THE ROLE OF CADHERIN-BASED JUNCTIONS IN THE FOCAL ADHESIONS
OF KERATINOCYTES
A Report of a Senior Study
by
Stephen Revilla
Major: Biochemistry
Maryville College
Spring, 2014
Date Approved _____________, by ________________________
Faculty Supervisor
Date Approved _____________, by ________________________
Division Chair, Natural Sciences
iv
ABSTRACT
Forces that are translated from the cytoskeleton through the matrix of a cell regulate
functions such as cellular spreading, proliferation, and wound healing. The human body
responds to injuries by increasing calcium ion concentrations throughout keratinocyte cells,
and higher calcium levels can activate cellular components such as E-cadherin, whereas low
levels of calcium have been shown to inhibit E-cadherin. Previous studies have shown
through Traction Force Microscopy that the force distribution patterns observed in cell
colonies vary greatly when E-cadherin functionality is switched on or off. However, the
causes for this at the cellular level had not yet been documented. Using fluorescence
microscopy, keratinocyte colonies were imaged to view their focal adhesions and compare
the distributions of these linking proteins. We found high calcium colonies (with functional
E-cadherin) had a significantly denser per unit length ratio of peripheral focal adhesions to
inner-colony adhesions than that of low calcium colonies (which lack E-cadherin). The
average ratios for high calcium and low calcium colonies were 1.85 to 0.90, respectively.
Since focal adhesions display force into the extracellular matrix, and our study showed a
distribution of focal adhesions correlating to the force distribution patterns that have been
previously shown, this study provides evidence that E-cadherin affects focal adhesion
distribution which then influences strain patterns that have been observed in other studies.
iii
TABLE OF CONTENTS
Page
Chapter
I Introduction and Background
Cellular Components
Immunostaining
1
2
9
II Materials and Methods
Creating of substrates for TFM
Primary mouse keratinocyte
Cell Staining
14
14
15
16
III Results
Cellular images
Focal adhesion quantification
18
29
26
IV Discussion
Quantitative explanations
Future Studies
29
30
38
Appendices
Appendix A – IACUC Approval of Study
Appendix B – Quantification of Focal Adhesions
References
40
41
43
47
iv
LIST OF FIGURES
Figure
Page
1. E-cadherin Junction Illustration
3
2. TFM Substrate Illustration
6
3.
7
TFM Bead Distribution Under Keratinocyte Colony Stress
4. Traction Stress Mapping of Keratinocyte Colonies
8
5. Immunostaining Flow Chart
12
6. Keratinocyte Colony in High Calcium
29
7. Keratinocyte Colony in Low Calcium
20
8. Keratinocyte Colony in DECMA High Calcium
21
9. Keratinocyte Colony in KO Low Calcium
22
10. Keratinocyte Colony in KO High Calcium
23
11. Distance Measurement Around Colony Periphery
24
12. Distance Measurement of Intra-Cellular Boundaries
25
13. Ratios of Peripheral Adhesions to Inner Colony Adhesions
26
v
14. Average Density Ratios around Peripheral Focal Adhesions
28
vi
ACKNOWLEDGEMENTS
This thesis was made possible by the generous Sackler donation / NSF REU grant,
the Horsley Lab, and Dr. Noble. I would like to give special thanks to Aaron Mertz, Yonglu
Che, and Valerie Horsely of Yale University for their unfaltering assistance and guidance
through my research. I would also like to thank Dr. Swann of Maryville College for her
assistance in not only the writing of this paper, but also her guidance throughout the process.
vii
CHAPTER 1
INTRODUCTION AND BACKGROUND
Cellular shape, as well as cellular spreading, affects cellular proliferation in tissues.
More specifically, forces that are translated through the cytoskeleton of a cell to the matrix of
the cell partake in this regulation of proliferation.1 These forces have been termed traction
forces and can be quantified. In response to interest in cellular forces, many researchers have
been investigating the physical forces that a cell displays onto a substratum. The field of
mechanobiology has been focused for more than a decade on the mechanics of single,
isolated cells.2 Unfortunately, by only studying single cells little information is gained on
how the cells act together to give tissues the physical properties they are seen to display.
Investigation of the physical properties of colonies of should provide a better understanding
of the mechanical properties of entire tissues. This information will improve our
understanding of how these cellular forces regulate tissue spreading and proliferation
processes which play fundamental roles in wound healing and embryonic development.1
To study these physical properties of cell colonies is helpful to investigate the traction
forces that cells display on the extracellular matrix (ECM) through adhesion proteins, along
with forces created from interactions with adjacent cells. Cells generate these traction forces
1
though myosin-generated tension in the actin cytoskeleton.3 The forces displayed from the
cells are actually transmitted through the cytoskeleton.4 In epithelial cells the ECM helps to
“link” adjacent cells together in colonies, and the forces exerted through the ECM have been
a major topic of interest. In fact it has been determined that properties of the ECM modulate
cellular forces.5 The matrix takes advantage of linker proteins to hold epithelial cells
together; these are called adhesion proteins.
Adhesions are formed by cadherin proteins which rely on calcium ion (Ca2+)
interactions for functionality. Cadherin proteins are the primary cell-to-cell adhesion
molecules observed in epithelial tissue. 3,5 Cadherin has been found in all multicellular
animals, as well as in many bacteria. When calcium is removed from the medium where
these cells grow, the cells lose their ability to form cadherin bindings and form less cohesive
junctions with each other. Interestingly enough though, these cadherin proteins have been
shown to regain function6 if placed back into normal calcium environments, such as that seen
in normal culture media (high calcium media).7
Cadherin proteins are transmembrane adhesion proteins, meaning they span the full
width of cellular membranes. Inside the cell these transmembrane proteins bind to the
cytoskeletal actin filaments.7 Outside the cell these transmembrane proteins can bind to other
structures, including other transmembrane proteins. The adherens junction is a cell-to-cell
adhesion where cadherins in one cell bind to and form stable attachments with other cadherin
proteins in adjacent cells. In this manner the caderins connect actin filaments from the
cytoskeletons of adjacent cells. There are various types of cadherin proteins, most of which
are named according to where they are found in the body. The specific cadherin of interest in
this study is E-cadherin, which is primarily found in epithelial tissue, such as skin cells.
2
Shown in Figure 1 is a representation of the bonding formed by an E-cadherin
transmembrane protein linking two adjacent cells.
Modified from:
Figure 1: E-cadherin is a transmembrane protein that binds to other E-cadherin,
forming a junction from the actin of one cell to the actin of a neighboring cell. Cadherin is
the primary linker of cell-to-cell contacts.
The other adherin proteins of interest are those of the integrin superfamily. Cadherin
proteins are primarily involved in cell to cell adhesions, but integrin proteins mediate the
linkage of cells to matrix.7 Integrins, like cadherins, are transmembrane proteins that are
linked to the cytoskeleton. They are also affected by Ca2+ concentrations and, like
calmodulin, can be switched between an active and inactive state (although through different
means). Integrins can create strong adhesions in culture dishes known as focal adhesions.
These integrins are also involved in signaling pathways, and the cell itself is often times
dependent on these adhesions to the matrix. The intracellular signals that have been
3
associated with these integrin proteins can influence many properties of the behavior of cells,
including survival and proliferation.8
Cells, specifically focal adhesions, exert traction forces onto the ECM to which they
adhere.9 The ECM is known to play an integral role in cellular migration and proliferation.4
Therefore the focal adhesions, through modifying the ECM , play a direct role in cellular
migration, proliferation, and wound healing. These focal adhesions are often found at the end
of actin stress fibers that are concentrated at the peripheries of epithelial cells. It is known
that focal adhesions function as adherent structures that a cell can use to bind to fibronectin.
