In situ and real-time readout of nuclear mechanotransduction via single cell mechanics and site-specific fluorescence reporting - PROJECT SUMMARY
The goal of this R21 is to develop new tools for unveiling force-initiated activation of the gene transcription ma-
chinery during nuclear mechanotransduction, using combined single-cell mechanics and site-specific fluores-
cence reporting. Nuclear mechanotransduction is defined as direct gene activation by external force via cyto-
skeletal force propagation to the chromatin. This process is critically important for the development and
maintenance of weight-bearing structural tissues such as cartilage, tendons, ligaments, and associated con-
nective tissues. Aberrant mechanotransduction contributes to the pathophysiology of osteoarthritis. Significant
voids exist in our understanding of nuclear mechanotransduction, especially at the molecular level in real
time. The most significant event is the critical moment at which mechanical force is translated into transcrip-
tional activation of genes at the junction where chromatin is anchored to the inner nuclear membrane.
Knowledge of this event would significantly advance our understanding of nuclear mechanotransduction and
establish systematic correlation of mechanical cues with downstream gene activation. This would have wide
impacts and novel applications in programming cell phenotype via mechanical cues and development of new
theragnostic tools.
Many mechanoresponsive genes are rapidly activated within a short timeframe, often seconds or
minutes, suggesting that they are transcriptionally paused. It is established that rapid activation of transcrip-
tionally paused genes requires the transcription factor cyclin-dependent kinase 9 (Cdk9). Recently, Cdk9 has
been localized to the inner nuclear membrane. Our central hypothesis is that the kinase activity of Cdk9 can
be used as a surrogate for imaging the activation of mechano-responsive genes in real time in-situ (Fig 1).
Here we propose to develop new fluorescent reporters to monitor Cdk9 activity, localized at the inner nuclear
membrane, upon activation using single-cell mechanics.
Our new tools will enable us to visualize gene activation in situ, in real time among living cells via our
site-specific fluorescence reporters upon mechanical perturbation at single cell level. Two specific aims are:
(SA1) To develop and validate Cdk9 activity reporters as surrogates for imaging gene activation. (SA2) Corre-
late mechanical cue parameters with gene activation in living cells utilizing the Cdk9 reporters, using single-cell
mechanics and varying the location, magnitude, duration, direction, and frequency of the applied force.
The conceptual innovation is that a reporter for Cdk9 activity serves a surrogate for visualizing force-initi-
ated gene activation. The technical innovations include the design and production of site-specific Cdk9 fluores-
cence reporters, the combined approach of using single-cell mechanics to deliver the designed mechanical cues,
and AFM/confocal imaging to monitor the subsequent gene activation in real time and in-situ.
The outcomes include: (a) visualizing real time and in situ nuclear mechanotransduction in living cells;
(b) capturing the transcriptional activation of genes; (c) revealing signal network, including linker of nucleoskel-
eton and cytoskeleton (LINC) complexes, nuclear envelope proteins and lamins that anchor chromatin to the
perinuclear matrix; and (d) establishing correlation of transcriptional activation with mechanical cues e.g., mag-
nitude, duration, direction (shear versus normal) of force, force modulation frequency and amplitude. These
outcomes support our long-term objectives to (a) program gene activation and cellular signaling via me-
chanical cues, and to generate a new and impactful tool for the scientific community to probe the cellular pro-
cesses involved in nuclear mechanotransduction.
