Microglia podosomes:

Characterization, Ca2+ regulation and potential role in migration

by

Tamjeed Ahmed Siddiqui

A thesis submitted in conformity with the requirements

for the degree of Master of Science

Graduate Department of Physiology

University of Toronto

© Copyright by Tamjeed Ahmed Siddiqui 2012

Microglia podosomes: Characterization, Ca2+ regulation and potential role in

migration

Tamjeed Ahmed Siddiqui

Master of Science

Graduate Department of Physiology University of Toronto

2012

ABSTRACT

Microglia, immune cells of the central nervous system, activate in response to

pathophysiological stimuli. One of their reactive phenotypes is to migrate to site

of injury where they could have either beneficial or detrimental effects. However,

little is known regarding the mechanisms underlying microglial migration and how

they traverse the unique extracellular environment in brain tissue to reach their

destination. Our laboratory first discovered that microglia express structures

called podosomes, which can adhere to as well as degrade extracellular matrix.

In this study, I further characterize microglial podosomes, and show that they

associate with Iba1, Orai1 and calmodulin, proteins not yet observed in

podosomes of other cell types. I also present evidence that podosome formation

depends on Ca2+ and its entry through store-operated Ca2+ channels. The

findings in this thesis contribute to a better understanding of podosome dynamics

and their probable roles in microglia migration.

ii

ACKNOWLEDGEMENTS

I would like to begin by expressing my gratitude towards my supervisor, Dr

Lyanne C Schlichter. Her support, dedication, enthusiasm and advice among

many other attributes made working with her an educational yet fun experience.

Throughout my master’s program, she taught me many valuable lessons that

allowed me to evolve and mature into a better scientist. Dr Schlichter continues

to be a wonderful mentor and I am looking forward to working with her.

I extend thanks to my committee members, Dr Milton Charlton and Dr

Rene Harrison, and examination committee members, especially Dr Christopher

McCulloch, for their time and valued suggestions.

My gratitude also goes to my peers at the lab as well as staff members at

the department who always helped me with any inquiries or issues that I would

face. Specifically, Iska and Star for being patient and kind enough to discuss

topics with me and review my writing. Baosong’s invaluable help with so many

experiments and techniques as well as wonderful “thought experiments” that he

came up with. To Cat, for taking the time to teach me immunocytochemistry

techniques and imaging and Roger being a good person to bounce ideas off of. I

would especially like to thank Xiaoping Zhu. With an infectious smile, she has

been a trusted and reliable colleague, a wonderful friend and someone I can

always count on. I am truly indebted to her for everything she has done and for

teaching me so many techniques.

Special thanks go to my parents for their self-sacrifice to send their

teenage son to Toronto to ensure I received one of the best educations available.

My mother, who provides endless love to the family, taught me that through hard

work anything can be achieved and to never give up. My father, who has always

been patient and understanding, taught me to be honest, tolerant and have an

open mind. They continue to be my role models and are the architects of my

personality. Thanks to both my brothers as well, my partners in crime, for always

being there and sharing the good times with me.

iii

To my one and only Kiruththiga Sures, my companion, my best friend, my

love. Like an angel, she has always been by my side through thick and thin,

always encouraging and motivating me to strive for the best and never settle for

less. She has been so caring, patient and understanding for the countless

number of times that I was late (and after hearing every excuse in the book that I

could imagine). I may never be able to repay her for everything she has done for

my family and especially for me. Thank you.

iv

TABLE OF CONTENTS

Abstract ii

Acknowledgements iii

List of Figures vi List of Tables viii Introduction

Cell migration 1

Invadosomes 4

Introduction to Monocytic Immune Cells 10

Microglia – Immune cells of the CNS 12

Podosomes in microglia 16

Thesis Objectives 20

Materials and Methods

Microglia culture 21

Immunocytochemistry (ICC) 22

Ca2+ and podonuts (podosomes) 24

Image Analysis 26

Results

Previously known podosome components found in microglial podosomes

29

Ca2+ and podosome (podonut) formation 36

Novel expression of several molecules in microglia podosomes 42

Discussion 48

Working Model 59

Future studies 64

References cited 66

v

LIST OF FIGURES

Figure 1. Schematic diagram of migrating cell (Chhabra and Higgs, 2007)

Figure 2. (Top) Electron micrograph showing distinct core and ring structural

arrangement in smooth muscle cell podosome. (Bottom) Schematic diagram

showing localization of common podosome-associated molecules

Figure 3. Schematic diagram illustrating development of immune cells in blood and CNS

Figure 4. Primary rat microglia express podosomes

Figure 5. Functional podosomes expressed in microglia with SK3 localized in podosome

core

Figure 6. Microglia in vitro express podonuts after 20 hr in culture

Figure 7. Heat induced antigen retrieval (HIAR) enhances antibody specificity for

immunogen

Figure 8. Illustration of fluorescence intensity normalization of immunocytochemistry

images

Figure 9. Phosphotyrosine staining show localization to microglial podosomes

Figure 10. Immunostaining of Tks5 show localization to podosome ring in microglia

Figure 11. Phospho-caveolin-1 (pCav1) localizes to podosome ring in microglia

Figure 12. Immunolabeling of Nox1 shows localization to podosome ring in microglia

Figure 13. Ca2+ plays crucial role in podosome (podonut) formation

Figure 14. Importance of Ca2+ for podonut formation

Figure 15. Podosome (podonut) formation required Ca2+ entry through CRAC channels

Figure 16. Blocking CRAC channels results in loss of podosome (podonut) distribution

Figure 17. Immunolabeling of Orai1 show localization to podosome core in microglia

vi

Figure 18. Immunostaining of calmodulin (CaM) mostly shows localization to podosome

ring in microglia

Figure 19. Iba1 staining show localization to core in microglial podosomes

Figure 20. Immunolabeling of TRPM7 and podosome core in microglia

Figure 21. Updated schematic diagram showing podosome-associated molecules in

microglia based on findings in this thesis

vii

LIST OF TABLES

Table 1. Components that constitute podosomes, invadopodia and focal adhesions

Table 2. Summary of podosome components found in microglia and their corresponding

localization

viii

INTRODUCTION

Cell Migration

Cell migration is an important phenomenon that aids in various processes like

development of tissues, immune system function, angiogenesis and wound healing (Alberts

et al., 2008). It can also have a negative influence in pathological processes like tumour

metastasis. Cell migration is the result of a complex interplay between various intracellular

signalling pathways (e.g. Rho family GTPases, Cdc42, protein tyrosine kinases)(Ridley et al.,

2003; Alberts et al., 2008). Most of our understanding comes from studies based on different

cell types such as neutrophils, Dictyostelium, and fibroblasts (see reviews Lauffenburger and

Horwitz, 1996; Weiner, 2002; Le Clainche and Carlier, 2008; Vicente-Manzanares et al.,

2009). It is important to note that although cell migration is a well-studied phenomenon,

detailed mechanisms vary between different cell types and species (Becchetti and Arcangeli,

2010). Migration can be classified into two basic forms: random migration and directed

migration (Ridley et al., 2003; Gilbert, 2006). Random migration (chemokinesis) involves

intrinsic factors being activated that initiate migration without responding to any external

cues, such that cells migrate spontaneously in any direction. Directed migration, however,

adds more complexity to the intricate processes underlying cell migration. Migration

following an external cue, also known as chemotaxis, involves not only regulating the

migration machinery but also responding to the external stimuli via receptor signalling that

continually influences the migration machinery. In either case, cell migration is initiated by

inducing cell polarity and extension of the cell membrane and cytoplasm in the direction of

migration (Ridley et al., 2003). This establishes a leading edge at the cell front and a uropod

at the rear. The leading edge is the site of extensive assembly of cytoskeleton, mainly actin

1

polymerization, protruding lamellipodia and a lamellum (Figure 1). Lamellipodia are sites of

rapid actin polymerization and protrusion that form membrane ruffles that have not yet

adhered to substrate. The lamellum, on the other hand, is located dorsal to lamellipodia, and

provides stability and traction by adhering to the extracellular matrix (ECM). The uropod in

the trailing end of migrating cells is a site of disassembly of cytoskeletal elements like

microtubules, and of retraction and detachment from ECM that allows cells to move forward.