Fibronectin is a glycoprotein common in the ECM of connective tissues that underlie
epithelial tissues. In nature fibronectin is found as a dimer composed of two similar subunits
linked by disulfide bonds. Fibronectin is a ligand for many integrins which then link the
ECM to the cytoskeleton of the cell. This means that fibronectin is actually capable of
binding to the surfaces of cells though the integrins to which they are linked.10 Fibronectin
can be thought of as a general binding/linking molecule, as it also contains fibrin-binding
sites. This binding is said to play a role in cell adhesion and even cell migration. This linkage
is often stimulated by fibrin clots, and is likely involved in post-inflammatory macrophage
clearance.10 Fibronectin exists in soluble and insoluble forms in the body, which allows it to
circulate though the blood in its soluble form (inactive) and be part of the ECM (active
form). Fibronectin fibrils then are specifically attached by the body to the ECM in a highly
regulated process. This protein functions in the ECM and affects cell adhesion, migration,
and differentiation.10
Wound healing is accomplished through four phases: hemostasis, inflammation,
proliferation, and remodeling.11 Hemostasis is noted by fibrin clot formation, which is in part
4
achieved through forming fibronectin lingages. Many cell signaling molecules are then
released in a cascading fashion, recruiting white blood cells, macrophages, and other cells to
deal with the wound in the inflammation stage. Macrophages are then later involved in the
transition to the proliferative phase. The proliferative phase is noted by epithelial cell
proliferation and migration through the ECM surrounding the wound. Around the wound,
fibroblasts secrete collagen and fibronectin. These substances help make up the ECM and
allow for new cells to interact with the healing area. The cellular structures that link directly
with the ECM are termed focal adhesions. Focal adhesions are located where the
cytoskeleton interacts with the ECM. These focal adhesions have groups of integrins which
directly impact the ECM.12 Through recruiting of focal adhesion kinase many cellular
downstream events are regulated. Some of these responses include cell differentiation and
migration. By regulating differentiation and migration focal adhesions then have a direct
impact upon wound healing.
While fibronectin is of importance in the body, its binding properties make it quite
useful in a laboratory setting as well. In a lab setting fibronectin can be used as a cell binding
protein to cause a cell to bind to a substrate to which it would not bind normally, such as a
silicone gel.6 Specifically, the active form of fibronectin is spread thinly across a silicone gel
substrate, promoting attachment of cell colonies to the gel. Cell binding deforms the silicone
due to the traction stresses produced through the ECM into the substrate. One method of
measuring the traction stresses that a cell exerts onto its substrate is though traction force
microscopy (TFM).6
TFM works through quantifying the gel deformation of a substrate covered in
silicone. It is necessary to use an elastic silicone that is consistently predictable in its stressed
5
positions if forces are to be quantified from TFM images. The substrates are made from a
glass cover slip that is covered in a layer of fluorescent beads. These beads are then covered
in 25 ᶙm of the silicone gel. This first layer of beads is used purely as a reference point; they
are not affected by traction forces of cellular activity. A second layer of beads is added onto
the initial layer of silicone, and is then covered in a thin layer of the silicone gel (3 ᶙm).
These beads will serve as indicators for how far the traction forces of the cell(s) have
deformed the substratum. A layer of fibronectin is then added to the top layer of gel6 to
promote binding of a cell (or cells) to the substrate.9 A schematic of the TFM substrate is
shown in Figure 2.6
Indicator Beads
Reference Beads
Figure 2: A representation of a substratum used in TFM, where a layer of
fluorescently tagged beads are covered in a silicone gel. The bottom layer of beads is used
for referencing position, and the top layer of beads is what actually will become displaced
when traction forces from the cell colony act upon the gel. A layer of fibronectin coats the
top of the silicone gel to activate binding of the cell colony to the substrate. Modified from
Mertz et. al.6
Previous work showed through TFM that a cell displays a large amount of its traction
force at the cell periphery, and that the force is directed to the center of the cell. 5,6 This
points to cells being contractile, almost as if they are pulling themselves into the substrate.
Shown in Figure 3 is the outline of a cell colony with arrows pointing inward representing
the direction of the gel displacement caused by the colonies traction forces against the
substrate.6
6
Figure 3: Highlighted in green is the outline of a cell colony that was on a silicone
gel substrate. Fluorescently tagged microscopic beads placed in the gel were displaced by the
traction forces generated by the colony onto the substrate. Arrows are shown to represent
their direction of displacement of the gel by the colony. (Mertz et al.6)
As previously stated, focal adhesions are often found at the end of actin stress fibers
that are concentrated at the peripheries of epithelial cells. At the Horsely lab and Dufresne
lab (Yale University), researchers are looking into these focal adhesions and their
involvement in observed traction forces. The focal adhesions are of particular interest, as
they seem to have a direct impact on the force that is transmitted to the ECM. In fact, focal
adhesions function as linkages to the ECM. The basic overview is that a significant amount
of strain energy forces that cells exert to a substratum are generated through non-muscle
myosin-generated tension in the actin cytoskeleton.5 Through intercellular adhesion
(specifically E-cadherin 6) and focal adhesions, these forces are transmitted to the ECM and
traction stress is displayed into the substrate.
Only recently have studies investigated the collective mechanical properties of
colonies of cells.5,6 This inquiry will help bridge the gap in understanding of the mechanics
of cellular systems between single cells and whole tissues. Recent work in this field has also
used TFM to attempt to quantify the mechanical forces and strain energies exerted by
7
colonies of keratinocytes onto their substrate.6 Primary mouse keratinocytes are used in
contrast to human skin cells for obvious reasons. Investigating colonies of these cells, as
opposed to just single cells, is likely to give much more understanding into the properties of
tissues.
Previous work has shown that the traction stress exerted by colonies of cells is
concentrated at the periphery of colonies in high-calcium environments in which the cells
exhibit strong intercellular adhesions. However, traction stress appears to distribute in more
random areas throughout the cell in low-calcium environments in which the cells do not have
strong intercellular adhesions.6 Figure 4 shows the density strain created by cell colonies onto
substrates using TFM.
Figure 4: TFM imaging and density mapping of keratinocyte colonies plated in both low
calcium (0.05 mM CaCl2) and high calcium (1.5 mM CaCl2) environments. Orange areas
indicate areas of high stress.
Figure 4 show higher strain densities at the colony peripheral in the high calcium plated
colonies, but shows strain distributed more evenly throughout the colony when cells are
plated in low calcium environments. This means that calcium plays a vital role in traction
forces, especially in relation to the distribution of forces.
8
Calcium is known to play a large role in signaling, and is therefore important in
wound healing. Understanding how calcium affects the traction forces and ECM of cells is
vital to understanding the effects of cellular forces in cellular proliferation and wound
healing. We hypothesize E-cadherin (modulated by calcium concentration) influences focal
adhesion distribution. Since focal adhesions are discussed to create stress into the ECM,
distribution patterns observed in low/high calcium environments of these adhesions are
viewed as a plausible cause for the force distribution variations (through the ECM) observed
in the substrates as seen in figure 4.
