Transcript Cell Effects on Mechanical Properties of Environment

Cell Effects on Mechanical
Properties of Environment
Morgan Boes
Sources
• Beningo, K. A., Dembo, M., Kaverina, I., Small, J. V., & Wang,
Y.-l. (2001). Nascent Focal Adhesions are Responsible for
the Generation of Strong Propulsive Forces in Migrating
Fibroblasts. The Journal of Cell Biology , 153 (4), 881-7.
• Dobereiner, H.-G., Dubin-Thaler, B. J., Giannone, G., &
Sheetz, M. P. (2005). Force sensing and generation in cell
phases: analysis of complex functions. Journal of Applied
Physiology , 98, 1542-6.
• Wang, J. H., & Li, B. (2010). Mechanics rules cell biology.
Sports Medicine, Arthroscopy, Rehabilitation, Therapy, &
Technology , 2 (16).
Mechanical Forces
• a push or pull exerted by the cell
• a push or pull encountered by the cell
“Mechanics rules cell biology”
Cells in the musculoskeletal system are subjected to various
mechanical forces in vivo. Years of research have shown
that these mechanical forces, including tension and compression,
greatly influence various cellular functions such as
gene expression, cell proliferation and differentiation, and
secretion of matrix proteins. Cells also use
mechanotransduction mechanisms to convert mechanical signals
into a cascade of cellular and molecular events.
An overview of cell mechanobiology to highlight the notion that
mechanics, mainly in the form
of mechanical forces, dictates cell behaviors in terms of both
cellular mechanobiological responses and
mechanotransduction.
Cell Types that encounter forces
• Fibroblasts in tendons and ligaments are
under tensile stress
• Chondrocytes and osteocytes are subjected to
compression and shear stress
Internal Mechanical Forces
• Forces generated by the cells themselves
– Considered intracellular tension
– In non-muscle cells this is created by cross-linking of
actomyosin.
• These forces are then transmitted to the ECM via
focal adhesions
– These forces are called cell traction forces (CTFs)
– CTFs direct ECM assembly, control cell shape, permit
cell movement, and maintain cellular tensional
homeostasis.
Cellular Traction Forces (CTF)
• Deform the ECM and cause stress and strain in
the network, which then in turn modulate
cellular functions such as gene expression and
protein secretion.
• Cells can also use their internal contractile forces
to regulate their own proliferation and
differentiation.
• Internal mechanical forces generated by cells
themselves regulate cell biology in terms of
metabolic state, cell proliferation and
differentiation, etc. Especially when these CTFs
are transmitted to the ECM, where they regulated
many vital cellular functions such as migration
and ECM assembly
Cellular functions affected by Cellular
forces
•
•
•
•
•
Cell proliferation
Differentiation
Gene expression
Protein synthesis of ECM components
Production of cytokines and growth
factors
• Therefore CTFs are important in
fundamental biological processes
such as embryogenesis, angiogenesis,
and wound healing.
Force sensing and generation in cell
phases: analyses of complex functions
• Cellular morphology is determined by
– Motility
– Force sensing
– Force generation
Two major explanations for motility
• Either cellular motility depends in a
continuous fashion on cell composition
• Or it exhibits phases wherein only a few
protein modules are activated locally for a
given time.
• Observations of the behavior of cells can be
dissected into functional pathways involving
key proteins or protein groups that contribute
specific function to the overall behavior
• Cells in suspension have a basal level of
motility that enables the to probe their
immediate environment.
Phase 1- “early spreading”
• Cell goes from a rough sphere to a thick disk on a 2D
surface with about the same cross-sectional area
• During this phase there are three necessary steps
• This phase is activated by a cell-wide process that
induces disassembly of filaments generally and
spreading locally.
Phase 1 Step 1
• Local sensing of the matrix coating of the surface
which activates
– General breakdown of cortical actin filaments and
cortical structure
– Local assembly of actin filaments at matrix-coated
surfaces
• In this step there is a threshold to the activation
that is a function of both fibronectin density and
time.