In addition to signalling pathways that regulate cytoskeletal rearrangements,

regulation of intracellular Ca2+ is vital for cell migration (Evans and Falke, 2007). There is

evidence that many migrating cells maintain a descending Ca2+ gradient from rear-to-front of

the cell (Brundage et al., 1991; Laffafian and Hallett, 1995; Xu et al., 2004; Hoffmann et al.,

2009). Removal of extracellular Ca2+ impedes or stops cell migration, illustrating the

importance of local-, global- or spatiotemporally regulated Ca2+ concentrations during

migration (Brundage et al., 1991; Mandeville et al., 1995). Although it is believed that

migrating cells maintain a relatively low Ca2+ concentration at the cell front, Wei and

colleagues showed that directed migration is the result of localized Ca2+ rises in

microdomains (termed Ca2+ flickers) at cell front, which are short-lived and depend on

chemoattractant receptor signalling and membrane stretch (Wei et al., 2009). The authors

suggest that having a low Ca2+ background helps maintain a chemical driving force for Ca2+

entry through Ca2+ permeable channels at the cell front for subsequent initiation of Ca2+

signalling cascades necessary to steer the cells in a particular direction.

2

Figure 1. Schematic diagram of a migrating cell (Chhabra and Higgs, 2007).

Reprinted by permission from Macmillan Publishers Ltd: Nature Cell Biology. Chhabra ES, Higgs HN. The

many faces of actin: matching assembly factors with cellular structures. Copyright 2007

3

As important as regulation of signalling pathways and Ca2+ is to cell migration,

migrating cells also need to remodel the matrix in order to traverse through tissues (Alberts,

2009), which are made up of many cells that are held together by cell-cell interactions (e.g.

tight junctions) as well as cell-ECM contacts (Alberts, 2009). The ECM surrounding cells

serves not only as “glue” that allows cells to adhere for anchorage, but provides additional

support to keep the cells bound together. Characteristics of ECM in different tissues also

differ (Alberts, 2009); the components that make up the overall matrix are variable, leading

to differing rigidity, organization and fluidity. ECM typically consists of fibrillar collagens,

laminin and fibronectin, along with proteoglycans, glycosaminoglycans (GAGs) and

glycoproteins. The brain ECM, however, is very different from peripheral tissues, in that has

very little collagen and other commonly found ECM proteins (Bellail et al., 2004). Instead,

brain ECM primarily consists of glycosaminoglycan hyaluronan, proteoglycans, some of

which are specific for brain ECM (e.g. neurocan), and glycoproteins like tenascin-C. The

highly anionic hyaluronan, which is the major brain ECM component, binds hygroscopic

molecules (e.g., the cations, Na+ and Ca2+) and water (Bellail et al., 2004). The brain matrix

is water-rich and lacks rigid fibrillar collagens, making it softer than the ECM in other tissues

(Bellail et al., 2004). Taken together, this information means that cell migration through the

brain will require that cells degrade a specialized extracellular matrix and navigate through

an environment where cells are densely packed.

Invadosomes

Cell adhesion to the ECM substrate is also important for cell stability, anchorage and

traction during migration. Cell adhesion structures are sites where cells attach to the

4

underlying substratum. Focal complexes and focal adhesions are well-documented and

extensively studied adhesion structures found in many cell types, which under some

circumstances provide traction for cell migration. Integrins at the cell-ECM contact sites are

adhesion molecules that mediate communication with the cell regarding its interaction with

the ECM, and influence many signalling cascades, including cytoskeletal rearrangements

(Caswell et al., 2009). In the 1980s, a new type of adhesion structure was discovered: the

‘podosome’ (David-Pfeuty and Singer, 1980; Wolosewick, 1984; Tarone et al., 1985; Linder

and Aepfelbacher, 2003), which was later proposed to play a role in tissue invasion.

Monocyte-derived cells (e.g. macrophages) (Messier et al., 1993), constitutively active Src-

transformed cell lines (David-Pfeuty and Singer, 1980; Tarone et al., 1985; Abram et al.,

2003), endothelial cells (Osiak et al., 2005), and vascular smooth muscle cells (Burgstaller

and Gimona, 2005) are now known to express podosomes. Invasive cancer cells (e.g. human

breast cancer) (Mandal et al., 2008) possess similar structures, which were named

‘invadopodia’ (the combined term, invadosomes, is often used for invadopodia and

podosomes) (Murphy and Courtneidge, 2011). Podosomes were recently discovered in vivo

in transforming growth factor beta (TGFβ)-stimulated endothelial cells in mice (Rottiers et

al., 2009) and also in 3D cultures of macrophages in vitro (Rottiers et al., 2009; Cougoule et

al., 2010) .

Because many components of invadosomes are also present in other cell-ECM

adhesion structures, it was initially difficult to distinguish invadosomes, and determine

whether they are indeed discrete adhesion structures. It is now clear that they exhibit some

morphological, protein composition, and functional similarities and differences (reviewed in

(Block et al., 2008). Both podosomes and invadopodia are F-actin rich structures that

5

displays a unique ring and core structural arrangement (Figure 2) (Gimona, 2008). The ring

seems to have an adhesive nature and contains most of the integrins and adhesion-associated

molecules (e.g. talin, vinculin, paxillin); whereas, the core is rich in F-actin, and actin-

regulating molecules like Arp2/3 complex, WASP, and cortactin. Besides these cytoskeletal

regulators, other molecules have also been detected in invadosomes (Table 1) (Block et al.,

2008). Src tyrosine kinase signalling, a common signalling pathway involved in many

cellular processes including cell migration, is pivotal for the formation and regulation of

invadosomes (Linder and Aepfelbacher, 2003). Immunostaining for tyrosine phosphorylated

proteins shows localization to both the core and ring, suggesting their presence throughout

the structure. Unlike other adhesion structures, invadosomes can also degrade matrix due to

their association with matrix metalloproteinases (MMPs), and ADAMs (‘a disintegrin and

metalloproteinase’). There are several differences between the two subtypes of invadisomes.

Invadopodia have high degradative capability due to their slow turnover (>1 hr), whereas

podosomes are much more dynamic with turnover rates between 2 and 20 min, and shallower

matrix degradation. Invadopodia are larger structures (~5-8 µm in diameter) compared to

podosomes (~0.5-1 µm). Invadopodia are also sparsely distributed in cells (<10) while

podosomes are more compact and numerous (~20-100) (Murphy and Courtneidge, 2011).

Podosomes are protrusive adhesion structures primarily found at the ventral side of

polarised cells of the monocytic lineage, such as macrophages (Messier et al., 1993),

immature dendritic cells (Binks et al., 1998), and osteoclasts (Zambonin-Zallone et al., 1988;

Calle et al., 2006). Due to their adhesive properties, high turnover rates, and degradative

capacity, podosomes are well designed to aid highly migratory cells like macrophages in

traversing barriers like tissue layers (Carman et al., 2007). Indeed, when podosomes were

6

discovered, the initial opinion was that they functioned in transcellular migration

(Wolosewick, 1984).