Immunostaining
Using fluorescent microscopy to take images of these keratinocytes in both low (0.05
mM) and high calcium (1.50 mM) variations is used to show observable variation in focal
adhesion appearances. Fluorescent staining allows particular proteins of interest to be easily
viewed in cells. While variations of traction forces observed in colonies of cells in low and
high calcium environments have been shown6,13, the reasons for the differences are not well
defined. Imaging of these keratinocyte colonies allows one to see the affects that calcium
plays on proteins of the cell, particularly those involved in adhesions. Fluorescent staining
uses specific antibodies (which are tagged with fluorescent proteins) to bind to proteins. This
is very effective because antibodies are highly specific and can be used to bind to only one
particular ligand. A fluorescent molecule can be labeled to the antibody of choice, and in this
way it can cause specific areas of interest to fluoresce under the right wavelength. A fixative
is used to preserve the cell of interest for later viewing before the staining process.
Formaldehyde is a fixative that works by cross linking the proteins in the cell.14 After fixing
9
a cell it is then possible to add the fluorescent dyes which will bind to specific proteins
allowing for fluorescent microscopy images to be attained.
Use of three different stains allowed for observation of cellular components of
interest. One compound that was useful to immunostain in this study was paxillin. Paxillin is
a protein associated with focal adhesions and is likely to play a role in multiple signaling
pathways.15 The protein contains a multitude of motifs which affect protein interactions. The
paxillin protein itself, when complexed with other enzymes, attracts signaling molecules to
specific cellular areas.15 One of the locations this protein attracts signaling molecules to are
the focal adhesions. Since this protein is well described as a signaling molecule that often
complexes with focal adhesions, it has suggested that possible functions of this protein
include cell spreading and motility.15 This makes the paxillin protein of great interest to this
study which looks to increase understanding of keratinocyte colony adhesions. Therefore
using anti-paxillin antibodies attached with a fluorescent stain caused the binding to and
illumination of focal adhesion sites. This seems logical given that paxillin is associated with
molecular signaling at these adhesions.16 By adding fluorescently tagged anti-paxillin
antibodies to keratinocyte colonies it was possible to view the focal adhesions stemming
from the cytoskeleton. Paxillin has a multitude of motifs and locations where it functions as a
cell signaling recruiter, and therefore it is important to differentiate an actual focal adhesion
from an area of background noise. Focal adhesions were noted when the anti-paxillin stain
was fluorescing at the end of an actin fibers stained with a protein fluorescing at a different
wavelength. This technique has been used before in viewing keratinocytes.6
The second molecule of interest for immunostaining was phalloidin. The antiphalloiden stain fluoresced in the red wavelength, and it makes the actin cytoskeleton
10
observable. Phalloidin is a molecule that binds to the actin cytoskeleton, and by staining this
molecule in a different color than paxillin, it allowed us to see the focal adhesions on the end
of actin filaments circling the keratinocyte cells. Being able to view the actin cytoskeleton
allowed the determination of the focal adhesions to be more accurate.
The third and final stain that was used in this experiment was DAPI. DAPI is a bluefluorescent stain that is used to tag nucleic acids.17 The DAPI stain that was used in the
immune-stainings binds strongly to A-T rich regions of DNA. This allowed for the
observation of the nucleus in a cell, and was very beneficial when we compared colonies of
cells. This stain was of particular use because when observing multiple cells interacting
together through binding proteins, it is often challenging to find where one cell ends and
another begins. This study detailed how many cells were observed to be in each individual
colony, and the nucleotide stain helped make that possible. This stain fluoresced around 460
nm, creating the appearance of blue colored nucleus which is observed in the images. The
ability to locate the nucleus was necessary for distinguishing cells within colonies. Each cell
colony then had three images of fluorescence at these described wavelengths and the images
were overlaid into a composite picture that allowed for quantification of focal adhesions.
As previously stated, cells exert traction forces on the ECM through the focal
adhesions they are using for adhering purposes.9 Focal adhesion distribution was studied in
low calcium and high calcium environments. This study looks to find more data on the
affects Ca2+ has on these integrin proteins of interest. Shown in Figure 5 is a flow chart
depicting the direction this study went with regards to the fluorescent staining of colonies of
skin cells.
11
Immunostaining flow chart
Culture keratinocyte cells
Fix and preserve cells at a desired densityusing low and high calcium media
Stain cells for actin, focal adhesions, and nucleus
Image cells, looking for trends in focal
adhesion formation
Figure 5: A flow chart showing how this study was conducted.
Using primary cultures of mouse keratinocytes (skin epithelial cells), this study
compared the focal adhesions between high and low calcium environments, along with other
environmental variants which inhibited E-cadherin through other means than Ca2+
concentration. This study attempted to ascertain a better understanding of the role Ca2+ in the
differentiation of skin cells through the observation of cellular component differences due to
environmental variations. Deductive observation of fluorescently tagged cells showing focal
adhesions allowed for qualitative and quantitative differentiations between cells in various
environments.
Control tests were used for comparison of low calcium vs. high calcium
environments. If E-cadherin formation between cell colonies in a high calcium environment
alleviates the need for focal adhesions between colonies (lowering the tension observed
between cell colonies) then inhibiting E-cadherin in high calcium may reduce keratinocyte
12
proliferation as is seen in low calcium environmental conditions. One way we tested this was
by using an antibody against E-cadherin which competitively inhibited E-cadherin’s ability
to form junctions with neighboring cells in the colony. The DECMA environmental variation
tested did just this. DECMA is an anti-body that inhibits E-cadherin hemophilic binding by
blocking the extracellular domain of E-cadherin.18 The monoclonal antibody then disrupts the
adhesion in mouse cells, but does not completely destroy junctions. Therefore it was
hypothesized that the focal adhesions between cell junctions would be seen as something
between a high calcium environment which stimulates E-cadherins functions of intracellular
binding, and a low calcium environment where E-cadherin is not observed.
We also used a knockout (KO) mouse to derive E-cadherin deficient keratinocytes
(even in high calcium environments) to study the formation of focal adhesions. It was
hypothesized that KO cells in high calcium should behave similarly to wild type (WT) cells
in low calcium media.
13
CHAPTER II
MATERIALS AND METHODS
Creating of substrates for TFM
Five hundred ᶙL silane was added to 4 caps spaced around a dish containing 6
substrates. Silane (3-aminopropyl triethoxysilane) was vaporated onto 35-mm glass-bottom
dishes (WillCo Wells GWSt-3552) for an incubation of 10 minutes to bind florescent beads
to surface of dish. Each substrate had added to it 750 ᶙL of Flourescent Bead Solution (FBS)
with evaporated silane, and was incubated in closed container for 5 min. To create FBS, nine
ml of borate buffer (deionized water with 3.8 mg/mL sodium tetraborate and 5 mg/mL boric
acid) was mixed with 5 ᶙL fluorescent beads (dark-red florescent (660/680) carboxylatemodified microspheres (with radius of 0.1 ᶙm) and150 ᶙL 1-ethyl-3-(3dimethylaminopropyl) carbodiimide (EDC) and was vortexed. After incubation with FBS,
the supernatent was poured off the substrates. Pour off liquid-this step was discovered to be
very important. If the liquid was pipeted off, or poured off too slowly the beads would stick
in an uneven distribution. It was best to pour off the liquid as quickly as possible to get the
14
fluorescent tags to form an even distribution across the substrates. This was to create the base
layer of reference beads.
