation in Sun-1%2 depleted samples. In isolated nuclei, identical depletions led to decreased stiffness and significantly higher chromatin decondensation levels (47% & 39% increase for LaminA/C and Sun-1&2 nuclei respectively). When LIV was applied in series (0.7g, 90Hz, 20min) either twice (2x) or four times (4x), increased nuclear stiffness of intact MSCs showed dose dependency while stiffness changes in isolated nuclei was only detectable at the 4x LIV dose. Changes in isolated nuclear stiffness was not accompanied by changes in LaminA/C or Sun-1&2 protein levels. Interestingly, chromatin measurements in isolated nuclei showed a 25.4% smaller chromatin to nuclear area size in 4x LIV nuclei compared to controls