7

Figure 2. (Top) Electron micrograph showing the distinct core and ring arrangement in a smooth muscle cell podosome (Gimona, 2008). (Bottom) Schematic diagram showing localization of common podosome-associated molecules (Linder and Aepfelbacher, 2003). Reprinted from The Lancet, Vol. 18, Gimona M, The microfilament system in the formation of invasive adhesions, 23-34, Copyright (2008), with permission from Elsevier Reprinted from The Lancet, Vol. 13, Linder S, Aepfelbacher M, Podosomes: adhesion hot-spots of invasive cells, 376-385, Copyright (2003), with permission from Elsevier

8

Table 1. Components that constitute podosomes, invadopodia and focal adhesions (Block et

al., 2008). Reprinted from The Lancet, Vol. 87, Block MR, Badowski C, Millon-Fremillon A, Bouvard D, Bouin AP,

Faurobert E, Gerber-Scokaert D, Planus E, Albiges-Rizo C, Podosome-type adhesions and focal adhesions, so

alike yet so different, 491-506, Copyright (2008), with permission from Elsevier

9

Introduction to monocytic immune cells

The immune system is a complex defensive array made up of several cell types with

different phenotypes (Alberts et al., 2008). All immune cells recognise and react to protect

the host from many pathophysiological circumstances, such as cancer, infection, and injury

(Kindt et al., 2007). Blood immune cells develop from a common hematopoietic stem cell,

through a process called hematopoiesis that can give rise to several blood cell types (Figure

3)(Kindt et al., 2007). One type of progenitor immune cell, the granulocyte-monocyte

progenitor cell (Kindt et al., 2007), can give rise to monocytes that can be released into the

blood circulation from the bone marrow. Circulating monocytes are undifferentiated immune

cells that can migrate into various tissues (a process called extravasation) to differentiate into

resident macrophages (as reviewed in Gordon and Taylor, 2005; Kindt et al., 2007). Gordon

and Taylor, in their review, highlight that these tissue macrophages are of heterogeneous

phenotypes, forming subpopulations with unique functions within their corresponding

microenvironments. For example, osteoclasts have the ability to remodel bone tissue,

alveolar macrophages in lungs remove debris and pathogens due to high expression of

pattern recognition receptors and scavenger receptors, and microglia in the adult central

nervous system (CNS) predominantly represent the innate immune system.

In this thesis, I investigated several physiological aspects of rat microglia in vitro.

10

Figure 3. Schematic diagram illustrating development of immune cells in blood and CNS (Ransohoff and Cardona, 2010) Reprinted by permission from Macmillan Publishers Ltd: Nature. Ransohoff RM, Cardona AE. The myeloid cells of the central nervous system parenchyma. Copyright 2010

11

Microglia – Immune cells of the CNS Microglia are the resident sentinels of the CNS. Recently it has been shown that

microglia precursor cells of monocyte-lineage develop from mesodermal hematopoietic

cells–originating from the yolk sac– that enter and remain in the developing fetal brain (as

reviewed in Chan et al., 2007). It has been suggested that peri-natally and post-natally,

circulating monocytes in blood can also infiltrate into brain parenchyma (Figure 3)(reviews

Chan et al., 2007; Ransohoff and Cardona, 2010; Kettenmann et al., 2011). These cells then

proliferate, migrate to different regions of the brain and differentiate into microglial cells.

Microglia reside in an immune privileged environment, due to the blood brain barrier (BBB)

that separates the brain anatomically and physiologically, and strictly regulates substances

that cross the barrier (Alberts et al., 2008). Microglia are thought to form 5% to 20% of the

total glial cell population in the CNS (Kaur et al., 2010).

During post-natal development, microglia further differentiate within the brain, and

enter a quiescent state with a ramified morphology (Schlichter et al., 2010; Kettenmann et al.,

2011). In this non-activated state, microglia are non-migratory but have many processes

extending from the soma that are motile, constantly monitoring their environment through

pinocytosis, and interacting with other cells in CNS (Davalos et al., 2005; Nimmerjahn et al.,

2005; Kaur et al., 2010). Microglia respond first to perturbations of CNS homeostasis

(Miyake et al., 1988; Schilling et al., 2003). In response to a pathophysiological event,

microglia undergo a complex transformation process from the non-activated “resting” state to

“activated”. Once activated, microglia can exhibit one or more new phenotypes; e.g.,

increased proliferation, migration, phagocytosis, production of interleukins, cytokines and

chemokines (Hanisch and Kettenmann, 2007; Kaushal et al., 2007; Schlichter et al., 2010;

Kettenmann et al., 2011). The outcome of the microglial response is dependent on its

12

activation state, i.e., microglia react differently depending on the stimuli, factors released by

surrounding neuronal/glial tissue, and the pathophysiological context (Schwartz et al., 2006;

Carson et al., 2007; Colton, 2009; Kettenmann et al., 2011). Microglia are sometimes

referred to as brain macrophages, and can exhibit a wide range of immune functions that are

similar to peripheral macrophages; e.g., phagocytosis, free radical production, secretion of

chemokines and cytokines, and communication with rest of the immune system (Tambuyzer

et al., 2009). However, unlike macrophages, the response of activated microglia can be

immunologically silent, in which a system-wide immune reaction is not activated (Galea et

al., 2007). Along with the versatile nature of microglial activation, there is evidence of

microglial involvement in all neuropathologies (Kreutzberg, 1996; Streit et al., 2005; Block

et al., 2007; Hanisch and Kettenmann, 2007; Davoust et al., 2008; Colton and Wilcock, 2010;

Graeber and Streit, 2010; Kaur et al., 2010).

This thesis will focus on one of the reactive microglial phenotypes, which involves

cells migrating to sites of injury (e.g. to the lesion after intracerebral hemorrhage or ischemic

stroke) (Brockhaus et al., 1996; Zhang et al., 1997; Schlichter et al., 2010). Shortly after

stroke, our lab and other groups have shown that microglia/macrophages respond to the

injury by entering an activated state that includes a change in morphology from ramified to

amoeboid (Brockhaus et al., 1996; Zhang et al., 1997; Wasserman et al., 2008; Moxon-Emre

and Schlichter, 2010). Microglia/macrophages were then observed to progressively migrate

toward the damaged core from surrounding tissue, reaching maximal infiltration by 7 days

after both forms of stroke. Functionally, microglial migration can be either beneficial or

detrimental (Tambuyzer et al., 2009). For instance, beneficial effects include removal of

damaged cells and debris by phagocytosis, and secreting neurotrophic factors that aid in

13

repair processes. It has even been suggested that activated microglia secrete soluble factors

that attract neural precursor cells to the site of injury where they can differentiate to produce

neurons and/or glial cells that aid in the repair process (Aarum et al., 2003). On the other

hand, microglial activation and migration can also be detrimental and cause secondary

damage after the initial insult; e.g., due to production of highly reactive oxygen species

(ROS) and nitric oxide-derived species like peroxynitrite (see review Boje and Arora, 1992;

Kettenmann et al., 2011). Furthermore, production of inflammatory factors, such as

interleukins and tissue necrosis factor (TNF-α), can cause additional neuro-inflammatory

damage that propagates from the site of injury to surrounding healthy tissue (see reviews

Chakraborty et al., 2010; Kettenmann et al., 2011).

Although microglial migration to the site of injury is a well-documented response that

is common to most of its reactive phenotypes, the mechanisms have not been studied

extensively. During development, microglia migrate to different regions of the brain,

(investigated mostly in quails), with migration tangentially at first, and then radially (Cuadros

et al., 1994; Cuadros et al., 1997; Marin-Teva et al., 1998; Rezaie and Male, 1999;

Navascues et al., 2000; Sanchez-Lopez et al., 2004). In their non-activated, quiescent,

ramified state, microglia motility is mainly seen as extension of the many processes arising

from their somata, without overall cell displacement (Davalos et al., 2005; Nimmerjahn et al.,

2005). However, factors like ATP released from necrotic cells in the damaged brain act as

chemo-attractants, activate microglia and alter their morphology to the amoeboid form

(Honda et al., 2001), which is associated with the migratory phenotype (Kettenmann et al.,

2011).