PDMS silicone was prepared by mixing a 1:1 weight ratio of CY52-276A and CY52276B (Dow Corning Toray CY 52-276 KIT). Silicone, before being added to substrate, was
degassed (10 min). Three hundred μl silicone was then added to fluorescent bead infused
substrate to be spin-coated onto glass substrates at 2,000 rpm for 60 s with a PWM32 spinner
(Headway Research). The dish was heated at 50 °C for 3 min and resulted in a 21 μm thick
cross-linked elastomer. Once dried, silane was vaporated onto the elastomer-coated dish. A
second layer of florescent polystyrene beads was placed at a higher concentration, volume
ratios of 1:1,000 beads and 1:100 EDC in borate buffer. A second layer of degassed silicone
was spin-coated onto the glass substrates at 10,000 rpm for 90 s with a PWM32 spinner
resulting in a layer 3 μm thick. The substrates were left at room temperature overnight.
Before cells were plated, the silane surface was coated with fibronectin from bovine plasma
at a concentration of 0.2 mg/mL, which sat for 20 min at RT before being washed off with
PBS, as described in Mertz et al.13
Primary mouse keratinocytes:
Cells were extracted from primary mouse keratinocytes developed at Yale University
from the mouse lab. The IACUC form that allows the lab to use mice is attached in the
appendix of this paper. The isolated back skin of newborn CD1 mice was floated on dispase
overnight at 4°C. The epidermis was separated from the dermis with forceps and incubated in
0.25% trypsin for 10 min at room temperature. Individual cells were released by trituration
and plated on mitomycin-C–treated J2 fibroblasts in low-calcium medium (0.05 mM CaCl2).
After two to four passages, cells were plated on plastic dishes without feeder cells. KO cells
15
were generated through immunolocalization of cadherin by lentiviral transduction of Ecadherin–deficient keratinocytes using shRNA directed against P-cadherin, as described.13,19
DECMA images were acquired from cells that were treated with anti E-cadherin antibody
DECMA-1 (Abcam) at 6 ᶙg/mL in high calcium media. High calcium concentration was
defined by raising the concentration of CaCl2 of the low-calcium medium to 1.5 mM.
Cells were then kept in RV media (high glucose DMEM + 10% FBS) and media was
regularly refreshed. For subculturing, trypsin was used to break apart cell binding7 and to
split apart colonies. Short exposures of the protease of around 6-8 (prolonged subjection of
cells to trypsin can kill them) loosed the cells from the so that the dense cultures could be
subdivided to increase the life of the culture. The cells were then centrifuged to remove the
trypsin supernatant and were fed with fresh media. At this point the cells could be split into
multiple cultures to be grown to a desired density.
Keratinocytes were cultured as previously described in Mertz et. al.13 All animal care
and experiments were approved by the Institutional Animal Care and Use Committee of Yale
University.
Cell Staining:
Keratinocytes were fixed in 3.7% (vol) formaldehyde for 10 min, followed by
washing twice in PBS for 2 minutes each. A blocking solution made of normal donkey
serum, normal goat serum, gelatin, BSA, and Triton X dissolved in PBS solvent was used to
prevent nonspecific binding. Cells were stained using Alexa Fluor 594 phalloidin (3,000
units/μL) (Life Technologies), 8 ng/ᶙL monoclonal mouse anti-E-cadherin primary
antibodies (TaKaRa), and 4 ng/ᶙL rabbit anti-paxillin primary antibodies (Sigma-Aldrich).
After primary antibody incubation, cells were again washed in PBS. Following this PBS
16
wash, cells were incubated with secondary antibodies, 8 nm/ᶙL goat anti-rabbit Alexa Fluor
488 (Life Technologies) and 8 ng/ᶙL goat anti-rat Alexa Fluor 647 (Life Technologies).
Finally, a second coat of Alexa Fluor 594 phalloidin (3,000 units/μL) was incubated with
cells to bind actin. Cells were then mounted using ProLong Gold with DAPI (Life
Technologies) to stain the nucleus, as previously described.13
Fluorescently stained images were acquired using confocal laser-scanning microscope
equipped with Ar, HeNe 543, and HeNe 633 laser lines (Zeiss LSM 510 system). Images
fluoresced from the use of lasers with wavelengths 488, 568, and 633 nm. Keratinocyte
Images were acquired with a 40X zoom objective (Zeiss Plan Apo). This allowed for a field
of view of 313 x 313 ᶙm2 and a maximum resolution of 2,048 x 2048 pixels.
After staining the cells observations were made noting differences in focal adhesion
formation. Focal adhesions in cells grown in high calcium vs. low calcium media solutions
were quantified and recorded. First, the total number of adhesions formed by the cell were
counted and recorded. Second, adhesions formed at periphery of cells were counted and
recorded. Finally, adhesions formed at cell-cell contacts within the cell were counted and
recorded. Cell barriers were measured relative to zoomed images taken from the fluorescent
microscope using ImageJ software.
Statistical analyses were completed using Microsoft Excel software, and p values of
less than 0.05 were considered significant for two sample t-test assuming unequal variances
and using two tailed values.
17
CHAPTER III
RESULTS
A representative image of a cell colony fluorescing at the described wavelengths is
shown in Figure 6. The colony is in a high calcium environment, meaning E-cadherin is
functional within the colony. Figure 6 shows a typical example of a keratinocyte colony that
was living in a high calcium environment with E-cadherin function turned on, this is opposed
to a low calcium environment in which E-cadherin function is turned off. For reference, an
area determined to be with focal adhesions is highlighted, along with an area determined to
be just noise.
18
Figure 6: (image code: High cal 16): Image is a colony of what has been determined to be 6
cells placed in 1.5mM CaCl2. Blue fluorescence highlights nuclei, red highlights actin
cytoskeleton, and green highlights paxillin (a signal protein that associates with focal
adhesions). Circled in solid line are areas where clear focal adhesions can be observed, where
these structures stem from the actin cytoskeleton. Circled in dashed line are example areas
determined to be noise.
The colony seen in figure 6 shows a typical distribution of focal adhesions, as well as
typical junctions, for high calcium images. The density of focal adhesions in this colony is
far more concentrated around the periphery of the colony than within it. The peripheral focal
adhesions to inner colony adhesions (per unit length) ratio was determined to be 2.37 for this
particular colony. The high calcium colonies were very cohesive and had rounded edges.
Staining and imaging were also done for four other environmental variants (low calcium,
DECMA high calcium, KO high calcium, and KO low calcium) to compare the colony’s
19
focal adhesion locations in high calcium environments with functional e-cadherin properties
to colonies lacking e-cadherin function.
Cell colonies placed in low calcium environments lacked E-cadherin function.6 This
creates radically different appearances in colony spreading as is seen in Figure 7, which
shows a keratinocyte colony made up of 6 cells in a low calcium environment.
Figure 7: (image code Low cal 4) A keratinocyte colony made up of 6 cells colony plated in
low calcium medium (0.05 mM CaCl2).
This low calcium colony displays a typical distribution pattern observed in other low calcium
colony images, where focal adhesions are distributed in high density not only around the
periphery, but also throughout intra-colony boundaries. The peripheral focal adhesions to
inner colony adhesions ratio (per unit length) was determined to be 0.70 for this particular
colony. Qualitatively speaking the colony was much less cohesive, and even appears to be
20
broken up at some points. The edges are no longer smooth and rounded, but rather jut out in
certain areas.
