Cells are intricately connected to

Cells are intricately connected to the external environment through their
cytoskeleton. Whether in direct contact with neighbouring cells or with
a dense meshwork of polymers known as the extracellular matrix, cells
receive external signals that guide complex behaviours such as motility
and, in some cases, differentiation (for example from stem cells into cells
of a specific lineage). Whereas the contribution of chemical signals haslong been understood, physical signals have only recently been widely
recognized to be pervasive and powerful. The observation that physical
properties of the microenvironment can affect cell shape and behaviour
dates to the 1920s, when studies showed that mesenchymal cells embedded
in clots of various stiffnesses had different shapes (see ref. 46 for a
review). More recent studies have shown that the tension generated by
a contracting cytoskeleton can be used to sense the mechanical properties
of the extracellular matrix, which in turn have been shown to affect
cytoskeletal organization and cell behaviour47, although whether stiffness
or force is the most important signal remains a subject of debate48.
Of particular interest is how cell–extracellular matrix and cell–cell
interactions can lead to long-lived changes in cellular organization in tissues
and cell behaviour. Several studies have highlighted the importance
of physical cues in the organization of tissues during development. For
example, Thery and colleagues found that the orientation of the mitotic
spindle in dividing cells, and hence the location of the division plane and
the spatial arrangement of the daughter cells, is affected by the spatial distribution
of extracellular matrix proteins49. Using microcontact printing
to define patterns of extracellular matrix proteins to which cells adhere,
they found that the cells divided with predictable orientations controlled
by cortical contacts with the extracellular matrix. In addition to the pattern
of adhesion sites, the mechanical properties of cells themselves also
contribute to tissue organization. As one example, in gastrulating zebrafish
embryos, actin- and myosin-dependent plasma-membrane tension and
differential adhesion among cells drives the sorting of germ-layer progenitor
cells50. Cell proliferation can even be affected when external forces are
applied to tissue (Fig. 4). When a tumour spheroid, formed from murine
mammary carcinoma cells grown in a cluster within an agarose gel, is
exposed to compressive loads during growth, the cells proliferate more
slowly at regions of high stress and undergo programmed cell death when
exposed to sufficiently high stress51.
When the ‘normal’ mechanical properties of tissue are disrupted, the
effects can be considerable. In epithelial cell layers, altered stiffness of the
supporting tissue disrupts morphogenesis and drives the epithelial cells
towards a malignant phenotype52. In fact, the stiffness of the substrate
seems to be important for stem cells to differentiate properly. A substrate
with a stiffness that emulates normal tissue can function as a developmental
cue that directs stem cells to differentiate into cells of specific lineages,
including mesenchymal stem cells53 and neural stem cells54. How substrate stiffness, as well as growth factors and matrix properties, affect stem-cell
differentiation is reviewed in more detail in ref.

Emulating the native properties of a cell’s environment is an important
consideration that is often overlooked when studying cells ex vivo.
For example, the traditional method of culturing cells on stiff substrates,
which has been used for decades, can itself drive changes in the mechanical
properties and gene-expression profiles of the cells. Primary human
foreskin epithelial cells cultured in plastic dishes were found to increase
in stiffness with passaging, with cells being twofold to fourfold stiffer after
eight passages than were cells passaged fewer than three times56. Similarly,
human epithelial breast carcinoma (MCF7) cells were found to stiffen
with increasing passage number when cultured on glass coverslips57 and
endometrial adenocarcinoma cells cultured in plastic dishes expressed
more α-actin as a function of passage number, as they moved towards
a stromal phenotype58. In each of these cases, the cells were grown on
substrates with markedly different mechanical properties from native
tissue, and long-lived changes in cytoskeletal properties and cytoskeletal
organization were observed.
