MORPHOLOGICAL STUDY OF CELL PROTRUSIONS DURING REDIRECTED MIGRATION IN HUMAN CELLS

Congyingzi Zhang

A Thesis

Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of

MASTER OF SCIENCE

August 2013

Committee:

Dr. Carol Heckman, Advisor

Dr. Roudabeh Jamasbi

Dr. Peter Gorsevski ii ABSTRACT

Carol A. Heckman, Advisor

From the perspective of cell mechanisms, migration patterns arise from two

opposing sources which can be viewed as forces. One, called intrinsic, maintains the cell persistence. The extrinsic arises from signals (repulsive or attractive) exerted by an external stimulus. The extrinsic force is stronger than the intrinsic, since it can overcome the intrinsic force and cause the cell to change direction. The current studies were designed to determine whether these forces were associated with different protrusions. I studied human fibroblast cells that collide with a haptotactic boundary between an adhesive substrate (germanium) and a non- adhesive substrate (plastic) in a chemokinesis system. The morphologies of cells migrating on

the two substrates reflected the cells’ preference for the adhesive substrate. I measured the

prevalence of various protrusions during the process of cells turning away from the boundary and

reorienting their direction of travel. Classes that corresponded to protrusive features were

identified by extracting latent factors from a number of primary, geometric variables, and

included factor 4 (filopodia), factor 5 (cell mass displacement), and factor 7 (nascent neurites).

The data showed that as cells moved further and further from the boundary, they had

progressively lower values of factor 5. The correlation coefficient between the values is -0.4924.

Factor 4 appeared to decrease near the boundary and recover as cells migrated onto the adhesive

substrate. The results suggested that reorientation caused by the extrinsic force occurs by

reducing filopodia and increasing cell mass displacement. It can be suggested that the simplest iii explanation for turning would be that the cell takes directional cues from the remaining filopodia and obtains redirecting force from displacements of cell mass (factor 5).

iv

DEDICATION

To my parents, Mr. Yuwei Zhang and Mrs. Qi Zhang for their love, support, and encouragement. v ACKNOWLEDGMENTS

I want to express my sincere gratitude to my advisor Dr. Carol Heckman for her continuous support of my project. She is a great advisor who always helpful and patient to me, passionate and devoted to the research, cheerful and encouraging to everyone around her. She guided me through all the stages in my research and helped me writing this report for my project.

I am lucky to have her as my advisor and enjoyed all the fun from my study.

I sincerely thank Dr. Roudabeh Jamasbi from the Department of Biology and Dr. Peter

Gorsevski from the Department of Geology for their tremendous help in developing research methods and suggestions in writing this report. My sincere thanks to Dr. Marilyn Cayer for her help in my experiments.

I also would like to thank my lab mates Dr. Mita Varghese, Dr. Surya Amarachintha, and Francis Bugyei for their cooperation in the lab.

Most importantly, I want to thank my family members for their love, support, and encouragement. vi TABLE OF CONTENTS

Page

1. INTRODUCTION ...... 1

1.1 Roles of ...... 1

1.2 Mechanisms of cell migration and the related membrane structures ...... 2

1.3 Studies on cell redirection ...... 4

1.3.1 ...... 7

1.3.2 Chemokinesis and ...... 9

1.3.2.1 Chemokinesis vs. Chemotaxis ...... 9

1.3.2.2 Studies on chemotactic attractants ...... 9

1.3.3 Durotaxis ...... 11

1.4 Factors and shape analysis ...... 11

1.5 Haptotactic cell migration re-orientation model ...... 15

2. MATERIALS AND METHODS ...... 17

2.1 Cells and cell culture ...... 17

2.2 Creating the boundary between two different substrate ...... 18

2.3 Making cell migration tracking system ...... 18

2.4 Additives to DMEM for chemokinesis ...... 19

2.5 Microscopy for track and trace ...... 19

2.6 Shape analysis of track and trace ...... 20

2.7 Statistical analysis ...... 21

3. RESULTS ...... 22

3.1 Cell morphology on preferred and non-preferred substrate ...... 22 vii 3.2 Cell migration on preferred and non-preferred substrate ...... 24

3.3 Cell migration redirected at haptotactic boundary ...... 25

3.4 Protrusions and their dependence on the distance migrated after turning ...... 29

3.5 Protrusions and their dependence on the net distance from the boundary ...... 32

4. DISCUSSION ...... 35

4.1 Re-orientation is characterized by cell mass displacement ...... 35

4.2 Cell does not generate extra filopodia during re-orientation ...... 36

4.3 Further study of filopodia sensitivity distribution from angular data ...... 38

REFERENCES ...... 40

viii LIST OF FIGURES

Figure Page

1 Cell migration pattern ...... 5

2 3T3 cells crowding in the gold area of a dual substratum system ...... 8

3 Substrate with certain elasticity mathematically modeled as a spring ...... 13

4 Micrographs of 1000w cells with high and low factor values ...... 15

5 Haptotactic model on a culture plate ...... 16

6 SEM micrographs of migrating on germanium substrate ...... 23

7 SEM micrograph of fibroblasts on plastic substrate...... 24

8 SEM micrograph of fibroblasts growing in culture dish after 48 hours ...... 25

9 Fibroblasts from the germanium side reoriented at the haptotactic boundary ...... 27

10 Fibroblasts from the plastic side migrated across the haptotactic boundary...... 28

11 Schematic diagram of track length after turning

and net distance from the haptotactic boundary...... 30

12 Four factor values at cells front when the cells migrated away from the boundary 31

13 Four factor values at cells rear when the cells migrated away from the boundary .... 32

14 Four factor values at cells front with net distance between cell

and the haptotactic boundary ...... 33

15 Four factor values at cells rear with net distance between cell

and the haptotactic boundary ...... 33

Fibroblasts from the germanium side reoriented at the haptotactic boundary ...... 27

16 A model showing the most possible turning angles for a cell

colliding with a haptotactic boundary ...... 37 ix 17 A fibroblast hit the haptotactic boundary with nearly 90 degree angle ...... 38

x LIST OF TABLES

Table Page

1 Definition of factors representing cell protrusions ...... 14

1

1. INTRODUCTION

1.1 Roles of cell migration

Cell migration is an important cell behavior orchestrating various developmental, physiological, and pathological processes. For example, during gastrulation in embryogenesis, cells migrate as sheet forming new layers in embryo so as to form specialized tissue in the new sites. Additionally, neurogenesis and development are closely linked with cell migration.