The DECMA inhibited variants had their adhesion functions inhibited, but not
knocked out. An image of a keratinocyte colony inhibited by DECMA is shown in Figure 8.
Figure 8: (image code Decma 9): A 6-cell colony of cells plated in high calcium medium
(1.5 mM CaCl2) with 6 μg/ml DECMA antibody.
The DECMA high calcium colony (Figure 8) displays a typical distribution pattern observed
in other DECMA images, where focal adhesions are distributed in high density around the
periphery, and throughout intra-colony boundaries. The peripheral focal adhesions to inner
colony adhesions ratio (per unit length) was determined to be 1.50 for this particular colony.
The DECMA colonies were fairly cohesive, and the edges around the colony were smooth
for the most part.
21
To ensure E-cadherin junctions were the cause for variations in focal adhesion
distributions, KO keratinocytes were tested. Both high calcium KO and low calcium KO
colonies were imaged to decipher if E-cadherin function was indeed the cause for focal
adhesion distribution, or if the high/low calcium environment was having other effects on the
colonies. The KO cell colonies no longer had abilities to form E-cadherin junctions. An
image of a KO colony plated in low calcium is shown in Figure 9.
Figure 9: (image code KO low 13): A 4-cell colony of KO cells plated in low calcium
medium (0.05 mM CaCl2) and stained as previously described.
The KO low calcium colony show in Figure 9 displays a typical distribution pattern observed
for this cellular environmental variation. The focal adhesions are distributed in high density
around the periphery, as well as throughout intra-colony boundaries. The peripheral focal
adhesions to inner colony adhesions ratio (per unit length) was determined to be 1.22 for this
22
particular colony. The KO low calcium colonies were not tightly cohesive, and the edges
were rough.
KO cells in high calcium are shown in Figure 10, which displays a 4-cell colony.
Figure 10: (image code: KO high 13) A 4-cell colony of KO cells plated in high calcium
medium (1.5 mM CaCl2) and stained as previously described.
KO cells in high calcium showed the focal adhesions distributed in high density around the
periphery of the colony, as well as throughout colony cell-cell borders. The peripheral focal
adhesions to inner colony adhesions ratio (per unit length) was determined to be 1.29 for this
particular colony. The KO high calcium colonies were relatively cohesive (in comparison to
the KO low calcium colonies) and the edges were somewhat rough.
A system had to be devised in order for not only qualitative observations to be made,
but also so that quantifiable data could also be collected from the images taken. To
accomplish this feat a system of quantifying focal adhesions per unit length was used.
23
Since the experiment focused on finding an explanation for the strain difference
between the dominantly peripheral strain seen in high calcium models and that of the
seemingly unspecific strain seen in low calcium models, a system was used to compare this
situation. A first set of measurements calculated the number of focal adhesions seen around
the peripheral of a colony. The distance around the peripheral of a given colony was then
measured (as described in the methods) and a focal adhesion per unit length was established.
Figure 11 shows an example of how the peripheral of a colony was measured.
Figure 11: The blue line marks what was considered the periphery of a cell colony. This
colony is stained as previously described.
The focal adhesions that formed around this line were counted and were used for
comparison of the focal adhesions that were observed with cell colonies. Figure 12 shows the
lines representing the internal boundaries where focal adhesions were counted.
24
Figure 12: The blue line marks what was considered the internal length of a cell colony. This
colony was stained as previously described.
After the focal adhesions were counted per unit length for the interior of the colony
they were compared to the number attained from the peripheral focal adhesions per unit
length. These numbers were used to calculate a ratio comparing peripheral focal adhesions
per unit length (around the periphery) to internal focal adhesions per unit length (throughout
internal boundaries. Therefore if the ratio was found to be 2, it would suggest that the density
of focal adhesions (per unit length) around the periphery of the colony was twice that which
was throughout the internal cell-cell boundaries of the colony. These calculations were done
for images on each of the five colony environmental variations. These data sets are presented
in Figure 13.
25
Ratio of Peripheral Adhesions to inner
colony adhesion (per unit length)
2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
High Cal
Low Cal
DECMA (hi cal)
KO high
KO low
Figure 13: The ratio of peripheral focal adhesions per unit length (around peripheral of
keratinocyte colony) to internal focal adhesions per unit length (around internal junctions of
keratinocyte colony) for all 5 environmental condition variations. N for high calcium = 17; N
for low calcium = 14; N for DECMA = 24; N for KO high calcium = 11; N for KO low
calcium = 12. Standard error bars are included.
The average peripheral focal adhesions to inner colony adhesions ratios (per unit
length) for the high calcium, low calcium, DECMA, KO low calcium, and KO high calcium
were 1.85 ± 0.5, 0.90 ± 0.2, 1.70 ± .56, 1.09 ± 1.10, and 1.20 ± 0.60, respectively. Although
the average ratios had variations between each of the environments, not all of the differences
were significant. High calcium colonies had a significantly higher ratio than that of low
calcium (p = < 0.001), KO high calcium (p = 0.0078), and KO low calcium (p = < 0.001).
DECMA colonies had significantly different ratios from low calcium (p = 0.001), KO (p =
0.0278), and KO low (p = < 0.001)
High calcium focal adhesion ratios were not significantly different from distribution
ratios observed in DECMA colonies (p = 0.41). Low calcium colonies were not significantly
26
different from KO high calcium colonies (p = 0.128), nor were they significantly different
from KO low calcium colonies (p = 0.10). KO high colonies were not significant from KO
low colonies (0.585).
The next set of calculations looked to test the hypothesis that some of the focal
adhesions seen throughout colonies in low calcium environments actually displace
themselves when moved to high calcium environments from the internal areas of colonies to
the peripheral portions of the colonies. To get an idea if this was actually occurring the
peripheral adhesions per unit length of the high calcium images were compared to the
peripheral adhesions per unit length of the low calcium images. In this way a sort of average
focal adhesion density around colony peripheral was attained and used for comparison of the
high and low calcium variants. The ratio therefore shows the peripheral adhesion density of
the high calcium images vs. the peripheral adhesion density of the low calcium images. In
essence, a ratio of 2 to 1 would mean high calcium colonies had double the density of focal
adhesions (per unit length) than low calcium colonies. This data is shown in Figure 14.
27
Average density ratio around peripheral
focal adhesions (per unit length)
1
0.8
0.6
0.4
0.2
0
High Cal Density
Low Cal Density
Figure 14: Ratio of peripheral focal adhesions per unit length of high calcium images vs.
peripheral focal adhesion per unit length of low calcium images. Standard error bars are
included.
The focal adhesion density around the periphery of keratinocyte colonies showed a ratio of
1.00 ± 0.10, to 1.06 ± 0.13 between the high and low calcium variants. This resulted in not
significant difference between the high and low calcium variants in regard to periphery focal
adhesion density (p = 0.503).
28
CHAPTER IV
DISCUSION
Previous papers have shown that when observing the cellular mechanobiology of
keratinocytes the force distribution is dependent on E-cadherin junctions. It is also well
described that E-cadherin function is directly regulated through the concentration of
environmental Ca2+.6,13 In the human body calcium gradients regularly increase from 0.5mM
to greater than 1.4mM in the basal layer as a response to minor injuries such as abrasions.20
This Ca2+ increase plays major roles in keratinocyte proliferation and differentiation. The
results of this study though help to explain a previously undefined role of calcium through
way of E-cadherin junctions that actually impact the distribution of focal adhesions.