– Higher concentrations decrease the lag time before
spreading instead of increasing the rate of spreading
Phase 1 Step 2
• Continued actin filament assembly that
depends on the new binding of surface
integrins to new regions of the surface
Phase 1 Step 3
• Slow rearward transport of the newly
assembled actin filaments.
• This phase will automatically stop when either
the cell reaches a critical area or receives
another dominant signal
Phase 2: Contractile Phase of
Spreading
• Distinguished by a dramatic increase in the
rate of rearward actin movement.
• Required for continued spreading
• Rigid substrates are also required for
continued spreading
• But how does the cell know if the substrate is
rigid?
Rigidity Sensing
• At these early stages of spreading, there are no
stress fibers, thus it is difficult to understand how
the cytoskeleton is organized to support force
generation from one side to the other.
• The tension that the cell creates is clearly due to
myosin, and the major question is how the myosin is
organized to enable it to generate force to move
actin inward and ultimately to generate force on the
surround matrix
Ridgidity Sensing
• Rigidity sensing is a major aspect of the
contractile phase, and fibroblasts need a rigid
substrate to spread fully.
• Neurons prefer a softer substrate.
• There are two theories on how the cell senses
the rigidity of the substrate nearby.
Rigidity Sensing
Phase 3
• On sufficiently stiff substrates, the cell
continues spreading, approaching its maximal
area of contact. After which the cell moves
forward in a particular direction after
polarization is triggered by either internal
signals or external chemical gradients
• These phases are relatively general and
applies to fibroblasts, endothelial cells, and
presumably in a similar form to even neuronal
growth cones
• Motility phases involve a characteristic subset
of functions that are organized in specific
spatial and temporal order. They involve
distinct sets of protein modules
Phase Transitions
• How does the cell control the transitions
between the fast spreading and retraction
phases?
Initiating Cell spreading
• Characterized by an increase in the actin
polymerization velocity at the leading edge of
the lamellipodium, pushing the membrane
forward. Increased polymerization is triggered
by favorable contact with the ECM.
• Time from contact to spreading decreases
with fibronectin density
Transition to periodic contractile phase
• Linked to the activity of myosin light chain
kinase (MLCK), a protein control in turn the
activity of myosin motors. But there is no
direct evidence yet for the involvement of
myosin.
Global Cell Phases
• The evolutionary pressure to survive preserves
function but not necessarily the associated
protein modules.
• So by understanding cell mechanisms and
their phases, we can compare across cell types
even if the proteins involved vary.
• To better define these phases, define basic
functional protein modules
Basic functional modules example
• Regulatory proteins – controlled by a signaling
network coordinating spatially distant and/or
logically separate functional events in a cell
– Must not interact directly with the structural
proteins
Nascent Focal Adhesions Are
Responsible for the Generation
of Strong Propulsive Forces in
Migrating Fibroblasts
Focal Adhesions
• We know that focal adhesions tightly adhere
to the extracellular matrix
• But what is their role in force transduction?
The role of focal adhesions in force
transduction?
• To figure this out the researchers
– Mapped traction stress generated by fibroblasts
expressing GFP-zyxin.
• They found
– The overall distribution of focal adhesions only
partially resembles the distribution of traction
stresses
Leading edge of cells
• Faint small adhesions transmit strong forces
• Large, bright, mature focal adhesions exert
weaker forces
• This relationship is unique to the leading edge
of motile cells.
• Also traction forces decrease soon after the
appearance of focal adhesions
• As focal adhesions mature, changes in
structure, protein content, or phsophorylation
may cause the focal adhesion to change its
function from the transmission of strong
propulsive forces, to a passive anchorage
device for maintaining a spread cell
morphology.
Focal Adhesions
• Involved in anchoring cells to the substrate
• What about contractile forces that might be
transmitted through these structures to
propel directional movements
• Hundreds of focal adhesions must be
coordinated in order to maintain both the
direction of migration and the morphology of
the cell in an efficient manner. HOW?
• Solution – to generate maps of both dynamic
focal adhesions under a migrating fibroblast,
and traction forces that a cell exerts on the
substrate
Traction stress Measurement
• Tracked by embedding beads into a flexible
substrate that the cells are grown on.