Neuron cells have to migrate from their birth site hundreds of cell-body distances to reach the

final position within the correct cortical layers which is facilitated by continual reconstruction of

the network [1]. Cell migration in adults is integral to tissue repairing, wound

healing, immune response, tissue differentiation. In disease processes, cell migration is playing

crucial role regardless of whether the mechanisms of migration are normal or abnormal [2].

In any multicellular organism, the sustenance of system integrity is important, and a good example is found in wound healing. Once a wound is created on a surface, which is ordinarily covered by an epithelial layer, the cells will have the potential to close the gap through cell migration and cell proliferation. This process is meaningful to the maintenance of any living system, while also being exploited for research on cell migration and biomaterials applications in tissue engineering [3]. However, over-active cell migration could also be dangerous. Drivers of cell migration help to convert cells to metastasizing cells [4]. In tumor ,

invasive tumor cells migrate through the (ECM) and move into the blood

vessels for proliferation and translocation, which is termed intravasation. Translocated cancer

cells can again escape the blood vessels and penetrate the surrounding tissue, termed

extravasation. Two proteases secreting adhesive structures, podosomes and invadopodia, are 2 related to the penetration process, while the other two filament structures, filopodia and lamellipodia, and actin-rich adhesive structures attaching to the extracellular matrix are required for cell migration [5].

In early studies in cell migration, various cell lines and single cell microorganisms were

studied by a typical locomotion parameter, speed. For example, neutrophils migrated at 10 µm

per minute, polymorphnuclear leukocytes migrated at 3 – 30 µm per minute, macrophages at 2

µm per minute, astrocytes at 0.1 – 0.2 µm per minute, and fibroblasts at 0.08 – 1 µm per minute

[6-10].

To further understand the cell migration processes and study its mechanisms, systems

with various environmental cues were frequently used. These cues are categorized by their

physical and chemical properties: haptotaxis, chemotaxis and chemokinesis, durotaxis, and so on.

Haptotaxis refers to cells’ relative preference for different substrates. Chemotaxis and

chemokinesis are both related to motogenic factors but differ by the presence or absence of a

concentration gradient. Durotaxis focuses on the substrate rigidity (Young’s modulus) and its

effect on cell migration.

1.2 Mechanisms of cell migration and the related membrane structures

The initial requirement for the migration process is a spatial asymmetry which could be

induced by applying microscopic nonuniformities including in material composition of the

substratum, temporal stimulus gradients, or directional signals [11]. Distinct front and rear

regions could be identified on polarized cells during migration because of the shape of the cells.

Two of the protrusions at the cell leading edge, filopodia and lamellipodia, are the results of 3 dynamic actin assemblies underneath the cell membrane although they differ morphologically in actin assembly mechanisms [12].

The thin, needle-like filopodia are filled with tight, parallel bundles of filamentous (F)-

actin, which could be identified in cell types such as fibroblasts, keratinocytes, and migrating

neuron growth cones. The dynamics of filopodia growth are a balance between the actin

extension at the fast-growing barbed end and the retraction by the retrograde flow of the actin

bundle [13, 14]. The polymerization of actin bundle in filopodia is regulated by Cdc42, a Rho

subfamily GTPase, which activates IRSp53 (Insulin Receptor Substrate p53) causing it to bind

Mena (mammalian Ena/VASP (Enabled/Vasodilator-Stimulated Phophoprotein)) [15]. As a key

regulator of filopodia induction, directional migration and cell cycle progress, Cdc42 loss causes

defects in filopodia formation, adhesion to , wound-healing, polarization, and

response to stimulus gradients[16]. The most acknowledged function of filopodia is its sensory

role working as antennae to explore the ECM or other environment cues [17]. In addition, it has

been known to explore for the target cell to fuse the epithelial sheets during dorsal closure and

wound repair [17-19].

The other protruding membrane structure on the cell leading edge is the sheet-like

lamellipodia regulated by Rac1, also a Rho subfamily GTPase, which stimulates actin network

polymerization forming membrane ruffles [20]. The lamellipodium actin network disassembles about 1-3 um behind the leading edge, and the lamella provides the scaffold for actomyosin integrating with substrate adhesion producing the force for migration [21].

Another key structure regulating the cell’s morphology, focal adhesions (FA) are the

adhesive interactions between a cell and the ECM. On the outside of the cell, the transmembrane 4 receptors for ECM () interact with ECM ligands for stability. This is called “ligation.”

On the inside of the cell, it forms clusters which integrate with the actin filaments to form

bundles called stress fibers. These fibers or cables provide the contractile force for migration

and anchorage to the substrate [22, 23]. Signaled by Rho, also a Rho subfamily GTPase, the

assembly, maintenance, and dissolution of FAs is mediated through the phosphorylation of the

II chain facilitated by Rho kinase p160 Rho-associated kinase (ROCK). The

dominant mutant of ROCK has been found to cause defects in the formation of FAs, and an selective inhibitor of the kinase suppressed FA activity in smooth-muscle cells [24, 25]. Notably,

the fine, linear contact sites associated with filopodia and the small, punctate ones with the

lamellipodia are the precursors of the prominent FAs at the lamellipodium-lamellum interface

[22]. It is widely accepted that activated Rac, often associated with ruffling and lamellipodia,

initiates new punctate contacts at the cell front while Rho matures and transits the nascent

contacts into elongated cell-matrix adhesions [26-28]. In what is known as the basic cell motility cycle, protrusions formed at the leading edge set up the orientation of the polarity, and then, the contact sites are laid down on the substrate underneath. The contraction and detachment orchestrated in specific regions by the actomyosin and the dynamics cause a cell to move without directional bias or, if chemotactic factors are present, up the stimulus gradient.

However, the machinery of how environmental factors reorient or regulate the cell migration is

not fully understood [29].

1.3 Studies on cell redirection

In the studies of mammalian cell migration behavior, cell trajectories have been

segmented and categorized into two alternative modes. In mode I, the directional-mode, cells 5 migrate with more directional persistency at a higher speed. In comparison, in mode II, the re-

orientation mode, cells migrate with a change in directions at a lower speed. This bimodal

paradigm provided a great entry point for our understanding of the different behaviors in cell

migration and the study of the correlative activities (Figure 1) [30].