The first observation that must be made is that the focal adhesion distribution is
significantly different for cell colonies that are living in a high calcium environment vs. those
that are in low calcium. To make this determination, a qualitative observation comparing a
high calcium colony from a low calcium colony was made. Figure 6 shows a typical image of
a keratinocyte colony that was imaged in a high calcium environment. Figure 7 then shows a
typical image of a keratinocyte colony living in a low calcium environment. It is important to
note that these are just two of many images taken of keratinocyte colonies in both low and
high calcium environments and are therefore just used as a good representation of typical
observed cellular properties.
29
This paper previously described what defined a focal adhesion based off the images
gathered, so now a direct comparison will be made. In Figure 6 a colony of 6 cells is shown
to have focal adhesions (bright green at the end of red actin filaments) that are concentrated
around the peripheral of the colony. Focal adhesions can still be observed between individual
cells, however we observe far fewer which is noted by the smoother color which is lacking
many bright green dashes. Figure 7, which displays a similar sized colony also with 6 cells, is
of a keratinocyte colony in low calcium. The first difference noticed is the shape of the
colony. The low calcium colony is less smooth and appears to have more edges as you travel
around the periphery. Focusing attention to the focal adhesions we also observe a distribution
change. Whereas in Figure 6 the majority of the adhesions circled around the outside of the
colony, the low calcium image has focal adhesions that appear not only in high number
around the periphery, but also randomly distributed throughout the internal boundaries of the
cells making up the colony.
To determine if these differences were consistent amongst other colonies quantitative
comparisons had to be made as well as the qualitative observations depicted. As was
described in the methods, imageJ software was used to compare how many focal adhesions
were observed (per unit length) around the peripheral boundaries of a colony to that of the
internal cell boundaries in a colony. The high calcium cells had a density of 1.85. This means
that on average the peripheral boundaries were nearly twice as dense with focal adhesions
(per unit distance) than the internal boundaries between cells in a colony. For the low
calcium colonies we hypothesized the ratio would be smaller, since it appeared based on the
qualitative observation that there were higher densities of focal adhesions spaced randomly
throughout the colony of cells in the low calcium variants. Indeed, the average ratio attained
30
was 0.90 (external : internal focal adhesion density). This implies that the spacing (in low
calcium cells) between focal adhesion is highly similar for both the external and internal
boundaries of keratinocyte colonies. This difference in densities between that of high calcium
and low calcium colonies was shown to be significant (p < 0.001). It was therefore
determined that there is a quantifiable and significant difference between focal adhesion
distribution between that of high calcium and low calcium cellular environments.
Next it needed to be determined whether or not the difference in focal adhesion
spacing between that of low and high calcium was due to E-cadherin function. As was
described in the introduction, E-cadherin does not function in low calcium environments
(0.05mM) but does in high calcium (1.5mM). To therefore determine if the difference was a
direct effect of E-cadherin, several other variants/control groups were observed, both
qualitative and quantitatively.
The first control used an E-cadherin KO variation in high calcium (KO high calcium)
that completely knocks out E-cadherins functionality. Therefore, if the cause of focal
adhesion distribution differences was due to calcium’s effect on E-cadherin, and not some
other indirect pathway, the focal adhesion spacing of the KO high calcium would be similar
to that of low calcium. Figure 10 of a typical KO high calcium image shows a 4-cell colony.
KO high calcium colonies had somewhat smooth edges such as that seen in high calcium
images, yet looked to have focal adhesions distributed not only in high quantity around the
colony periphery, but also largely between internal cell-cell boundaries, much like is
observed in low calcium cellular colonies. This indicates that calcium is likely to have more
effects on keratinocyte colonies than just the focal adhesion distribution. Ca2+ is in fact well
described to play roles in both cellular proliferation, differentiation, and cellular motility.20,21
31
However, as far as focal adhesions go, their distribution does appear similar to that of stress
locations, and quantitative data were gathered which shows a correlation between force and
focal adhesion distribution. The average ratio of peripheral focal adhesion density to intracolony focal adhesion density for KO high cells was 1.20. This does appear to be a slightly
higher ratio than was observed for that of low calcium images, however the difference was
not significant (p = 0.128). After determining there was no significant difference in focal
adhesion distribution between that of low calcium images and that of KO high images,
knockout in low calcium images (KO low) were observed to ensure that the KO did not just
drop the ratio by some undetermined means.
When we compared the image of KO high calcium (Fig 10) to that of the KO low
calcium (Fig 9) we saw some similarities and some differences. Again the high calcium
seemed to result in a colony with smoother edges, and the KO low had the typical rougher
and less rounded edges observed in low calcium variants. However, the focal adhesion
distribution appeared very similar for both of these images. Both had clearly visible focal
adhesions going around the peripheral of the colony, and also throughout the internal cellular
boundaries of the colonies. When we quantified the actual data we saw that the focal
adhesion densities are indeed very similar, as was expected. The average ratios of peripheral
focal adhesion density to intracellular focal adhesion densities for KO high and KO low was
1.20 and 1.09, respectively. These numbers were statistically insignificant (p = 0.585), and
further imply that E-cadherin (which is regulated through calcium) is the cause for the focal
adhesion distribution variations observed.
The third variable of interest was termed DECMA and was grown in high calcium
media. This environment used a competitive antibody (as described in the introduction)
32
which inhibited E-cadherins functional ability, yet without completely removing it. We thus
hypothesized that these images would show a periphery to intra-colony focal adhesion
density ratio that was between that of low calcium and high calcium colonies. Looking at
Figure 8, which is the DECMA colony, we see a fairly rounded colony, with a somewhat
substantial amount of intra cellular and peripheral adhesions. The colony appears more
similar to that of the high calcium images, but no consistent qualitative comparisons were
able to be made for the DECMA images. The quantifiable data showed that the DECMA
images were indeed more similar to the high calcium colonies. The density ratio
(periphery:intracellular per unit length as previously described) for DECMA keratinocyte
colonies was 1.71, as compared to 1.85 for the high calcium images and 0.90 for low
calcium. This number does appear as though it was between the high calcium and the low
calcium as expected, however it was not significantly different from the high calcium ratio (p
= 0.41). Sense DECMA inhibits E-cadherin, if it was used in higher concentrations than was
in this experiment (6 ᶙg/mL) it could further inhibit E-cadherin and would likely cause focal
adhesion distribution to be less similar to that of the high calcium colonies.18 Perhaps then
through experimentation with higher concentrations of DEMCA antibodies these colonies
might be significantly different from not only the low calcium, but also the high calcium
colonies.
The next observation this study made was to determine if these focal adhesion
distribution variances could be the cause of the traction force distribution differences seen
between low and high calcium colonies of keratinocytes as has been shown by Mertz et al.13
Our study used low calcium (0.05 mM) and high calcium (1.5mM) media concentrations for
keratinocyte colonies as the experiments used in Mertz6 paper did. Mertz showed that
33
traction stresses, measured through TFM, of colonies in high calcium media distribute their
stress into their substrate primarily around the colony periphery, whereas in low calcium
images the stress is displayed throughout the entirety of the colony. Figure 4 shows a
representation Mertz et al. used to describe where the traction forces concentrate for both low
and high calcium colonies. This distribution does appear similar to the distribution observed
from the focal adhesion concentrations. However, the difference in focal adhesion
distribution is not nearly as intense a difference as the stress difference Mertz et al.6
observed.