• Then using large-scale matrix computations,
convert bead movements of substrate
deformation to maps of traction stress.
Traction stress calculations
• Plated cells on collagen I –coated flexible
polyacrylamide substrates.
• The confinement of traction stress was within
the cell boundary, global balance of forces and
torques was required
• Used a Monte Carlo simulation to determine
force balance.
Visualizing the location of Focal Adehsions
Results
• Small, nascet focal adhesions at the leading
edge exert transient forces to move the cell
forward.
• Mature focal adhesions serve primarily as
anchors to the substrate.
• Allows fibroblasts to migrate efficiently and
responsively without complex coordination of
the mechanical output among the adhesion
foci
Figure 5. Relationship between focal adhesions and mechanical
forces during fibroblast migration. The formation of focal adhesions,
accompanied by the generation of a pulse of propulsive
forces, drives the forward movement. Cell migration is sustained
by repeated formation of nascent focal adhesions, and thus repeated
pulses of propulsive forces. Mature focal adhesions play
only a passive role in anchoring cells to the substrate.
Advantages to this approach 1
• A division of labor between propulsive
adhesions and anchorage at the leading edge
which allows the cell to migrate while
maintaining its spread morphology.
Advantages to this approach 2
• Since cell migration is driven by transient
pulses of propulsive forces in the leading
lamella, minimal coordination is required
among mechanical interactions at a multitude
of focal adhesions
Advantages to this approach 3
• This mechanism facilitates rapid reorientation
in response to environmental cues, simply by
shifting assembly of nascent focal adhesions
to a new protrusive region.
Questions?
Sources
• Beningo, K. A., Dembo, M., Kaverina, I., Small, J. V., & Wang,
Y.-l. (2001). Nascent Focal Adhesions are Responsible for
the Generation of Strong Propulsive Forces in Migrating
Fibroblasts. The Journal of Cell Biology , 153 (4), 881-7.
• Dobereiner, H.-G., Dubin-Thaler, B. J., Giannone, G., &
Sheetz, M. P. (2005). Force sensing and generation in cell
phases: analysis of complex functions. Journal of Applied
Physiology , 98, 1542-6.
• Wang, J. H., & Li, B. (2010). Mechanics rules cell biology.
Sports Medicine, Arthroscopy, Rehabilitation, Therapy, &
Technology , 2 (16).
Figure 1. Monte Carlo simulation of traction stress
analysis. Small patches of traction stress were first
assigned at random locations within a square area (a
and b). Exact deformation matrix was generated
from this map at a finite resolution and density (c).
After applying random noise and neighborhood averaging
to mimic the resolution limit of the measurements
(d), the modified deformation matrix was
used to calculate the original traction stress (e and
f). A pair of forces separated by z4.4 mm appears as
a single large patch (arrowheads), whereas a pair
separated by z8.0 mm is clearly resolved (arrows).
Panels b and f show color rendering of the magnitude,
with red corresponding to strong traction
stress and blue corresponding to weak traction
stress
Figure 2. GFP-zyxin as a marker for focal adhesions.
Fish fin fibroblasts transfected with GFPzyxin
were plated on collagen-coated coverslips.
IRM (b) shows the localization of GFP-zyxin at
both large and small focal adhesions (a).
Immunofluorescence
staining of paxillin (d) shows a similar
colocalization with GFP-zyxin (c) at the leading
edge. Bars, 10 mm
Figure 3. Differences between the distribution of
traction stress and focal adhesions.
Distributions of traction stress at 0, 6, and 10 min
are shown as either vector maps (a, d, and g), or color
images after converting the magnitude into colors (b, e,
and h). The corresponding distributions of GFP-zyxin
show only a limited correlation with traction stress (c, f,
and i). Some focal adhesions contain a low concentration
of GFP-zyxin but generate strong forces (open arrow),
whereas other focal adhesions show strong GFP-zyxin
localization but generate relatively
weak forces (filled arrows). Arrow in g, 105 dyn/cm2. Bar,
20 mm.