ATP allows Ca2+ entry through receptor signalling via metabotropic P2Y receptors or

14

ionotropic P2X receptors that modulate intracellular pathways, such as PI3K/Akt signalling

(Ohsawa et al., 2007; Lalo et al., 2008). Some other physiological factors that activate and

attract microglia include ectonucleotidase-derived adenosine (Farber et al., 2008), glutamate

(Kim and Ko, 1998; Liu et al., 2009), chemokines (Biber et al., 2001; Liang et al., 2009),

bradykinin (Ifuku et al., 2007), growth factors (Bonifati and Kishore, 2007; Kettenmann et

al., 2011) and beta-amyloid (Stuart et al., 2007). There is evidence that signalling pathways

such as PI3K/Akt, Erk, non-receptor tyrosine kinases (e.g. Pyk2) are involved in microglial

migration (see review Kettenmann et al., 2011). Some ion channels proposed to be involved

in microglial migration are stretch-activated Ca2+ permeable channels, Ca2+-activated

potassium channels, the reversed mode of the Na+/Ca2+ exchanger (NCX), and Cl- channels

(see review Kettenmann et al., 2011). It is interesting that many of the molecules thought to

influence microglial migration are involved in Ca2+ signalling cascades; which suggests a

conserved, vital role for Ca2+ in migration and an important contribution of intracellular Ca2+

regulation (Evans and Falke, 2007), and a complex interplay between ligands, receptors, ion

channels, and the cytoskeleton. Microglia also need to migrate through the unique brain

ECM and tightly packed dense brain tissue without causing damage to normal cells. In this

context, there is very little known about migration mechanisms in microglia.

15

Podosomes in microglia

With Dr. Schlichter, a former graduate student, Catherine Vincent, discovered that

untreated microglia cells grown on glass cover slips show dot-like structures that are

abundant in F-actin, and enriched in the lamella region. Further investigation showed that

these structures possessed a ring pattern of talin staining and F-actin cores, and had

podosome-like dimensions, suggesting that microglia express podosomes in vitro (Figure 4).

Podosomes in macrophages also seem to be localized at the leading edge (Evans et al., 2003).

In addition, microglial podosomes often formed a larger structure that we called a ‘podonut’,

which is made up of many individual podosomes in an open ring formation. Functional

studies showed these structures have the ability to degrade fibronectin, a common non-brain

specific ECM substrate (Figure 5), which is a well-known function of podosomes (Chen et

al., 1985; Seals et al., 2005). Podonuts were observed after microglia were in culture for 20

hours (Figure 6) and formed at the cell-substrate attachment interface. It was also discovered

that SK3, a Ca2+ activated K+ channel, localized to the core of podosomes (Figure 5). In

preliminary studies (Vincent and Schlichter, 2010), blocking SK3 channel activity did not

affect podonut/podosome formation so its function in relation to these structures still requires

further investigation.

Activated microglia that are undergoing migration in the damaged CNS could benefit

from expression of these microscopic structures (podosomes), to provide the means for

localized degradation of brain ECM and to navigate through dense CNS tissue to reach the

site of injury.

16

A B

Figure 4. Primary rat microglia express podosomes. (A) Microglia immunolabeled for the

podosome core marker, Arp2 (red), and the podosome ring marker, talin (green), and the cell

nucleus (DAPI; blue) (B) Microglia immunolabeled for the podosome ring (talin; red), F-

actin in the core (phalloidin; green), and the nucleus (DAPI; blue). Boxes represent areas

chosen for higher magnification and colour separated on the right. Arrows show the

podosome ring and core structural arrangement. Scale bars = 5 µm. (Images from (Vincent

and Schlichter, 2010)

17

Figure 5. Functional podosomes expressed in microglia, with SK3 localized in podosome

core. Microglia stained for SK3 (red) show that it co-localizes with the podosome core

marker, F-actin (phalloidin; blue). Images on the right are the boxed areas, magnified and

colour separated. F-actin dots co-localize with regions of degradation of FITC-labeled

fibronectin (green). Arrowheads show co-localization of SK3, F-actin and fibronectin-

degraded spots. Scale bar = 5 µm. (Images from (Vincent and Schlichter, 2010)

18

Figure 6. Microglia in vitro express podonuts at 20 hr in culture. Images were acquired after

cells were in culture for the indicated number of hours; labelled for F-actin (phalloidin; red),

a podosome core marker. The large actin rings in the lamella region (podonuts) appeared at

the 20 hr time point. Scale bar = 20 µm. (Images from (Vincent and Schlichter, 2010)

19

Thesis Objectives

Podosome components are fairly conserved among different cell types; however,

some reports have shown apparent differences in their molecular make up. For example, in

smooth muscle cells, SM22α protein was shown to associate with the podosomal core

(Gimona et al., 2003). In dendritic cells, there is evidence that podosome assembly occurs in

a gelsolin-independent manner (Hammarfjord et al., 2011); contrary to the requirement for

gelsolin in osteoclasts (Ma et al., 2008; Crowley et al., 2009; Ma et al., 2010; Van Goethem

et al., 2010). With our lab first discovering podosomes in microglia (Vincent and Schlichter,

2010), and given that podosome components could vary in different cell types, I wanted to

further characterize podosomes in microglia using immunostaining and some functional

studies. In this thesis, I show that although some components are conserved between

podosomes in microglia and other cells; I have identified some molecules not previously

shown to associate with podosomes. Then, because Ca2+ plays a key role in cell migration,

and SK3 —a Ca2+ responsive channel— was found in the podosome core in microglia

(Vincent and Schlichter, 2010), I hypothesized that the regulation of podosomes is dependent

on Ca2+. Overall, the findings in this thesis contribute toward a better understanding of

podosomes, their features in microglia, and their Ca2+-dependence.

20

MATERIALS AND METHODS

Microglia cell cultures. Giulian and Baker (1986) were the first to develop

isolation and culturing protocols for microglia. The Schlichter lab published it’s first

microglia paper in 1996, and since then has refined the protocol such that after isolation,

neonatal rat microglia remain in a relatively resting state, as judged by very low expression

of many inflammatory mediators (Sivagnanam et al., 2010). Isolation of rat microglia was

done by Dr. Schlichter’s technician, Xiaoping Zhu, following standard protocols as described

previously (Kaushal et al., 2007; Ohana et al., 2009; Sivagnanam et al., 2010). Briefly,

primary microglia cultures were derived from 1 to 2 day old Sprague-Dawley rat pups. After

removing the meninges, the brain was dissected and mashed through a stainless steel sieve in

Minimum Essential Medium (MEM; Invitrogen, Burlington, Canada), and then centrifuged

at 1000g for 10 min. The supernatant was removed and the pellet was re-suspended in MEM

supplemented with 10% fetal bovine serum (FBS; Wisent, St-Bruno, Canada) and 0.05

mg/ml gentamycin (Invitrogen), and plated on flasks to maintain at 37ºC, 5% CO2

atmosphere. After 48 hr, cellular debris, non-adherent cells and supernatant were removed

and fresh medium was added. The mixed cell cultures were then maintained for another 5 to

6 days. Microglial suspensions were then obtained by shaking the mixed cultures on an

orbital shaker for 3-4 hours in 37ºC at 60 to 65 rpm. For my experiments, the suspension

containing microglial cells was then centrifuged for 10 min at 1000g and the cell pellet was

re-suspended in MEM supplemented with 2% FBS and 100 µM gentamycin. Cells were

plated at 60,000 cells per 15 mm diameter glass cover slip, incubated in the 2% FBS

supplemented MEM at 37ºC, 5% CO2, and used within 24 to 48 hr. The Schlichter lab has

repeatedly shown that primary microglia cultures obtained with this protocol are ≥99% pure,

21

based on labelling with several microglia markers: FITC-conjugated tomato lectin, or

antibodies against isolectin B4, ionized Ca2+ binding adaptor-1(Iba1) or CD11b (OX-42)

(Kaushal et al., 2007; Ohana et al., 2009; Sivagnanam et al., 2010).