Although there are statistically significant differences in focal adhesion distribution
between that of high calcium and low calcium environments, both appear to show more
concentrated densities of focal adhesions around the peripheries of their respective colonies
as opposed to internal adhesions. While it does appear that the focal adhesions play a part in
the stress distribution differences that have been previously shown6, it is unlikely the only
cause for the extreme variation in stress observed through the colonies. If they were the
major cause it would imply that the displayed traction forces would have a significant stress
at the periphery for not only the high calcium, but also the low calcium colonies.
Furthermore, being that there were still significant numbers of focal adhesions observed in
the interior in high calcium cell colonies (granted the number about half that of low calcium)
there should still be a significant traction force seen throughout the internal area of high
calcium colonies. However, this is not observed, and in fact there is hardly any strain energy
internally in high calcium colonies.13
Additionally, other evidence implies that the focal adhesions are unlikely to be the
only cause of the force distribution through traction forces that have been previously
34
described. It has been shown that low and high calcium colonies do not exhibit different
amounts of total average strain density from that of similar sized low calcium colonies.
Rather, the total work done appeared to be more of a result of the size of the colony, with
larger colonies performing a greater total work on the substrate, regardless of external
calcium concentration.22 What this then means is that when added together, both the internal
and peripheral area force that low calcium colonies display into their substratum is nearly
equivalent with the force that is primarily going around the periphery of the high calcium
images. In other words, the traction force around the peripheral area of a given high calcium
colony should be larger than the peripheral area traction force of a low calcium cell (sense a
low calcium colony also has internal stress adding to its total stress). What this means is that
if the focal adhesions are the main cause of the traction stress distribution differences, there
should be more dense focal adhesion populations surrounding high density colonies than that
of low density colonies.
To test whether high calcium colonies did have higher focal adhesion densities that
that of low calcium images, the ratio of peripheral focal adhesions per unit length of high
calcium images vs. peripheral focal adhesion per unit length of low calcium images was
determined. This allowed a direct interpretation to determine if the average spacing between
focal adhesions around the periphery of keratinocyte colonies would be different for low and
high calcium images. Figure 14 showed the ratio for high calcium to low calcium peripheries
cell density was 1.00 to 1.06, which showed no significant difference (p = 0.503) in focal
adhesion density around the peripheries of high and low calcium images. If focal adhesions
were the major cause for traction stress variations, there should have been a greater average
density of them surrounding high calcium colonies which display higher traction stress
35
around the peripheries of their colonies. Further, this paper has shown a greater density of
intra-colony focal adhesions in low calcium colonies than for high calcium colonies. Since
high and low calcium colonies have relatively equivalent peripheral densities, this implies
that low calcium colonies would have a greater total density (combining both internal and
external focal adhesions) than that of high calcium variants. Yet, this average overall higher
focal adhesion density did not result in a higher average total traction force distribution into
substrates in low calcium colonies.
Although perhaps not the only factor, it is likely that the focal adhesions play a role in
the stress distribution from the cell ECM. Realizing that focal adhesion have been shown to
be involved with many downstream signaling specifically related to the ECM12, combined
with the correlation of higher focal adhesion densities around high calcium colonies, it is
likely that the focal adhesions do still play some role in the differences in traction forces
observed between low and high calcium keratinocyte colonies, though this role is not yet
well understood. The focal adhesions distributions showed a strong correlation in densities to
where stress was strongly displayed into substrates of past experiments.6,13 Since focal
adhesions exert traction forces onto the ECM to which they adhere,9 and the ECM is
responsible for displaying traction stresses into the substratum,6 it is sensible that the focal
adhesions play a role in the observed traction stresses. The observed correlations between
areas of high stress and high focal adhesion density further support this assumption. A
separate cause for the difference of traction strain has been linked to the physical
cohesiveness of cell colonies, and other studies have used physical models to show this.13
Another hypothesis this paper investigated was whether focal adhesions were actually
being relocated from under the cells to the periphery of cells when E-cadherin function was
36
regained. It has previously been shown that focal adhesion under cell colonies (intra-colony)
actually disappear when E-cadherin function is re-gained,13 but it has not been determined if
the adhesions relocate. Comparing the ratio of peripheral focal adhesions (per unit length) in
high calcium images vs. peripheral focal adhesion (per unit length) of low calcium images as
shown in Figure 14 give the impression that is was not the case. While the internal adhesions
have been shown to disappear when E-cadherin function is regained, based off the data
shown if figure 14 there was no significant difference between high calcium and low calcium
peripheral focal adhesion density, and therefore it is unlikely that these adhesions were
relocating to the colony periphery. It would be expected that if these adhesions were
relocating to the colony peripheries in high calcium cells, the peripheral focal adhesion
density would be higher for high calcium colonies.
A final observation noted differences in actin formation between that of colonies
growing in high calcium to those in low calcium. In the high calcium colonies the actin
appears to actually work together and the colony seems to act more as one large cell. Figure 6
of a high calcium colony shows actin filaments that actually appear to line up with each other
going from cell to cell. The KO high calcium image (figure 10) appears to display a similar
trend. However, the low calcium and KO low calcium images (Figure 7 and figure 9) show
actin filaments that just circle individual cells that are making up the colony. It is possible
that the calcium plays a role in the actin formation within colonies, which could also have an
effect in the cellular distribution of traction forces that have been previously described.
37
Future studies:
Future studies could use a cloning project inserting GFP into a vector which could
eventually be used to implant into a cell of interest as a retrovirus. This would create a
lineage of cells that would have a fluorescent protein tagged to a specific gene of interest.
Since calcium plays such a large role in traction forces, along with many biological
processes that take place in keratinocytes, it would be very interesting to view calcium
binding taking place in the cell. To view binding of calcium one could image the calcium
binding protein calmodulin, but the cell would have to be alive. Calmodulin is a calcium
binding protein that plays a major part in many physiological processes.22 When calmodulin
binds Ca2+ it then stimulates the protein to bind to other target proteins in a cell that are then
found to influence many factors of cell growth and proliferation.21 Calcium ions play a large
role in signaling in many tissues, and there are multiple techniques for observing the
dynamics of calcium in living cells.23 Many of these techniques involve procedures that can
be damaging to the cell and could possibly affect results. One emerging technique uses
genetically encoded calcium indicators (GECIs) to overcome some of the drawbacks of
otherwise invasive techniques. One way these GECIs work is through fusing a fluorescent
protein, such as green fluorescent protein (GFP), to calmodulin. When the calmodulin then
binds to calcium and undergoes conformational changes it causes a change in the calmodulin
GFP complex that causes a change in fluorescence.23 This change produces a more intense
fluorescence from GFP. There are many benefits to using GECIs, the main one being that the
gene becomes encoded in the cell. This means that as the cell reproduces it will continue to
make cells that have a GFP-calmodulin complex and can fluoresce in the desired fashion.
Using this technique, it would theoretically be possible to see where in a cell calcium is
38
binding to the calmodulin protein by viewing though a fluorescence microscope while
calcium is added to the solution in which the cells are living.
Encoding the GECI will require biological techniques such as cloning and infection
of cells. This will no doubt be a long process but the information that could be acquired using
these GECIs could prove to be very beneficial. One hope could even be to work with in vivo
experimentation since the technique is intended for live cells and actually affects the genome
of the cell.
