(500e) The Role of the Cytoskeleton in Focal Adhesion Development and Migration of Cells Attached On 3D Aligned Fibrous Scaffolds

The Role of the
Cytoskeleton in Focal Adhesion Development and Migration of Cells Attached on
3D Aligned Fibrous Scaffolds

Kevin Sheets¹, Amrinder Nain^1,2

1.        STEP Lab, School of Biomedical
Engineering & Sciences, Virginia Tech, Blacksburg, VA

2.       Department of Mechanical Engineering,
Virginia Tech, Blacksburg, VA

Introduction

In the human
body, cells attach to and receive biophysical and biochemical cues from their
immediate fibrous microenvironment known as the extracellular matrix (ECM), which
helps drive overall cell behavior.  Cells form attachments to the ECM through
multi-protein complexes known as focal adhesions (FACs), whose size and
orientation dynamics (spatial and temporal) adapt with changes in surrounding
geometry^1?3. Likewise, other aspects of the cytoskeleton including f-actin stress fibers, myosin IIa motors, and microtubules adjust according to the makeup of their extracellular environment and cooperatively interact to produce locomotive force against the ECM via FACs^4,5.  While this phenomenon is well-understood on flat substrate conditions, recent work has shown that cell attachment profiles contain considerable differences when cultured on three dimensional fibrous substrates mimicking the native ECM^6?8.  Using the STEP (Spinneret-based Tunable Engineered Parameters) platform, which generates fibrous scaffolds of high aspect ratio nano/micro fibers with tightly controlled diameters and spacing, we examine FAC behavior on ECM-mimicking scaffolds in both normal cells and cells with cytoskeletal knockdowns^9?12.  By administering the drugs blebbistatin, nocodazole, and cytochalasin-D, cytoskeletal elements myosin IIa, microtubules, and actin are depolymerized respectively, causing notable changes to migration speed and FAC behavior on these scaffolds. The efficacy of these drugs in changing the FAC dynamics can be used for developing drug testing platforms, arresting cellular migration of metastatic cancerous cells and developing more effective tissue engineering scaffolds.

Materials and Methods

In addition to
using flat glass substrates, suspended polystyrene (PS) fibers of approximately
500 nm diameter were spun onto hollow STEP substrates. Scaffolds were coated
with fibronectin and incubated overnight prior to cell seeding using standard
cell culture techniques. Seeded mouse C2C12myoblasts attached to
and spread on the fibers within 4-6 hours.  Cells on fiber networks were then time-lapse
imaged using an incubating microscope (Zeiss) with a digitally-controlled X-Y-Z
stage.  Cell migration speeds were calculated via time-lapse by tracking the
position of the cell's nucleus every hour and noting the maximum distance
traveled per hour in a 12-hour window.  Blebbistatin (50 μM), nocodazole (10 μM), and cytochalasin-D
(5 μM) were added
directly to media at typically-noted concentrations 6 hours after initial
seeding and were time-lapse imaged immediately¹³. 

Fluorescence
microscopy was used to locate focal adhesion proteins as well as the f-actin
stress fibers and cell nuclei.  Cells were fixed in 4% paraformaldehyde for 15
minutes at room temperature, permeabilized in a 0.1% solution of Triton X100,
and then blocked in 10% goat serum for 30 minutes.  Primary goat antibodies
against paxillin (Invitrogen) were diluted 1:250 followed by GFP secondary
antibodies also at 1:250 to visualize FACs.  Rhodamine phalloidin was used
1:100 to stain stress fibers, and DAPI was used to stain the nuclei.

Results and Discussion

PS fibers of 500
nm diameter were deposited in single, parallel and orthogonal configurations
with cells responding to change in orientation and topography accordingly
(Fig.1A-C). After seeding cells on STEP scaffolds, cells were found to attach
within six hours.  Immediately following this time, cells were treated with the
drugs in aforementioned concentrations and then observed for changes to
migration behavior and cytoskeleton arrangement.  As can be seen in Figure 1,
migration speeds were found to vary according to both cell shape and
administered drug. 