Chemicals and antibodies. I used the channel blockers: 2-aminoethyl

diphenylborinate (2-APB; Sigma-Aldrich, Oakville, Canada), and spermine

tetrahydrochloride (Calbiochem, San Diego, CA). Several antibodies were used: rabbit

polyclonal anti-phosphotyrosine-14-caveolin 1 (Signalway Antibody; #11090, Pearland, TX),

goat polyclonal anti-Orai1 (Santa Cruz Biotechnology; sc-74778, Santa Cruz, CA), rabbit

polyclonal anti-SK3 (Alomone labs; APC-025, Jerusalem, Israel), rabbit monoclonal anti-

calmodulin (Abcam; ab45689, Cambridge, MA), rabbit polyclonal anti-Arp2 (Santa Cruz

Biotechnology; sc-15389), mouse monoclonal anti-talin 1/2 (Abcam; ab11188), rabbit

polyclonal anti-Tks5 (Santa Cruz Biotechnology; sc-30122), mouse monoclonal anti-

phosphotyrosine (Abcam; ab10321), rabbit polyclonal anti-Iba1 (Wako Chemicals; #019-

19741, Richmond, VA) and goat polyclonal anti-Nox1 (Santa Cruz Biotechnology; sc-5821).

Immunocytochemistry (ICC). Cover slips bearing microglia were fixed in 4%

paraformaldehyde (PFA) (Electron Microscopy Sciences, Hatfield, PA) in PBS for 10 to 15

min at room temperature, and then permeabilized for 5 min with 0.2% Triton X-100. To

block non-specific staining, cells were incubated in blocking solution (4% donkey serum in

PBS; Jackson Immunoresearch, West Grove, PA) for 1 hr at room temperature. The blocking

solution was replaced and the cells were then incubated overnight at 4ºC with a primary

antibody in blocking solution. After another 1 hr of blocking, a secondary donkey antibody in

22

blocking solution (conjugated to Dylight 488 or Dylight 594; 1:250; Jackson

Immunoresearch) was added to label the corresponding primary antibody, and incubated for

1 hr at room temperature in the dark. F-actin was visualized by incubating cells with Alexa

488-conjugated phalloidin (1:50 in block solution, Invitrogen) for 15 min at room

temperature in the dark. Cell nuclei were stained by incubating with 4',6-diamidino-2-

phenylindole (DAPI; 1:3000 in PBS) for 5 min. Cover slips were mounted on glass slides

with mounting medium (Dako, Glostrup, Denmark) for viewing using epifluorescence

widefield microscopy. Negative controls were prepared for each ICC preparation using the

aforementioned protocol, except that primary antibodies were omitted. Before use, antibodies

in blocking solution were centrifuged at 10,000 rpm for 10 min to remove any aggregates

that might bind non-specifically and introduce artefacts.

For some proteins, heat induced antigen retrieval (HIAR) was required (see example

in Figure 7). That is, when paraformaldehyde fixation immobilizes proteins by chemical

cross-linking, an unwanted side-effect can be that an epitope normally recognized by a

primary antibody becomes masked by the cross-linking (Fox et al., 1985; Mason and

O'Leary, 1991; Dapson, 1993). With HIAR, the chemical masking is essentially removed,

permitting the normal interaction between the antibody and corresponding epitope on the

protein of interest. HIAR was performed before the cell permeabilization step, using a

sodium citrate-based buffer that contained 10 mM trisodium citrate with 0.05% Tween-20

adjusted to pH 6.0 with HCl. The slides were heated in a microwave oven for 4 to 5 min,

with excess citrate buffer to prevent samples from drying, and then allowed to cool to room

temperature before being washed with PBS. Although HIAR is a useful technique to reduce

the undesirable effect of formaldehyde-based fixation, it is challenging, and results can be

23

inconsistent. During my thesis research, I tried several antigen retrieval techniques (e.g.

hydrochloric acid-based, Tris-EDTA-based) and found that citrate-based HIAR yielded the

best results.

Some antibodies did not stain after formaldehyde fixation and/or HIAR. In those

cases, I fixed the microglia with cold methanol, which precipitates proteins by dehydration.

Methanol (HPLC grade) was pre-chilled overnight at -40ºC, and then microglia on cover

slips were incubated for 5 min at -20ºC. Because this fixation technique does not require cell

permeabilization, the immunostaining protocol continued from the first blocking step. Note

that the methanol-fixed cells could not be labelled for F-actin using phalloidin (North, 2006).

Ca2+ and podonuts (podosomes). After microglia were cultured for 24-48 hr,

the tissue culture medium was replaced with either standard bath solution containing (in

mM): 125 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 5 D-glucose and 10 HEPES, adjusted to pH 7.4

(with NaOH) and 285-300 mOsm with sucrose; or Ca2+-free bath solution (prepared by

omitting CaCl2 and adding 1 mM EGTA). Bath solutions were sterilized by filtering through

0.2 µm filters, and all treatments were performed at 37°C. Prior to fixation of treated cells for

immunocytochemistry, cells were washed once with sterile PBS.

24

Figure 7. Heat induced antigen retrieval (HIAR) enhances antibody labelling of the

immunogen. Lower panels: Microglia immunostained for calmodulin (CaM; red), the

microglia marker, tomato lectin (TL, green), and the nucleus (DAPI; blue) show very low

CaM staining and nuclear localization. Upper panels: HIAR-treated microglia show an

improved staining pattern: higher CaM staining intensity, diffuse cytoplasmic localization,

and little nuclear staining. Scale bars = 20 µm (Images from (Vincent and Schlichter, 2010)

25

Image analysis. Microglia were imaged with a Zeiss Axioplan 2 widefield

epifluorescence microscope (Zeiss, Toronto, ON) equipped with a Zeiss Axiocam HR digital

camera. Images were then analyzed and deconvolved using Zeiss Axiovision 4.7 software. I

found that deconvolution was very helpful in analyzing the tiny podosome structures. Images

obtained from widefield microscopes are convoluted due to the thick optical section, in

which the microscope acquires light from the point source (fluorochrome), and from

peripheral ambient light emitting around the fluorochrome. The resulting image is a product

of the true image and the additional noise, which is defined by the point spread function

(PSF); i.e., the additional noise from a source of light above and/or below the plane of focus

(Campisi and Egiazarian, 2007). Deconvolution algorithms reduce the noise and distortions

introduced during image acquisition by using information about the optical system (e.g. type

of objective lens, refractory index of the immersion medium, PSF) (reviewed in Campisi and

Egiazarian, 2007). If the PSF function is unknown, blind deconvolution can be used, in

which the algorithm uses theoretical point-spread functions.

For images to be deconvolved after immunostaining, I acquired high magnification

epifluorescence images through the z-axis at 200 nm increments (z-stacks). The start-to-end

z-stacks corresponded to focal planes that included the entire cell volume. The same

microscope- and acquisition settings were used to obtain images of negative control cover

slips to detect autofluorescence and background signal from non-specific binding of each

fluorophore-conjugated secondary antibody (Figure 8). To remove artefacts introduced

during the staining procedures, this background signal was subtracted when acquiring z-stack

images, which were subsequently deconvolved using a theoretical PSF and the constrained

iterative maximum likelihood deconvolution algorithm in Axiovision 4.7. Automatic z-stack

26

correction was used to compensate for the decay of fluorescence signal due to

photobleaching, because the same sample was imaged along the z-axis numerous times to

acquire the z-stacks. For illustration, an image was extracted from the z-stack in which

podosomes were in focus.