Figure 1. Cell migration pattern falls between two extreme particle migration modes, ballistic and diffusive. Experimental mean squared displacement versus time for the MCF-10A human mammary epithelial cells expressing the plasmid vector alone (pbabe), the normal (neuN) or transforming (neuT) versions of the rat Her 2/Neu oncogene [30]. The neuN gene is commonly amplified in human . Reproduced from [30] by permission of Springer Press.

Among all the studies on aspects of cell reorientation or redirection, researchers have

focused on indexes to describe cell behavior during migration. Linear cell locomotion velocity

and directional persistence were frequently used in such studies [31, 32]. For example, in studies

of embryonic chick dorsal root ganglion growth cone turning, the growth cones were re-

orientated by the localized treatment of myosin 1c which caused lamellipodial expansion in a

certain range. The instantaneous velocity (as mean speed) and time between significant direction

changes (as directional persistence parameter) were measured and fit into the Persistence 6

Random Walk (PRW) model [33]. One of the problems of these models may be distinguishing

between the velocity-dependence and pattern-dependent variations in motility.

In addition to mean speed V and persistence time P, the cell polarization is another essential parameter revealing the instantaneous orientation which is highly related to the directional persistency. For example, the motility of mouse 3T3 and sarcoma cells was observed

and compared on substrates of different rigidity and topography. In this study, the cell

polarization was defined by the ratio of short to long cell axes. A threshold value (0.75) for the ratio was defined to categorize the polarized and non-polarized cells. The durotactic effect (see

Durotaxis) from different substrates was evaluated and compared by the counts of the polarized and the non-polarized cells showing increasing substrate rigidity influenced 3T3 cell polarization and it exhibited a persistent type of migration. However, sarcoma cell spreading was more modified by the substrate topography and it migration seemed to escape from ECM cues [34].

In the studies of migration persistence, turning angle is another parameter relied upon in

models of random walk migration and biased random walk migration. In Wang’s study, growth

cone movement in random directions has an average turning angle near zero, which shows the

conservation in turning [33]. Arrieumerlou and Meyer observed primary dendritic cells,

fibroblasts, and neutrophils changed direction by turning their leading edge. In a biased random

walk triggered by C5a, global cell orientation was shown as a result of small discrete turns

towards the chemoattractant led by local extension of a lamellipodium in the left and right leading edge [35]. In the case with no external cue, Li et al. found that amoeboid cells from

Dictyostelium discoideum migrated in a zig-zag manner and turned every 1 - 2 minutes on average. This is a result of the reorientation between characteristic ranges of –π/4 to π/4 within 2 7

- 3 minutes. The behavior of cell remembering its last turn and consistently turning away from it was considered as a strategy seeking for potential guidance cue or target [36].

Within all these quantitative cell migration models, researchers examine how cells

respond to the various stimulants being applied to manipulate or bias their pattern of migration.

In the following section, I review several types of models and the conclusions from those studies

that inspired me. This is where I began my research on the topic of cell migration and graphical and statistical methods to understand how cells use protrusions and how they contribute to the process of migration.

1.3.1 Haptotaxis

In previous work of S. B. Carter [37], a gradient of palladium coating on a cellulose acetate surface was applied to determine if it had an effect on cell motility. Compared with the

evenly coated surface, fibroblasts on the gradient showed a more uniform migration with longer tracks, and they moved towards the upper end of the gradient. Thus, the cells became dense at the upper gradient end. Carter used ‘haptotaxis’ to describe this phenomenon and suggested that the relative strength difference among peripheral adhesions contributed to directed migration and

arrangement of cell shape. Later, Carter separately studied different sites on leading and trailing

margins of the cell and pointed out the passive nature of cell mass, i.e. migration is not initiated by any force inside the cell but depends on the substrate on which cell is sitting [38]. In

Letourneau’s study on sensory ganglia from chicken embryo, the axonal growth cone also

showed relative preference for various substrata including petri dishes, palladium, dishes coated

for tissue culture, and polyornithine dishes. The author showed that the direction of the

elongating growth cones could be guided by the patterning of the culturing surface [39]. Similar 8 dual substratum systems were made to guide cell motility using electron microscope grids as

stencils and evaporating metal (palladium or gold) evenly on other substrata, i.e. agarose and

cellulose acetate [40, 41]. Making observations on 3T3 cells (Figure 2) [42], Albrecht-Buehler

found that filopodia served as a rapid scanner over the environment and led the lamellipodia to

spread behind it on its preferred substratum. The author’s explanation of this phenomenon was

that the firmness of attachment between filopodia and the substratum dictated the direction. After

filopodia retraction, only the filopodia still attached could lead the spreading of the

lamellipodium. Directed cell migration introduced by a haptotactic system was considered as a

result of uneven competition among different parts of lamellipodia in the cell periphery [43]

which was mainly associated with the filopodia activities. Clearly, filopodia can sense the more

adhesive substrate and set the direction to the favorable substrate. However the role of

lamellipodium is not clear. In the history of the field, the lamellipodium and especially the

ruffling membrane is the main motility organ [44].

Figure 2. 3T3 cells crowding in the gold area of a dual substratum system (dark: gold, bright: glass) 3.5 h after plating. Reproduced from [42] by permission of Rockefeller Press. 9

1.3.2 Chemokinesis and chemotaxis

1.3.2.1 Chemokinesis vs. Chemotaxis

Apart from seeking for guidance cues from the substratum underneath, the cell may also

recognize chemical cues defused in the liquid culture medium. Two terms were used to describe

the cell response, chemokinesis and chemotaxis. Chemo identifies the chemicals as stimulus and

kinesis refers to an undirected locomotion. “Chemotaxis” refers to the cells migrating in a

directional manner [45]. From the aspect of the arrangement of the stimuli, chemotaxis is the

response to a chemical concentration gradient. The difference between locomotion in chemotaxis

and chemokinesis is that in the chemotaxis, the cell migrates persistently towards the source of

the attractant [46]. Chemokinesis is said to be positive if displacement of cells moving at

random is increased and negative if displacement of cells is decreased [45]. This is discussed

further below (see Models of persistence).

1.3.2.2 Studies on chemotactic attractants

Over time, many growth factors have been found to have a chemotactic effect on various

cell lines. For my research in which I rely on the cell’s reflecting from the boundary between the

two substrates, cell locomotion was demanded because cells must travel to hit the boundary.