Fig. 1: A-C) STEP fiber layouts used to get cells to attach in D-F) spindle, parallel, and kite configurations, respectively. (G) C2C12 migration speeds as a function of cell shape and administered drug. With the exception of cytochalasin-D, the only significant change to migration speed was to spindle-shaped cells treated with nocodazole. * indicates statistical significance compared to no drug case (p<0.001, n = 5 cells each).
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Spindle cells,
which attach and elongate along a single fiber axis, form stable adhesion patches
(~ 11 µm in length) at the cell periphery (Fig.1D).  Due to their high
polarity, these cells are capable of migrating at high speeds (50+ μm/hr).  However, when
actin is depolymerized with cytochalasin-D, the cell is no longer able to
extend its membrane and form stable adhesion patches, which causes cells to be
non-migratory. On the other hand, nocodazole, which depolymerizes microtubules,
reduced spindle cell speed to approximately half the original value.  Since the
cell no longer contains microtubules, this drug prevents the cell from
splitting into daughter cells despite progressing through the cell cycle.  The
end result is a more massive cell containing multiple nuclei which remains
confined to the same attachment profile.  In theory, because the mass of the
cell increases without any visible change to the FAC, migration speed
decreases.  Lastly, blebbistatin-treated cells migrated at the same speed as
the wild type cells.  In the absence of myosin IIa, cells are unable to sense
the stiffness of the fiber matrix and thus do not develop mature adhesions. 
These temporally unstable FACs ultimately allow the cell to maintain normal
migration speeds.

Parallel cells
were found on STEP scaffolds which contained fibers that were spaced relatively
close together (<20 μm). 
At this separation distance, the cells were often found spanning the gap
between the two fibers and forming prominent adhesion clusters at the cell
periphery ? two per fiber for a total of four main clusters (Fig 1E).  Parallel
cells migrated about 40 μm/hr. 
Cytochalasin-D again stopped migration altogether.  Blebbistatin did not alter
migration speed and the effect of nocodazole was dampened for this shape, most likely
since parallel cells travel slower to begin with and have additional adhesion
sites compared to spindle cells, which only have one fiber to elongate on.

Kite-shaped cells,
which attach to intersecting fibers and form two adhesion clusters along each
fiber axis, migrate the slowest of the three shapes at about 30 μm/hr (Fig.1F).  Again,
cytochalasin-D stopped migration whereas blebbistatin and nocodazole did not
significantly change migration speed.  Cells travel slower in this shape
presumably due to the formation of four nearly equal FACs which each pull
towards the cell center.

Fig. 2: Fluorescence images showing paxillin (FACs, green), phalloidin (actin, red), and DAPI (nucleus, blue) on drug-treated cells for both the flat glass and spindle shape conditions. Blebbistatin causes shorter FACs to form. Nocodazole FACs are very similar to untreated cells. Using cytochalasin-D, FACs are no longer able to form.
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To further probe
the effect of drugs on the cytoskeleton, cells were fixed and stained to
measure changes in focal adhesion lengths, stress fiber formation, and nucleus
positioning (Fig 2).  This was performed on both flat glass as a control
substrate and on STEP fibers.  There were several notable changes to the
structure of the cells that were treated with drugs which confirm changes seen
in migration speeds.  First, blebbistatin-treated cells contained FACs that
were significantly shorter than untreated cells.  This is likely due to the
inability of the latent adhesions to mature by responding to the fiber beam
stiffness (N/m). Cells exposed to nocodazole were frequently observed to
contain multiple nuclei within the same cell body.  Since this drug depolymerizes
microtubules, a structural component key to mitosis, the cell continued to
progress throughout the cell cycle without forming daughter cells.  Despite
this extra cell matter, FACs were essentially unchanged.  Lastly,
cytochalasin-D treated cells contained no traces of actin or FACs whatsoever,
confirming its significance in the cell migration process. 

Fig. 3: FAC lengths and distances from nucleus for each cell shape and drug combination. * indicates statistical significance compared to no drug case (p<0.05, n = 3 for each cell/drug combination)
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Conclusions

            Using the STEP
platform for fiber manufacturing, cellular migratory behavior and cytoskeletal
arrangements were tracked for both normal and drug conditions.  While
fluorescence images demonstrated definitive changes to cytoskeletal appearance
and arrangements compared to the 2D case, overall migratory speeds were
observed to remain constant with treatment of blebbistatin, and nocodazole only
significantly affected spindle shaped cells.  Cytochalasin-D caused migration
to stop altogether.  In these cases, changes to the cytoskeletal proteins
caused subsequent changes to focal adhesion formation and function, especially
compared to the traditionally-studied 2D cases. This information will be used
in the future to further understand and predict cell motion based on attachment
profiles with the hopes of designing structures to either speed cell migration
for applications such as wound healing, or slow migration in cases such as
metastatic cancer cells.

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