Statistical Analysis. Quantitative data are presented as mean ± standard error of

the mean (SEM). One-way ANOVA statistical test was performed followed by Tukey’s

analysis. Results were considered significant if p<0.01.

27

Original Image Negative Control Final Image

Figure 8. An example showing subtraction of background fluorescence from

immunocytochemistry images. The same microscope and image acquisition settings used to

acquire raw images (left) were used to acquire images of negative control samples (middle).

Intensity settings of negative control images were then applied to raw images essentially

removing autofluorescence/background staining to get final immunocytochemistry images

(right).

28

RESULTS

Previously known podosome components found in microglial podosomes

The classical podosome structure consists of a distinct ring and core that differ in

molecular components. In this thesis, talin was used as a marker to identify the podosome

ring, while F-actin or Arp2 were used to identify the core. These markers are commonly used

in the podosome literature (Linder and Aepfelbacher, 2003; Murphy and Courtneidge, 2011).

Src family kinases (SFKs) form a large family of tyrosine kinases involved in

regulating many processes, including angiogenesis, cell proliferation, metastasis, and bone

metabolism (reviewed in Aleshin and Finn, 2010). Src kinase activity is also important for

regulation of podosomes. For instance, expressing constitutively active Src in fibroblasts

causes them to express podosomes (David-Pfeuty and Singer, 1980; Chen et al., 1985;

Tarone et al., 1985; Aleshin and Finn, 2010; Dovas and Cox, 2011); and pharmacological

inhibition of SFKs disrupts podosome formation in macrophages (Linder et al., 2000b;

Cougoule et al., 2005).

As a first test of whether microglial podosomes express similar signalling pathway, I

correlated labelling of some known Src substrates with podosomes. When an anti-

phosphotyrosine antibody was used to label microglia for tyrosine phosphorylated proteins, a

punctate staining pattern was seen at the leading edge of polarized cells. Double labelling

with F-actin and high-resolution imaging showed that phosphotyrosine proteins were

enriched in the podosome core, and in some cases, in the ring-like structure around the F-

actin staining (Figure 9).

29

Figure 9. Phosphotyrosine staining is enriched in microglial podosomes. Colour-separated

images of a representative microglial cell stained for the podosome core marker, F-actin

(phalloidin; green), and phosphotyrosine (pTyr, red), with a merged image shown below. The

boxed area is shown magnified and colour separated on the bottom right. Arrows and

arrowheads show co-localization of F-actin and pTyr. Scale bars = 5 µm n

30

The presence of tyrosine phosphorylated proteins in microglial podosomes suggested

that tyrosine kinase signalling molecules, such as Src, might be involved in podosome

dynamics. To further examine Src substrates in microglial podosomes, cells were

immunolabeled for tyrosine kinase substrate 5 (Tks5): a substrate of Src kinase previously

shown to be enriched in invadopodia and podosomes (Abram et al., 2003; Seals et al., 2005).

It has been suggested that Tks5 acts as an organizer in the initial stages of invadosome

assembly (Seals et al., 2005). In microglia, Tks5 and talin co-localized in the podosome ring

(Figure 10). Caveolin-1 is another tyrosine phosphorylated protein, which can be

phosphorylated at tyrosine 14 residue by the SFKs, Lyn, Hck and c-Src (Li et al., 1996;

Grande-Garcia et al., 2007). Caveolin-1 is a major structural protein specific to specialised

membrane domains, called caveolae (Alberts et al., 2008). In microglia, tyrosine 14

phosphorylated caveolin-1 (pCav1) showed extensive ring-like staining that was similar to

the talin staining pattern (Figure 11).

Together, these findings show that podosomes in microglia express several tyrosine

phosphorylated proteins that are known substrates for Src and Src-family tyrosine kinases.

31

Figure 10. Immunostaining shows localization of Tks5 in the podosome ring in microglia.

Colour-separated images of a representative microglial cell stained for the podosome ring

marker, talin (red) and Tks5 (green); merged image at right. The boxed area is shown

magnified and colour separated at the far right. Arrows and arrowheads show co-localization

of talin with Tks5. Scale bars = 5 µm

32

Figure 11. Phospho-caveolin-1 (pCav1) is enriched in the podosome ring in microglia.

Colour-separated images of a representative microglial cell immunostained for the podosome

ring marker, talin (red) and pCav1 (green); merged image at right. The boxed area is

magnified and colour separated at the top right. Arrows and arrowheads show co-localization

of talin and pCav1. Scale bars = 5 µm

33

There is evidence that Nox1 is localized to invadopodia, and required for their

formation (Seals et al., 2005). The Nox1 enzyme generates reactive oxygen species (ROS),

which have pleiotropic effects, including acting as a second messenger; e.g., inhibiting some

protein phosphatases, activating some protein kinases, and regulating some ion channels

(Bedard and Krause, 2007). Because ROS production by microglia is one important

component of their cytotoxic behaviours, its regulation within podosomes is of interest. In

order to address whether Nox1 is present in microglial podosomes, I found it necessary to use

methanol fixation for immunostaining. The observed co-localization with talin indicates that

Nox1 is present in the podosome ring in microglia (Figure 12).

To summarize this section, I found that podosomes in microglia stained for

phosphotyrosine, Tks5, pCav1 and Nox1. These components have been reported in

invadosomes of at least one other cell type (Tarone et al., 1985; Nakamura et al., 1993; Burns

et al., 2001; Pfaff and Jurdic, 2001; Abram et al., 2003; Colonna and Podesta, 2005; Seals et

al., 2005; Oikawa et al., 2008; Diaz et al., 2009; Gianni et al., 2009; Stylli et al., 2009).

34

Figure 12. Immunolabeling for Nox1 shows localization to the podosome ring in microglia.

Colour-separated images of a representative methanol-fixed microglial cell stained for the

podosome ring marker, talin (red) and Nox1 (green); merged image at right. The boxed area

is shown magnified and colour separated at the top right. Arrows and arrowheads show co-

localization of talin with Nox1. Scale bars = 5 µm.

35

Ca2+ and podosome (podonut) formation

There is little specific information about mechanisms that regulate ECM degradation

and migration of microglia. In several other cell types, migration depends on Ca2+, and is

impeded by removing extracellular Ca2+ (Brundage et al., 1991; Mandeville et al., 1995).

Polarized cells maintain a descending intracellular Ca2+ concentration gradient from the

uropod at the rear to the lamellum at the front (Brundage et al., 1991), and Ca2+

microdomains in lamellipodium has been proposed to aid cell steering during migration (Wei

et al., 2009). Interestingly, our lab discovered that SK3, a Ca2+-calmodulin-gated K+ channel,

localizes to the core of microglial podosomes (Vincent and Schlichter, 2010). Here, I tested

the hypothesis that Ca2+ regulates podosome formation in microglia. Apparently, this aspect

of podosome regulation has not been studied previously in any cell type.

I tested this hypothesis by visualizing the larger podonuts, which are made up of

many individual podosomes (typically >100 (Vincent and Schlichter, 2010). Either

extracellular Ca2+ was removed by replacing the culture medium with a Ca2+-free bath

solution, or intracellular Ca2+ was buffered to an abnormally low level by incubating the cells

with 10 μM BAPTA-AM in standard bath solution. All treatments were carried out for 30

min at 37ºC. Control or untreated cells were maintained in standard bath solution under

identical experimental conditions. An interesting observation when extracellular Ca2+ was

removed or intracellular Ca2+ was buffered, was the loss of the lamellar morphology

displayed by untreated cells (Figure 13).

36

Figure 13. Ca2+ regulates podosome (podonut) formation. Microglia were incubated (30 min,

37ºC) in standard bath solution (CTL), Ca2+-free bath solution with EGTA (EGTA), or with

10 μM BAPTA-AM in standard bath solution (BAPTA-AM). Cells were then fixed and

stained for podosomes: F-actin (phalloidin; green), talin (red), and cell nuclei (DAPI; blue).