Endothelial A and Fibroblast Growth Factor 2 attracted endothelial cells towards the high end of a test chamber in which a stable concentration gradient of growth factor was

maintained by a constant flow from the attractant source [47]. Insulin and insulin-like growth

factors (IGFs) were well known for their role in activating cell migration. Both insulin-like

growth factors (IGF-I/II) stimulated tissue invasion and tumor metastasis in cancer development

by promoting cell migration through binding on IGF and insulin receptors. The binding triggered 10

phosphatidylinositide 3-kinase (PI3-K) signaling and the extracellular-signal-regulated kinase

1/2 (ERK1/2) and mitogen-activated kinase (MAPK) cascades leading to one of the prerequisites of cell migration, the recruitment the small GTPase Ras to membrane [48]. Insulin shares much structural similarity with IGF-1 [49] and interacts with insulin receptor. Insulin has also been reported being chemotactic on human tissue cells [50] and causing in

insulin-resistant diabetic patients by increasing the level of ERK1/2 dependent MMP-9

production [51]. In the most controversial field, investigators tried to understand the role of

chemotactic cell migration in cancer development. Cancer cells were studied under chemotactic

environment in a Boyden chamber system [52]. Here, more cells reach the upper side of the

chamber where the concentration of the growth factors was higher. In a mesothelioma cell line,

the chemokinesis model was tested with various growth factors including EGF, transforming

growth factor-alpha, IGF-I/II, and stem cell factor. Treatment by IGF-I and IGF-II caused greater

random migrating capability when compared to those other growth factors [53]. In a study of the

chemotactic effect of EGF on breast cancer cell migration, a nonlinear concentration gradient of

EGF at 0-50ng/ml induced directional locomotion of cells. However, the author also shows that

EGF affected cell migration pattern differently for 25ng/ml and 50 ng/ml concentration. EGF

significantly boosted the cell migration speed and the directionality measured as chemotactic

index [54]. 10ng/ mL platelet-derived growth factor (PDGF-BB) has been tested for its

chemotactic effect in a scratch wound assay in vitro on rat marrow stromal cells. At the

presence of PDGF-BB, cells filled up the gap within 12 hours as a result of increased cell

migration while the gap was barely closed in the same amount of time at the absence of PDGF-

BB in a control group [55]. Although some investigators discussed chemotaxis and chemokinesis together and some discussed them separately, it is important to understand that the studies of 11

chemokinesis addressed the underlying mechanism of motility. Without motility the cells are

unable to do directional migration in a gradient. Therefore, chemokinesis is the more

fundamental mechanism for both non-directional and directional motility. My study addressed

this mechanistic aspect of how cells move.

1.3.3 Durotaxis

In normal tissue cells, growth is anchorage-dependent, which means they have to be attached to a solid substrate in order to grow. This property, called anchorage-dependence,

depends on the capability of sensing the rigidity and using the mechanical feedback from the substrate to regulate cell spreading, growth, and survival [56]. Some cells that don’t have this

property are oncogenically transformed. There are also some normal cells, especially in

hematopoietic lineages, that are anchorage-independent. The sensitivity to rigidity is reflected in

cell’s preference for a stiff substrate, which is a phenomenon named by Wang as “durotaxis”

[57].

When such physical and mechanical cell-substrate interaction was studied in cell migration, Wang et al. found that 3T3 fibroblast cells would easily migrate into an area with relatively higher when they approached from an area with lower stiffness. However, the cell would retract or turn away from the boundary between substrates of different stiffness when they approached the softer substrate. As a mechanical cue, durotaxis provides a different model system for looking into the migration mechanism during cell re-orientation and persistence.

Pelham and Wang initially found that on flexible substrate cell motility and lamellipodial activities were increased while cell spreading was decreased [58]. To understand the cell- 12

substrate mechanical interaction, Pelham and Wang cultured fibroblasts on an elastic substrate

embedded with fluorescent beads whose signal could be collected. The force exerted by the cell

was calculated by the displacement of the beads. They found that the forces near the leading

edge were stronger and more variable than those in the posterior area [59]. Furthermore, smaller

adhesions near the leading edge in motile cells were found to exert strong contractile tension

forces while those mature focal adhesions in the posterior area exert weaker force [60]. Since

lamellipodium provided almost all the forces for locomotion, the cumulative result from the

strong, variable, and dynamic traction forces provided by those smaller adhesions at the leading

edge should be responsible for steering the locomotion [61]. Later, Georges et al. discussed and

compared the cell’s morphological response to soft and rigid substrate pointing out the possible relation between the traction force and cell’s morphologically different behavior while crawling

on substrate with different stiffness [62]. According to Georges, the extent of cell spreading

resulted from the relative difference in stiffness between cell and the substrate, that is, with the

same traction force, if the cell was softer than its substrate, it would be easily deformed, and vice

versa. The authors model this arrangement using a spring to represent the cell mechanism of

testing the substrate stiffness (Figure 3). If the substrate is yielding when the cell exerts tension

on it through the focal contacts, the cell reduces the tension force and rounds up. If there is

resistance from the substrate, the cell spreads out and expands the area throughout which it

develops focal contacts. This model explains how the cell gauges substrate stiffness. The

deficiency in this explanation is that there is no knowledge of the surface morphology at a

molecular level. Therefore, roughness and smoothness are not distinguished from stiffness. 13

Figure 3. A substrate with certain elasticity can be mathematically modeled as a spring with a spring constant k. When a cell, represented as a gray block in (A), pulls the substrate with a force P and causes a displacement by distance x , the opposite force from the substrate F = -kx. On a soft substrate (B) with a lower k, cell can easily displace the substrate by x. However, on a stiff substrate (C) with higher k, the substrate can only be displaced by a smaller distance x with the same pulling force F. Figure reproduced from [62] with permission of American Physiological Society.

In Wang’s durotaxis experiment, 3T3 cells extend the cell edge towards the direction

where the substrate was pulled away from it mimicking migration onto a stiffer substrate.

Conversely, they migrate away from the direction where substrate was pushed towards it

mimicking migration onto a softer substrate. Cells produced significantly stronger traction forces near the leading edge on a rigid substrate, compared to a soft substrate [57]. However, the

substrates used in durotaxis may vary in degree of irregularity, which is not a problem in

haptotaxis system like Albrecht-Buehler’s [42]. The mechanisms might be similar but since it is not yet understood, this cannot be confirmed.