As more examples of cells responding to mechanical cues through
the cytoskeleton are found, the questions of how, what and where physical
inputs are sensed is becoming central. There is substantial evidence
implicating stress-induced changes in focal adhesions and adherens junctions26,
and several molecules have been identified as specific mediators
of mechanical inputs. For example, mesenchymal stem cells differentiate
into cells of various lineages depending on the substrate stiffness, and this
elasticity-sensitive lineage-specific differentiation is blocked by inhibiting
the protein non-muscle myosin53. A force-induced conformational
change in p130Cas (also known as BCAR1), a scaffolding protein that is
involved in focal adhesions, causes it to be more easily phosphorylated
by Src59. And sensitivity to the elasticity of the extracellular matrix during
angiogenesis is mediated by the Rho inhibitor p190RhoGAP (also known
as GRLF1), through its effect on two antagonistic transcription factors
Although changes in gene expression may be the end point of
mechanosensing, the process can take days for cells in culture, and it is
unclear how information about physical interactions with the mechanical
micro environment is stored. Are heritable changes in gene expression in
mechanically perturbed cells due only to changes in chromatin structure
and organization or other familiar epigenetic mechanisms? The heritability
of changes that arise from mechanical interactions — and that are
mediated by the cytoskeleton — raises the question of whether the organization
and reorganization of long-lived cytoskeletal structures themselves
might have a role in recording the cell’s mechanical ‘history’.
Cytoskeletal epigenetics
The idea that cellular structures can be passed on and influence the
behaviour of subsequent generations of cells is not new. In the 1930s,
embryologists recognized that regional differences in the molecular
composition of ova caused daughter cells to inherit different surface
molecules61. In the 1960s, Paramecium aurelia cells that were genetically
identical to wild-type cells were found to pass on alterations in the orientation
of their cilia for hundreds of generations62,63. Internal structures
of the cytoskeleton can also persist after cells divide. Daughter 3T3 fibroblast
cells have similar actin-filament stress-fibre organization and motile
behaviour64,65, and adjacent epidermal ‘siamese twin’ cells in Calpodes
ethlius caterpillars have the same number of actin-filament bundles66. The
emergence of primary cilia from sister cells was recently found to depend
on centriole age, with sister cells that inherit the old centriole growing a
primary cilium before sister cells that inherit the new centriole, which
was formed before cell division67.
As a look at any dish of cultured cells will confirm, genetically identical
cells can have markedly different cytoskeletal structures, presumably as
a result of random events as well as slight differences in external conditions.
Endothelial cells grown in vitro under similar conditions, for example,
contain stress fibres that are oriented randomly. But, if those cells are
exposed to shear stress from fluid flowing above them, they respond by
elongating and orienting their stress fibres in the direction of the flow. If
the shear stress is removed, then the variability in stress-fibre orientation
returns, but it does so slowly. Interestingly, if the elongated cells are
detached from the surface, then the elongated shape persists68. In this way,
the cytoskeleton can be a record of a cell’s past mechanical interactions.
Given the interconnectedness of the cytoskeleton and its role in the transduction
of mechanical signals from the external microenvironment, as
well as its role as a scaffold for many reactions69, the ability of cytoskeletal
structures to record the past may result in the cytoskeleton profoundly
affecting the cell’s future and even the future of the cell’s progeny70.
Given that cytoskeletal structures are often highly dynamic, with specific
factors that promote disassembly and recycling of the cytoskeletal
building blocks competing with factors that assemble and stabilize them,
is it possible for mechanical inputs to be recorded? During endocytosis,
actin-filament networks can assemble around clathrin-coated pits and
displace the invaginated membrane in less than 15 seconds71. But, when
the timescales for assembling and stabilizing a cytoskeletal structure are
longer than those for disassembling and recycling it, the result can be
a persistent structure that affects the behaviour of a cell over a longer
timescale than the initial signal. In essence, the system shows hysteresis,
a common feature of magnetic, electrical and elastic properties of materials,
in which there is a lag between application or removal of a stimulus,
such as a force, and its effect. In biological systems, this hysteresis seems
to involve active, energy consuming processes. In one example, a growing
actin-filament network that had been reconstituted in vitro was exposed
to a weak compressive force during growth. When the compressive force
on the network was gradually increased and then rapidly reduced to the
previous level of force, the velocity of network growth increased and persisted
at a rate that was significantly higher than the original velocity at that
force44. It is likely that this increase in velocity resulted from an increase in
actin-filament de