(19f) Confined Cell Migration Induces Nuclear Volume Expansion and Blebbing By Triggering RhoA-Mediated Nuclear Influx

Introduction:
color:black">Cell migration through tissues is a critical step during the
metastatic spread of cancerous cells from primary tumors to distal organs in
the body. Arial;color:black;background:transparent">Cancer cells in vivo must migrate
through complex confining microenvironments that initiate intracellular
signaling cascades distinct from those experienced during 2D migration¹.
As the largest and stiffest cellular component, the nucleus poses a significant
barrier to confined cell migration². As such, confined
migration exerts a mechanical stress on the nucleus, which can ultimately lead
to the blebbing and rupture of the nuclear envelope followed by DNA damage ^{background:transparent"> 3} line-height:150%;font-family:Arial;color:black;background:transparent">. While
actomyosin contractility has been implicated in regulating nuclear envelope
integrity, the exact mechanism of nuclear envelope blebbing and rupture remains
unknown.

background:transparent">Methods: line-height:150%;font-family:Arial;color:black">To explore the mechanism of confinement-induced
nuclear envelope bleb formation and rupture, we prompted cells to migrate
through PDMS-based microfluidic channels with fixed dimensions of 3 µm in
height, 10 µm in width, and 200 µm in length. 11.0pt;line-height:150%;font-family:Arial;color:black;background:transparent"> We
combined these microfluidic migration assays with molecular biology techniques,
high resolution imaging (confocal, FLIM FRET, optogenetics), and mathematical
modeling to elucidate how contractile forces specifically promote nuclear bleb
formation. 

background:transparent">Results and Discussion: background:transparent">We demonstrate that confinement-induced activation
of RhoA/myosin-II contractility at the cell posterior locally increases
cytoplasmic pressure, and through nucleo-cytoskeletal bridges formed by the
LINC complex, promotes passive influx of cytoplasmic constituents into the
nucleus without altering nuclear efflux. line-height:150%;font-family:Arial;color:black">Photoablation of cortical
actomyosin contractility or inhibition of the RhoA/ROCK pathway suppresses this
confined migration-induced nuclear influx. line-height:150%;font-family:Arial;color:black;background:transparent">Elevated
nuclear influx is accompanied by nuclear volume 11.0pt;line-height:150%;font-family:Arial;color:black;background:transparent">expansion,
blebbing and rupture. Moreover, inhibition of nuclear efflux is sufficient to
increase nuclear blebbing on two-dimensional surfaces, and acts synergistically
with color:black;background:transparent">RhoA/myosin-II contractility to further
augment blebbing in confinement 150%;font-family:Arial;color:black;background:transparent">. Nuclear volume
expansion and blebbing ultimately reduce cell motility, as predicted by
mathematical modeling based on lubrication theory.  Our results
demonstrate that confinement regulates RhoA activation and perturbs nuclear
flux homeostasis with significant consequences for nuclear size, integrity and
cell motility (Figure 1). line-height:150%;font-family:Arial;color:black">Overall, our work provides
additional insight into the processes of nuclear bleb formation and rupture,
which could aid in the development of novel therapeutics to combat metastasizing
cancer cells that experience this phenomenon.

background:transparent"> 

background:transparent">1. C.D. Paul et. al. Nat Rev Cancer, 17: 131-140
(2017). 2. K. Wolf et. al. JCB, 201: 1069-1084 (2013). 3. C.M. Denais
et. al. Science, 352: 353-358 (2016).