1.4 Factors and shape analysis

As discussed above, cell protrusive activities provide important information about how cells

explore their environment. Quantification and classification of protrusions will help to interpret 14

their role in cell migration. Otherwise, the observations rely on visual assessment of morphology,

which is subjective. Factor analysis is a statistical method used to generate theoretical variables

from observable variables [63]. Heckman’s laboratory has extracted and classified various

features or properties as 20 latent factors by sampling cell lines at various passage levels over a

period of time. The features can be used to define the cell phenotype. Among the 20 factors,

four of them, factor 4, 5, 7, 16, are predominantly composed from variables describing the cell

edge and therefore they are protrusions (Table 1). Figure 4 shows the morphological features of the cells with either high or low value for each factor. For my study, the edges were traced from

migrating cells on SEM photographs. The contours were extracted with a program compiled in

C++ on an FTP server, elvis.bgsu.edu. More programs generate observable variables from the

contour. Then, that output is submitted and the value for each latent factor was calculated from

the database computed in statistical analysis software (SAS).

Factor Number Definition

4 Prevalence of sharp, flat, and tapering features at the cell edge (filopodia)

Blunt or sprawling projections or enlarged invaginations at edge (mass 5 displacement)

7 Nascent neurites [64]

16 Pointed protrusions but wider and higher than filopodia

Table 1. Definition of factors representing cell protrusions [65] 15

Figure 4. Representative micrographs showing 1000w cells with high and low factor values, (A) Cell with high factor 4 value, 2.96, (B) Cell with low factor 4 value, -3.84, (C) Cell with high factor 5 value, 4.89, (D) Cell with low factor 5 value, -2.52, (E) Cell with high factor 7 value, 5.34, (F) Cell with low factor 7 value, -3.40, (G) Cell with high factor 16 value, 3.657, (H) Cell with low factor 16 value, -2.952. Arrows in frame C show multipolar nature thought to represent lamellae. The bar on each micrograph indicates the scale. Reproduced from [66] by permission of Elsevier.

1.5 Haptotactic cell migration re-orientation model

On a dual-substrate haptotactic surface, the cell would selectively migrate onto the preferred substrate. When choosing between two substrates, it receives the cue from the adhesiveness of the substrate at the boundary. Two things were known which I could depend on when designing the project. One is, the filopodia will be preferentially at the leading edge of a migrating cell because filopodia are known as a persistence mechanism (reviewed in [67]).

Another fact that was known from preliminary was that when the cell met with the non-preferred substrate they will turn away. Knowing all this, I could investigate the mechanism of turning.

My hypothesis was that cell would re-orientate at the boundary leading off to a different direction and putting filopodia at the leading edge. We also need to realize cells migrate differently on substrates with different rigidity. According to Guo et al., cell merged to form 16

tissue-like structure when they grow on a soft substrate while migrate away from each other

when grow on a stiff substrate [68]. In my study, I have not seen cells forming tissue-like clumps

when they were on either adhesive or non-adhesive substrate. So, I assumed these differences

were small on the dual-substrate system.

In my research, the haptotactic boundary or barrier was established by creating a surface

with alternating substrates, germanium and plastic (Figure 5). These substrates differed in charge

attracting different molecules from the culture medium which created a haptotactic boundary.

Figure 5. Haptotactic model on a culture plate. The brown shade represents the germanium and the white strips represent the plastic. Different may be attracted from the culture medium, which makes germanium a high adhesive surface and plastic a low adhesive surface to the cells.

Cells were given chemokinesis stimulus to make them move on a migration trajectory tracking system with nano-gold particles [69]. Then, images of the track and the contour were

made from the cells migrating near the haptotactic boundary. Values of the factor 4, 5, 7, and 16

were calculated for the leading edge and trailing edge (see Materials and Methods). These

statistical values for the factors were compared among the cells at various distances after turning

away. So, the most possible protrusion leading off after turning will be revealed. 17

2. MATERIALS AND METHODS

2.1 Cells and cell culture

20.0 g Dulbecco’s Modified Eagle Medium (DMEM) (Gibco, Grand Island, NY) powder

was initially dissolved in 1600 ml deionized water combined with 20 ml of penicillin-

streptomycin solution (Gibco, Grand Island, NY) and 11.2 ml of amphotericin B (Invitrogen,

Paisley, Scotland, UK). 10% NaHCO3 (Fisher Chemicals, Fair Lawn, New Jersey) was used to

adjust the pH of the solution to neutral when color of the solution changed from golden to ruby.

Deionized water was added to a final volume of 1800 ml. The solution was filtered with a

sterilized Millipore filter of 90 mm in diameter and 0.22 um in pore size (Millipore, Billerica,

MA) and collected in 4 sterilized 500-ml glass bottles, 450 ml in each. For cell culture, 50 ml of

FBS (fetal bovine serum) purchased from HyClone (Logan, Utah) was added into each bottle for

a 10% concentration of FBS. Part of the serum-free medium was kept for experimental study. A

small amount of medium from each bottle was tested for sterility before being used for cell

culture.

A calcium magnesium free Hanks balanced salt solution (CMF-HBSS) with trypsin and

EDTA was prepared for dissociating cells from the old dishes. 50 ml of 10X CMF-HBSS (Gibco,

Grand Island, NY) and 0.875 g Na-EDTA were dissolved in 100 ml of deionized water. 10 mg phenol red (Matheson, Cincinnati, OH) and 175 mg NaHCO3 (Matheson, Norwood, OH) were

dissolved in deionized water to a final volume of 400 ml and combined with the CMF-HBSS

EDTA solution. To adjust the pH, 1N NaOH was added until the liquid turned a cherry red color.

Deionized water then was added to a final volume of 437.5 ml. The solution was autoclaved. 18

For use, 12.5 ml of 0.5% trypsin (Gibco, Grand Island, NY) was added into 87.5 ml of CMF-

HBSS EDTA solution.

Human fibroblast cells (GM21808, Coriell Institute for Medical Research) of circumcised

newborn’s foreskin were cultured with DMEM 10% FBS made as above. The cells were cultured

with the medium at 37 ℃ with 5% CO2 and 95% air. For subculture, they were dissociated from the old dish by incubating with the trypsin CMF-HBSS EDTA for 4 minutes, and collected by centrifugation at 740rpm for 5 minutes. A sterilized glass tray containing distilled water was kept on the bottom rack of the incubator for humidity.