Arrowheads show examples of podonuts. Scale bars = 20 µm

37

Figure 14. Ca2+ and Ca2+-permeable ion channels are required for podonut formation. The

percentage of microglial cells expressing podonuts is shown in control microglia (CTL,

untreated), and after treatment with Ca2+-free bath solution (0 Ca2+) or 10 μM BAPTA-AM

as in Figure 13. In addition, two ion-channel blockers were used: 50 μM 2-APB, 100 μM

spermine. Podonuts were counted from three random fields on each immunostained cover

slip across microglia cultures prepared from 4 different animals. Data are shown as mean ±

standard error; and the asterisks indicate statistically significant differences compared to CTL

(untreated); **p<0.01.

38

Removal of external Ca2+ or buffering intracellular Ca2+ dramatically reduced

podonut formation in microglia (Figure 14), implying that their formation and/or persistence

depend on Ca2+. Because both treatments similarly inhibited podonut formation, I

hypothesized that Ca2+ entry, rather than external Ca2+, regulates the podosomes. The

Schlichter lab has shown that rat microglia express two functional Ca2+-permeable ion

channels: the non-selective cation channel, TRPM7, which is blocked by spermine; and

Orai1 (the pore forming subunit of the Calcium Release Activated Calcium [CRAC]

channel), which has a high selectivity for Ca2+ and is blocked by 2-APB (Jiang et al., 2003;

Ohana et al., 2009). Blocking TRPM7 channels with 100 µM or 1 mM spermine did not alter

the prevalence of podonuts (Figure 14, 15B). However, blocking CRAC channels with 50

µM 2-APB not only abolished podonut formation but significantly changed the microglial

morphology (Figure 14, 15A). High magnification images of 2-APB-treated microglia show

a lack of punctate F-actin staining, indicating a loss of podosomes as well as the podonut

superstructure (Figure 16). Control microglia treated with only DMSO (the vehicle for 2-

APB) did not differ from untreated cells. This result indicates that Ca2+ entry via CRAC is

required for expression of podonuts, and hence, podosomes. Interestingly, in time-lapse

images of microglia treated with 50 µM 2-APB (not shown), migration was reduced. These

preliminary data suggest that Ca2+ entry through CRAC channels contributes to microglia

migration.

39

A B

Figure 15. Podonut (podosome) formation requires Ca2+ entry, likely through CRAC

channels. Microglia were treated for 30 min at 37ºC with 2-APB (50 µM, A), a blocker of the

CRAC channel, or the TRPM7 blocker, spermine (100 µM, B) in standard bath solution.

Cells were then fixed and stained for podosomes: F-actin (phalloidin; green), talin (red),

nucleus (DAPI; blue). Arrows show podonuts. Scale bars = 20 µm

40

Figure 16. High-magnification images to show loss of podosomes after 2-APB treatment.

Colour-separated images of microglia cells labelled for podosomes: F-actin (phalloidin;

green) and talin (red). Cells were treated for 30 min at 37ºC with the CRAC channel blocker,

50 µM 2-APB. Scale bars = 5 µm

41

Novel expression of several molecules in microglia podosomes

Although many podosome components are conserved across different cell types, a

unique molecule, SM22α, was identified in podosomes of vascular smooth muscle cells

(Gimona et al., 2003). This suggested that some molecular components of podosomes could

depend on the cell type. Indeed, our lab was the first to identify the SK3 channel as a

component of invadipodia, specifically in microglial podosomes (Vincent and Schlichter,

2010).

Because I found that the Orai1/CRAC channel blocker, 2-APB, reduced number of

podonuts in microglia, I next tested the novel hypothesis that Orai1 is a podosome

component in microglia. After using heat-induced antigen retrieval (HIAR), I observed Orai1

immunolabeling in podonuts, specifically in core of individual podosomes (i.e., co-localized

with Arp2; Figure 17). Orai1 is the pore-forming subunit of the CRAC channel (which

requires the STIM1 protein for functionality). Together, the results in Figures 15–17 suggest

that Ca2+ entry through CRAC channels in podosomes regulates their formation and/or

stability.

The presence of SK3, a Ca2+-activated K+ channel, together with a source of Ca2+ (the

Orai1/CRAC channel) raised the possibility that these two channels interact in podosomes.

However, SK channels require calmodulin (CaM) binding in order to respond to Ca2+;

therefore, I next asked whether CaM is present in microglial podosomes. After performing

HIAR to obtain specific CaM immunolabeling, I found that CaM was enriched in podonuts,

and mainly co-localized with talin in individual podosome rings (Figure 18). Occasionally,

CaM staining was also seen in the centre of talin-stained rings; i.e., in the podosome core.

When using an antibody against ‘ionized calcium binding adaptor molecule 1’ (Iba1)

42

as a specific marker to label microglia, I made the surprising observation that it was enriched

in podonuts (Figure 19). Iba1 is present in individual podosomes, and double labelling for F-

actin (or talin) shows that it co-localizes with F-actin in the core structure. This is intriguing,

because Iba1 is a microglia-specific Ca2+-binding protein that is known to cross-link actin

filaments in the CNS (Imai et al., 1996; Sasaki et al., 2001; Imai and Kohsaka, 2002).

Table 2 summarizes all components found in microglial podosomes in this study, and

their localization to the ring versus core.

43

Figure 17. Immunolabeling of Orai1 in microglia shows enrichment in podonuts and

localization to the podosome core. Colour-separated images of a representative cell (after

heat-induced antigen retrieval) stained for Orai1 (green) and the podosome core marker,

Arp2 (red); merged image is at right. The boxed area is magnified and colour separated at the

far right. Arrows show co-localization of Orai1 with Arp2. Scale bars = 5 µm

44

Figure 18. Immunostaining shows calmodulin (CaM) association with microglia podosomes.

Left: After heat-induced antigen retrieval, colour-separated images show CaM (red) and the

podosome core marker, Arp2 (green). The arrows in the merged image (below) show

examples of CaM in podosomes. The boxed area is shown magnified and colour separated at

the right. Right: After HIAR, colour-separated images show staining for CaM (red) and the

podosome ring marker, talin (green). In the merged image (below), arrows show examples of

CaM in individual podosomes. The areas within solid and dotted boxes are shown magnified

and colour separated to the left and right, respectively. CaM was seen mainly in the

podosome ring structure, and less in the core. Scale bars = 5 µm

45

Figure 19. Iba1 staining is enriched in podonuts and present in the podosome core. Upper

panel: Colour-separated images of a microglial cell stained for Iba1 (red) and the podosome

core marker, F-actin (phallodin; green). In the merged image at right, the arrows indicate co-

localization of F-actin and Iba1 in individual podosomes. Lower panel: Colour-separated

images immunostained for Iba1 (green) and the podosome ring marker, talin (red). In the

merged image at right, arrows indicate co-localization of talin and Iba1 in individual

podosomes. The boxed area is magnified and colour separated to the right of each panel.

Scale bars = 5 µm

46

Table 2. Podosome components in microglia, and their localization to the core and ring.i1

Component Podosome Localization Talin Ring

F-actin Core Arp2 Core PTyr Core & ring Tks5 Ring

pCav1 Ring Nox1 Ring Orai1 Core CaM Primarily in ring, some in core Iba1 Core

47

DISCUSSION

Podosomes are dynamic microscopic structures that continually undergo assembly,

maturation and disassembly (Murphy and Courtneidge, 2011). Individual podosomes within

a cell might be in different stages of development depending on numerous factors, including

substrate attachment, protein kinase activity, and actin reorganization. Following the

discovery of podosomes in microglia in the Schlichter lab, SK3 was clearly shown to

associate with podosomes (Vincent and Schlichter, 2010). The co-localization of SK3 with

Arp2 and F-actin indicated that it is trafficked to the core region of podosomes. Following

the initial discovery of microglial podosomes, I wanted to determine whether they contain

several components that are conserved in podosomes of other cell types.