39
APPENDICES
APPENDIX A
42
APPENDIX B
QUANTIFICATIONS OF FOCAL ADHESIONS
Individual
Colony and cells
in colony
Total
periferial
adhesions
Total
innercolony
Adhesions
Length
around
colony
(pixels)
Total length
of innercolony
segments
(pixels)
hi1 (6 cells)
hi 1.1 (2 cells)
hi2 (8 cells)
hi5 (4 cells)
hi7 (4 cells)
hi8 (5 cells)
hi10 (4 cells)
hi11 (15 cells)
hi12 (6 cells)
hi13 (8 cells)
hi14 (6 cells)
hi15 (6 cells)
hi16 (7 cells)
hi 18 (7 cells)
hi 19 (8 cells)
hi 20 (2 cells)
hi 21 (2 cells)
lo1 (14 cells)
lo2 (14 cells)
lo4 (6 cells)
lo5 (7 cells)
lo6 (14 cells)
lo7 (11 cells)
lo8 (2 cells)
lo9 (7 cells)
lo15 (10 cells)
lo 16 (2 cells)
lo 18 (3 cells)
lo 19 (9 cells)
lo 21 (10 cells)
71
63
80
61
53
51
48
86
71
97
97
97
94
51
84
46
59
113
120
94
68
123
93
81
119
103
55
112
95
65
35
9
42
24
24
9
20
59
36
33
35
34
29
13
47
7
10
150
141
107
80
130
80
23
132
82
11
29
72
63
2340
1412
3355
2823
1651
2906
1581
3540
2552
3318
3943
3064
2837
1677
2892
1646
1945
3825
3933
3364
3303
4315
3567
2087
3357
2869
1870
3038
3706
3790
1643.5
346.4
2989.1
1531.6
1055.00
1400
1053.7
5560.3
2231.8
2861.3
2472.5
2076.7
2075.2
1199.3
1857.7
344.7
509.2
4799.9
4925.2
2683.3
2428.7
5564.4
2435.2
409.1
2580.6
2620.4
423.7
883.3
2064.4
3411.5
Periferial
adhesion
per unit
length
(pixels)
Inner
colony
adhesions
per unit
length
(pixels)
0.030
0.045
0.024
0.022
0.032
0.018
0.030
0.024
0.028
0.029
0.025
0.032
0.033
0.030
0.029
0.028
0.030
0.030
0.031
0.028
0.021
0.029
0.026
0.039
0.035
0.036
0.029
0.037
0.026
0.017
0.021
0.026
0.014
0.016
0.023
0.006
0.019
0.011
0.016
0.012
0.014
0.016
0.014
0.011
0.025
0.020
0.020
0.031
0.029
0.040
0.033
0.023
0.033
0.056
0.051
0.031
0.026
0.033
0.035
0.018
Perifereal
adhesions
to inner
colony
adhesions
per unit
length
1.425
1.717
1.697
1.379
1.411
2.730
1.600
2.290
1.725
2.535
1.738
1.934
2.371
2.806
1.148
1.377
1.544
0.945
1.066
0.701
0.625
1.220
0.794
0.690
0.693
1.147
1.133
1.123
0.735
0.929
44
lo 20 (3 cells)
decma 1 (6
cells)
decma 2 (6
cells)
decma 3 (8
cells)
decma 4 (8
cells)
decma 6 (8
cells)
decma 7 (5
cells)
decma 8 (7
cells)
decma 9 (6
cells)
decma 10 (3
cells)
decma 11 (6
cells)
decma 13 (8
cells)
decma 14 (9
cells)
decma 15 (10
cells)
decma 16 (2
cells)
decma 18 (5
cells)
decma 19 (4
cells)
decma 20 (4
cells)
decma 21 (2
cells)
decma 22 (4
cells)
decma 24 (12
cells)
decma 25 (5
cells)
decma 28 (4
cells)
decma 29 (5
cells)
149
63
3499
1126.6
0.043
0.056
0.761
98
22
3118
1997.7
0.031
0.011
2.854
64
31
2177
1726.4
0.029
0.018
1.637
70
42
2547
2647.6
0.027
0.016
1.733
104
58
2286
2371.9
0.045
0.024
1.860
102
60
2272
2411.7
0.045
0.025
1.804
52
25
2321
1457.3
0.022
0.017
1.306
112
53
2644
1770.7
0.042
0.030
1.415
91
43
2694
1902.8
0.034
0.023
1.495
67
20
2043
707.3
0.033
0.028
1.160
112
33
3178
1905.9
0.035
0.017
2.035
61
38
1980
1761.1
0.031
0.022
1.428
72
62
2381
2597.7
0.030
0.024
1.267
102
64
3058
3443.7
0.033
0.019
1.795
85
20
2389
715.7
0.036
0.028
1.273
68
61
3494
1820.7
0.019
0.034
0.581
52
16
1629
927.9
0.032
0.017
1.851
43
12
1748
997.8
0.025
0.012
2.046
46
10
1591
364.9
0.029
0.027
1.055
77
22
2316
1265.5
0.033
0.017
1.913
124
66
3141
3501
0.039
0.019
2.094
83
19
2559
1551.6
0.032
0.012
2.649
72
15
2670
1160.9
0.027
0.013
2.087
106
25
3277
2098.9
0.032
0.012
2.716
45
decma 30 (3
cells)
ko lo1 (5 cells)
ko lo3 (4 cells)
ko lo4 (5 cells)
ko lo 5 (2 cells)
ko lo7 (3 cells)
ko lo 8 (5 cells)
ko lo 10 (5 cells)
ko lo 11 (2 cells)
ko lo 12 (2 cells)
ko lo 13 (4 cells)
ko lo 14(4 cells)
ko lo 15 (3 cells)
ko hi 2 (3 cells)
ko hi 4 (5 cells)
ko hi 6 (2 cells)
ko hi 9 (3 cells)
ko hi 10 (2 cells)
ko hi 11 (5 cells)
ko hi 12 (8 cells)
ko hi 13 (4 cells)
ko hi 14 (4 cells)
ko hi 15 (2 cells)
ko hi 16 (2 cells)
90
35
1778
640.9
0.051
0.055
0.927
69
96
84
34
58
78
79
42
72
67
112
81
64
75
43
69
88
125
127
96
74
91
69
30
21
19
8
19
22
24
10
11
22
35
36
8
23
11
13
16
70
71
31
17
20
19
3360
2152
2498
1433
1682
1970
2407
1399
1725
1757
1916
2706
2116
2017
1397
1907
2041
2951
3419
2438
1978
2511
2275
1352.6
475.9
820.6
354
492.3
883.9
1052.7
164.8
250.7
703.5
806.9
846.3
551.5
1029.1
322.7
575.5
241.4
1202.1
1600.8
1016.8
991.4
372.1
383
0.021
0.045
0.034
0.024
0.034
0.040
0.033
0.030
0.042
0.038
0.058
0.030
0.030
0.037
0.031
0.036
0.043
0.042
0.037
0.039
0.037
0.036
0.030
0.022
0.044
0.023
0.023
0.039
0.025
0.023
0.061
0.044
0.031
0.043
0.043
0.015
0.022
0.034
0.023
0.066
0.058
0.044
0.030
0.017
0.054
0.050
0.926
1.011
1.452
1.050
0.893
1.591
1.439
0.495
0.951
1.220
1.347
0.704
2.085
1.664
0.903
1.602
0.650
0.727
0.838
1.291
2.182
0.674
0.611
46
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