2.2 Creating the boundary between two different substrate

Petri dishes were cleaned in absolute ethanol overnight and wiped with lens paper. In preliminary experiment, different shapes of boundary were created by lining strips parallel or intersecting at right angle. In the final experiment, strips of 1/16” wide tape separated by 1/16” partially covered the bottom of the culture dishes. The dishes with the tape were coated with

germanium in a Denton Vacuum BTT-IV. Before use, the bell jar of the coater was wiped with

ethanol to eliminate contamination. 0.025g of germanium in a tungsten basket was used for each

coating run. Evaporation was at a vacuum of 2x10-5 Pa, a rotating speed of 10% rpm, and

approximately 9 amps. The basket was checked through a piece of dark glass to make sure all the

germanium has been evaporated.

2.3 Making cell migration tracking system

1% BSA (Sigma-Aldrich, St. Louis, MO) in distilled water was dissolved overnight at 4℃

and filtered with a 0.22 um Millipore filter. The tape was peeled from the petri dishes to expose 19

germanium-plastic boundaries then the 1% BSA was poured into the dishes. The fluid was taken

off by aspiration, dried for 5 seconds, and denatured by absolute ethanol to enhance adhesiveness

for the nanogold particles. The fluid was taken off by aspiration and the dish was heated to 90℃

to denature the protein [43]. A mixture of 1.8 ml of 14.5 mM AuCl4H (Alfa Aesar, Ward Hill,

MA), 6 ml of 36.5 mM Na2CO3(Fisher Chemicals, Fair Lawn, New Jersey), and 11 ml of distilled water was brought to boiling, and 1.8 ml of 0.1% paraformaldehyde (Polysciences,

Warrington, PA) was added. The solution was ready for use when boiling and turning to a

muddy dark-blue color. The hot nanogold particle solution was poured onto the prepared petri

dishes. The dishes were opened for a short while to avoid steam build up. The dishes were then

closed and kept in the incubator under cell culture conditions as above for at least 45 minutes to

let the nanogold particles settle down. The solution was replaced with the culture medium and kept in the incubator overnight. The prepared dishes were gently rinsed in phosphate buffered saline (PBS) to eliminate any trace of paraformaldehyde on the nanogold particle-coated surface.

The dishes were kept in PBS at 4℃ for use within two weeks [70].

2.4 Additives to DMEM for chemokinesis

55 ng/ml dexamethasone 0.1% human serum albumin (HSA), 17 ng/ml PDGF-BB, 12 ng/ml insulin, and 12 ng/ml epidermal growth factor (EGF) were added into no-serum DMEM medium. All the additives were purchased from Sigma-Aldrich, St Louis, MO. The subcultured cells were added to the above medium and plated on the prepared dishes at a density of 5000

cell/dish and incubated for 48 hours.

2.5 Microscopy for track and trace 20

When the samples were collected for analysis, the dishes were emptied and fixed in 3%

paraformaldehyde in PBS for 10 minutes, then rinsed in PBS twice. The samples were kept in the

PBS until they were observed with a phase contrast inverted microscope. The cells and their tracks near the germanium boundary were photographed and numbered for track study. This

procedure was used to determine the right chemokinesis conditions so the cells would collide

with the boundary.

The sample dishes were refixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH

7.2, for 30 minutes, then rinsed with phosphate buffer twice, 15 minutes each. The dishes were

dehydrated in graded of series of ethanol: 40%, 60%, 80%, 95%, 100%, 100%, 100%, 15

minutes each and critical point dried. The samples were coated with 2 nm of gold-palladium

(Polaron E5100) and mounted on aluminum stubs with colloidal graphite (Electron Microscopy

Sciences, Hatfield, PA). The samples were examined in SEM (Hitachi S-2700) and digital

images were collected in Quartz PCI Digital Imaging System. For large cells, several pictures

were taken at a set magnification to show the edge of cells clearly.

2.6 Shape analysis of track and trace

The track images of the cells were loaded on the ArcGIS program for shape analysis.

Proper coordinate systems were established on the .tiff image for following measurements.

Polygons and polylines were determined by the edge of the tracks. The polygons allow measuring the area while the polylines allow synthesizing Euclidean distance from the edge into the polygonal track pattern for objectively defining the central lines. The lengths of the central

lines were measured in ArcGIS and then the orientation of the cells to the boundary was

measured with Image J. 21

To determine values for the different types of protrusions, several pictures of each cell

were merged in Photoshop to delineate the cell edge. Two transparent sheets were created with

manual tracings of the leading edge and trailing edge. The tracings were flattened separately

against a white background and sent as .tiff to an SGI computer running as an FTP server under

the IRIX operating system. This computer, , was employed for the initial

analysis of shape variables. Factor values 4, 5, 7, and 16 were determined for the leading and

trailing edge of the examined cells. For details of the methodology, see references [67, 71-75].

2.7 Statistical analysis

Best Subsets Regression was used to determine the most influential group of factors with

different independent variables. I also calculated the least mean squares regressions of factor

values with the independent variables and the correlation coefficients (CC) between independent

and dependent variables, using Excel software. In the results shown below, the correlations are

given as a metric for the variance of values within the samples. P values and R square values were also obtained but, since the number of cases included in the analysis was small, these significance and the regression results may need to be revised with an increase in sample size.

22

3. RESULTS

3.1 Cell morphology on preferred and non-preferred substrate

When observed by SEM, human fibroblast cells developed different morphological

features when being plated on the two types of substrates in my study. Cells plated on an

adhesive substrate (germanium) became polarized during migration, displaying the morphology

of a fan-shaped, broad area in the front and a pointed, narrow area in the rear (Figure 6).

However, when they were plated on a non-adhesive polystyrene plastic, the cells rounded up and the peripheral cell mass shrank as well. The area where the cell adhered to the substrate was

decreased as a result. The fact that microspikes were present on the sparse attachments to the

substrate suggested that focal adhesions were formed there but the adhesions could not develop

tension (Figure 7). 23

Figure 6. Three SEM micrographs showing fibroblasts migrating on substrate coated with germanium. The cells show a recognizable morphology with a broader area at the front, termed top (T), and a narrow area in the rear, termed bottom (B). The white floccules covering the substrate surface are nanogold particles. The area free from nanogold particles is the track where particles were picked up and attached on the cell membrane during cell migration. 24

Figure 7. A SEM micrograph showing human fibroblast on plastic substrate. The cell lost its typical morphological features described above (shown in Figure 6) and rounded up. Part of the cell protrusions were reaching out for adhesive substrate (G = germanium) which was separated from the non-adhesive substrate (P = plastic) by the haptotactic boundary (pointed out by the arrows).

3.2 Cell migration on preferred and non-preferred substrate

When the tracks of motile fibroblasts were observed by SEM, they were slightly different on germanium substrate over plastic substrate. From the micrograph (Figure 8), the tracks on the two substrates showed cells were migrating on both of them. However, most of the cells initially plated on plastic detached from the substrate and left empty tracks within 48 hours. In comparison, the cells that were initially plated on the germanium-coated substrate were still attached and migrating in their own tracks. 25

Figure 8. A SEM micrograph showing human fibroblast cells growing in culture dish for 48 hours. The arrows are pointing to the boundary between the two substrates, germanium (G) and plastic (P). The black patterns formed on the nanogold particles are the cell tracks. On the plastic side, there are few cells located within the tracks. On the germanium side, cells are still attached and sweeping away nanogold particles to make their tracks.

3.3 Cell migration redirected at haptotactic boundary

When cells and their tracks were studied under a light microscope (Olympus IX81, bright

field), some of them that initially migrated from the germanium towards the plastic were re-

orientated at the haptotactic boundary and never migrated into the plastic. Such cells typically

either turned to the right (J-shaped track) or to the left (L-shaped track) at the boundary (Figure

9). The majority of the cells continued to migrate along the boundary.

When cells hit the haptotactic boundary from the other direction, from plastic to germanium, I found the cells’ behavior was quite different. Cells starting on the plastic substrate easily penetrated the haptotactic boundary and kept migrating straight on to the germanium substrate. These cells show no tendency to migrate along the boundary. Their behavior was 26 totally distinct from that of cells on the adhesive substrate, a point which is further dealt with in the Discussion. From my observation in this section, the cells exclusively chose the germanium substrate (adhesive substrate) when the two substrates were available. Compared with the cells migrating on the germanium substrate, those orientated from plastic substrate (non-adhesive substrate) to germanium migrated with more persistency. This observation is also discussed below.

27

Figure 9. Two light micrographs taken a5on Olympus IX81 light microscope with bright field showing how fibroblasts from the germanium side are reoriented at the haptotactic boundary. In the micrographs, these two fibroblasts initially start migrating from the germanium towards the plastic surface. However, they change their direction at the haptotactic boundary and stop migrating further into the plastic. The upper cell turns right making a J-shape track and the lower cell turns left making an L-shape track. The light-scattering of gold causes the cells to appear dark and the germanium coating slightly darker than the plastic. The arrows are pointing to the haptotactic boundary between plastic (P) and germanium (G). 28

Figure 10. Two light micrographs taken at 400X Olympus IX81 with bright field showing fibroblasts from the plastic side migrated across the haptotactic boundary. In the micrographs, these two fibroblasts initially migrated on the plastic surface near the haptotactic boundary. They penetrated the boundary and kept on migrating onto the germanium surface. The arrows are pointing to the haptotactic boundary between plastic (P) and germanium (G).

29

3.4 Protrusions and their dependence on the distance migrated after turning

Using 20 cells that moved from the germanium surface and turned at the haptotactic

boundary, I analyzed the dependence of their factor 4, 5, 7, and 16 values on the distance they

travelled after turning (measured as TL in Figure 11). The result (Figure 12) showed that the cell

mass displacement increased during the re-orientation process at the haptotactic boundary. The

values for this variable, factor 5 (Table 1), were high for cells near the boundary and declined as

the cell finished the re-orientation step and migrated away from the boundary. Moreover, the CC

of the values with TL was -0.4924, which indicates that factor 5 values vary inversely with TL.

Cells did not put more filopodia at the front during the reorientation process as I

hypothesized. Instead, filopodia decreased when cells were changing their direction and the decrement was recovered after the cells migrated away from the boundary. This trend was indicated by a CC value equal to 0.2905 (Figure 12). This correlation suggested a positive trend

in filopodia. The result of factor 7 and 16 suggested that nascent neurites and wider pointed

protrusions at the cells front did not significantly increase or decrease during the re-orientation process.

In Figure 12, only the factor values from the top or the front edge of the cell are shown. I also measured factor values at the bottom or the tail of the cells but the correlation coefficients for the factors’ values with TL were invariably small except for factor 16. This is shown in

Figure 13. 30

Figure 11. Schematic diagram showing track length after turning and net distance from the haptotactic boundary. The haptotactic boundary separates the two substrates, plastic (P) and germanium (G). The diagram shows a cell encountering the boundary, which turned and kept migrating on the germanium. The two independent variables in my study are track length after turning (TL) and net distance from the boundary (DT). The thin blue line shows the central line of the cell track. 31

Figure 12. Increment or decrement in four factor values at cells front when the cells migrated away from the boundary. The TL (track length after turning) shows how far they migrated after leaving the barrier. Factor 4 values increased after turning away from the boundary (A). Factor 5 values decreased when cells migrated away from the boundary (B). There was no significant change in factor 7 and factor 16 values when the cells reoriented at the boundary (C, D) (N = 20). 32

Figure 13. Increment or decrement in four factor values at cells rear when the cells migrated away from the boundary. The track length after turning (TL) shows how far they migrated after leaving the barrier. Factor 16 values increased after turning away from the boundary (D). There was no significant change in factor 4, factor 5, and factor 7 values when the cell re-orientated at the boundary (A, B, C) (N = 20).

3.5 Protrusions and their dependence on the net distance from the boundary

Likewise, I also analyzed the 20 cells for the correlation between their factor 4, 5, 7, and

16 values and the net distance between them and the boundary (measured as DT in Figure 11).

The result (Figure 14) showed that the cell mass displacement decreased as the cell was getting farther away from the boundary. However, the filopodia, nascent neurites, and wider pointed protrusions were not significantly affected by the re-orientation process. The absolute values these correlation coefficients were low (<0.2) suggesting that very little of the variance in factor values could be explained by the independent variable. 33

In Figure 15, I also show the correlation coefficients of factor values at the bottom or tail of the cells with the distance (DT) between the cell and the boundary. However, the CC values were invariably small except for factor 16. The CC value for factor 16 indicates that it increased with the DT, similar to the trend I had observed with TL (cf. Figure 13 and Figure 15).

Figure 14. Increment or decrement in the four factor values at cells front with net distance between cell and the haptotactic boundary. The distance from the boundary represents the depth that cell migrates into the germanium after turning. Factor 5 value decreases when cells migrate further into the germanium (B). There was no significant change in factor 4, 7 and factor 16 values when the cell migrates further into the germanium (A, C, D) (N = 20). 34

Figure 15. Increment or decrement in the four factor values at cells rear with net distance between cell and the haptotactic boundary. The distance from the boundary represents the depth that cell migrates into the germanium after turning. Factor 16 values increased when cells migrate further into the germanium (D). There was no significant change in factor 4, factor 7, and factor 16 values when the cell migrates further into the germanium (A, B, C) (N = 20)

35

4. DISCUSSION

4.1 Re-orientation is characterized by cell mass displacement

My result showed that increase in cell mass displacement was inversely related to the

track length after turning. The re-orientation process is positively related to the cell mass

displacement. As the cells went further onto the adhesive substrate, the factor 5 values were

decreasing. From the literature review, we knew that spreading in the cell peripheral cytoplasm

was essential to generate enough contractile force for migration. In addition, the uneven traction

force in the periphery caused competition among different quarters in the cell edge. The

direction and speed of cell migration highly depended on the direction and magnitude of the

resultant force. So, changing the distribution of cell mass will alter or update the migration mode

(i.e. change in direction and speed). However, we need to understand that cell mass

displacement needs direction from filopodia to guide cells around the substrate. I use an analogy

of a car which represents the cell. Filopodia is the driver turning the steering wheel and sending

directions to the cell mass displacement, the motor, about where to go. Without the direction

from filopodia, cell mass displacement cannot tell which way to go on a substrate. This

resembles a report in the literature where Andrew et al. described the competition between a pair

of forked pseudopods [76].

My observation agreed with this model and pointed to a certain type of protrusion, cell

mass displacement (factor 5), which was the most influential protrusive structure during a re-

orientation process. When the migrating away from the boundary (i.e. increased net distance between the cell and boundary), the cell was recovering from the re-orientation and gaining persistence with decreasing factor 5 values. 36

4.2 Cell does not generate extra filopodia during re-orientation

According to my result, there was no increment of filopodia in the cell periphery during

the re-orientation, which could be interpreted according to two well-known principles of cell

behavior. First, the filopodia colliding with the haptotactic boundary were physically inhibited.

From the morphology of cells located on the non-adhesive surfaces, which suggested that their

periphery was not spreading out, I could conclude that there was a non-optimal environment for

filopodia to extend. Second, while this stopped filopodia extending further into the non-adhesive

surface, the cell tended to keep using the rest of its filopodia to scan across the available surface

instead of evolving nascent ones over a short time period. This influence at the boundary is

consistent with the declining value of factor 4 shown in Figure 12. A similar observation was

reported in a neuronal migration study and was termed “biased selection” [77]. Interneurons

were found responding to chemoattractant signals by selecting and strengthening among the

available branches. During this process, the branches not extending towards the signal were

inhibited and shortened, while the “selected” branch was strengthened and elongated further

toward the signal. In my research, I consider filopodia as the vision of a migrating cell. I use this analogy for several reasons. Comparing the front and rear edges of a polarized and persistently migrating cell, filopodia are in the front just like our eyes are in the front of our head,

and the visual field is limited to certain range of angles. Second, the strongest filopodia leads the

migration direction just like our eyes resolve better when focusing on an object. When a cell’s

“vision” is partially blocked by a haptotactic barrier, the surviving filopodia will be responsible for steering the cell. Depending on the angle it had collided with the boundary, the distribution

of the surviving filopodia may be varied. The locality retaining the most surviving filopodia will

win the contest and lead off according to the direction pointed by those filopodia (Figure 16). 37

This is consistent with the observation of L-shaped and J-shaped tracks, which suggested the cell

followed along the boundary rather than reflecting from it. In addition, when a cell comes from

the non-adhesive surface, the contact with the adhesive surface is enough to ensure it follows a

straight trajectory off the boundary (Figure 10). In an extreme case, the cell could migrate

towards the boundary with a straight angle (Figure 17), inhibiting all the filopodia at the front

and blinding the cell to the non-adhesive substrate. Then the cell mass could eventually migrate

into the non-adhesive substrate for a short while. Theoretically, such a “crucial mistake” could

be corrected if the cell has not migrated too far from the boundary before the nascent filopodia

extend and find the adhesive substrate again. It has been observed that the cell located on the

non-adhesive substrate still had the chance to sense the adhesive substrate and migrate into it if

the cell was close enough (Figure 7).

Figure 16. A model showing the most possible turning angles for a cell colliding with a haptotactic boundary. A fibroblast cell migrated from the adhesive substrate and met the haptotactic boundary. The elliptic fan-shaped area shows the hypothetical distribution pattern of the sensitivity and the strength of filopodia at the front (large radius = strong and sensitive, small radius = weak and insensitive). When colliding with the boundary, part of this area is blocked by the non-adhesive substrate (red region). The surviving filopodia make up the new strength 38

and sensitivity distribution pattern on the adhesive substrate (green region). The hypothesis shown in Figure 16 holds that the stronger filopodia have the most weight in decision- making. As perceived from the model, the density of the blue lines is in proportion to the cell’s likelihood of re- directing migration from low angle to higher angle.

Figure 17. A fibroblast that hit the haptotactic boundary with nearly 90 degree angle. This cell exceeded the boundary (pointed by arrows) between the adhesive substrate (G = germanium) and the non-adhesive boundary (P = plastic). Its cell mass evenly split into halves and tended to bend back to the germanium. This consistent with literature reports that the forked leading edges represents a competition representing pseudopodia, which results in choice of one side to set a new trajectory.

4.3 Further study of filopodia sensitivity distribution from angular data

In my study, I noticed that most of the cells preferred to migrate along the haptotactic boundary rather than leave and migrate into the germanium. It was consistent with the hypothesis that the cell would compare the two substrates and pick a side according to the

sensory signaling from the filopodia. Furthermore, my observation shows cells particularly

preferred to reflect from the boundary with small angles suggesting the uneven sensitivity to the

cues. As I discuss above in the “vision” analogy, this may be because the strongest filopodia at 39 the front will have the most influence on the migration pattern. To confirm this statement, an angular assessment could be conducted. Using the same method, one could take images of the tracks from cells colliding with the boundary. Then, the angle between the track after turning and the haptotactic boundary for all cell samples would be collected. From the distribution of the sample in a range from 0 (inclusive) to 90 degree (exclusive), we would be able to model the strength of the filopodia in the front edge. From the model, we can develop a test whether the filopodia sensing is more concentrated in the middle and gradually getting weakened on the sides.

This hypothesis does not, however, account fully for the role of displacements of cell mass in redirecting the path of locomotion.

40

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