In this thesis, I have shown that Ca2+ is involved in podosome regulation in microglia;

and I showed that several known podosome-associated proteins, as well as novel molecules

associate with microglial podosomes. This is a significant discovery because although

migration of activated microglia towards the site of injury in the damaged CNS is a well-

known phenomenon, it is not well understood how microglia perform the difficult task of

migrating through not only distinctive brain ECM but also tightly packed CNS tissue in order

to reach their destination. The expression of podosomes in microglia provides significant

insight into how microglia might move through CNS tissue. Podosomes have several

characteristics that could aid activated migratory microglia: (i) attachment to ECM to provide

anchorage and traction, (ii) fast turnover for quick assembly/disassembly of these structures

for fast migration, and (iii) localized ECM degradation to allow microglia to traverse cell

layers in a controlled manner.

The initial discovery of podosomes in microglia by the Schlichter lab was followed

48

by the discovery that the Ca2+-activated SK3 channel localizes to the podosome core.

Microglial podosomes also collectively form a characteristic superstructure that we have

termed a ‘podonut’; i.e., many podosomes forming a large ring, with a centre that is generally

devoid of podosomes. Osteoclasts form a similar structure, called a sealing zone, in which

podosomes form a belt along the outer edge of the entire cell, resulting in a tight seal (Jurdic

et al., 2006). The sealing zone forms a sealed cavity into which protons and various proteases

are secreted for bone resorption (Sasaki and Hong, 1993; Duong and Rodan, 1998; Mulari et

al., 2003). Podonuts in microglia were found only in the lamella region of polarized cells

(Figure 4; (Vincent and Schlichter, 2010). This places podosomes in the leading edge of

microglia and in an ideal location to help microglia migrate.

Although podosomes may provide microglia with the means to migrate, the

mechanisms that mediate cell migration of microglia is a poorly understood subject. It is a

well-known fact that Ca2+ and its spatiotemporal regulation are important for directed cell

migration (Becker and Showell, 1972; Gallin and Rosenthal, 1974; Wilkinson, 1975; Boucek

and Snyderman, 1976; Estensen et al., 1976; Evans and Falke, 2007). In this study, I tested

the hypothesis that Ca2+ plays a crucial role in regulating podosomes. I provide supporting

evidence that podonut and thus, podosome formation is dependent on Ca2+. The absence of

extracellular Ca2+ or chelation of intracellular Ca2+ with BAPTA significantly reduced the

number of podonuts. Furthermore, microglia in culture tend to show lamellar morphology.

However, removal of Ca2+, intracellular or extracellular, resulted in loss of the lamellae.

Using time-lapse microscopy, our lab showed that microglia undergoing migration all

expressed lamella at their leading edge (Vincent and Schlichter, 2010). Given that lamella

formation is a requirement for microglia migration and Ca2+ chelation leads to loss of

49

lamellae in microglia, this would suggest that microglia migration is dependent on Ca2+.

Further studies, such as live recordings with cell tracking or transwell migration assays need

to be done to confirm this finding.

Because removal of Ca2+ affected podonut stability, I hypothesised that Ca2+ entry

into microglia is required for podonut and podosome formation. Despite attempts by several

laboratories, including ours, only one study has shown any evidence of voltage-gated Ca2+

channels in microglia (Colton et al., 1994). Many laboratories, including ours, have shown

other primary Ca2+ influx pathways, including TRP and CRAC channels (Norenberg et al.,

1997; Hahn et al., 2000; Jiang et al., 2003; Kraft et al., 2004; Fonfria et al., 2006; Kim et al.,

2006; Ohana et al., 2009; see review Kettenmann et al., 2011). While microglia express

transcripts for several TRP channels, TRPM7 expression was shown to be the highest among

them (Ohana et al., 2009), and our lab showed that rat microglia have a large TRPM7 current

(Jiang et al., 2003). Evidence presented in this thesis shows that inhibition of CRAC channels

abolished podonuts and podosomes, while blocking TRPM7 channels had no effect on

podonut numbers. Our present findings suggest that CRAC channel-mediated Ca2+ entry is

needed for formation of podosomes in microglia. Biophysical analysis of the CRAC channel

shows that it is highly selective for Ca2+ (Zweifach and Lewis, 1993). Because Ca2+ is a well-

known second messenger, I speculate that localized Ca2+ entry through Ca2+ permeable

channels in podosomes could modulate downstream effectors that lead to the formation of

podonuts.

In this study of rat microglia, I discovered the novel association of some molecules

with podosomes. Much knowledge regarding podosomes comes from Src-transformed cell

lines and other cell lines (e.g. the monocyte/macrophage RAW cell line). In most of these

50

cases, endogenous factors have been significantly modified (e.g. over-expression of kinases)

that do not reflect the biochemistry of unmodified, untransformed cells. Nonetheless, a key

signalling pathway necessary in formation of podosomes is activation of Src tyrosine kinase

(Linder and Aepfelbacher, 2003). Inhibition of Src kinase activity disrupts podosome

formation in osteoclasts and primary macrophages (Tanaka et al., 1995; Linder et al., 2000a),

while conversely, inhibition of tyrosine phosphatases induces podosome formation in

fibroblasts and monocytes (Marchisio et al., 1988; Cory et al., 2002). In addition, many

substrates of Src were also found to associate with podosomes (e.g. Pyk2 tyrosine kinase,

Tks5, PI3K) indicating further that Src signalling is essential for podosome regulation

(Schaller et al., 1994; Qian et al., 1997; Lock et al., 1998; von Willebrand et al., 1998;

Chellaiah et al., 2001; Abram et al., 2003). Therefore, I tested the hypothesis that similar

signalling molecules are present in microglial podosomes, by staining for substrates of Src

kinases. Immunolabeling for phosphotyrosine showed localization mostly to podosome core,

and to a lesser extent, the podosome ring. This provides the first evidence that tyrosine kinase

signalling might be involved in podosome regulation in primary microglia. Future studies

employing pharmacological modulators of tyrosine kinase signalling can be used to test

whether tyrosine kinases, specifically Src tyrosine kinases, are required for podosome

formation in microglia. I then hypothesized that other substrates of tyrosine kinases might

localize to podosomes. Tks5, a Src kinase substrate, has recently been reported to be an

organizer for podosome formation (Abram et al., 2003; Seals et al., 2005; Burger et al.,

2011). Tks5 consists of 5 SH3 domains that allow it to interact with various proteins,

possibly recruiting them to podosomes. Tks5 also contains a PX domain that can bind to

phosphatidylinositol bisphosphate (PIP2) – a commonly found membrane phospholipid in

51

podosomes that might help Tks5 localize to podosomes (Sechi and Wehland, 2000). In

microglial podosomes, I show that Tks5 localizes to podosomes; specifically to the

podosome ring. Other reports have shown association of Tks5 with podosomes in

transformed cell lines (Abram et al., 2003; Courtneidge et al., 2005), myoblasts (Thompson

et al., 2008), and macrophages (Burger et al., 2011). Tks5 can also associate with other

proteins, including the matrix degrading proteins, ADAMs 12, 15, 19 (Abram et al., 2003)

and MMP9 (Burger et al., 2011), the cytoskeletal regulating proteins, N-WASp, dynamin and

focal adhesion kinase (Courtneidge et al., 2005), and dystroglycan (Thompson et al., 2008).

The structure of Tks5 and its ability to interact with diverse molecules suggests that it

functions as a scaffolding protein that recruits and restricts localization of other proteins to

podosomes, resulting in a signalling nexus for the podosome machinery.

In this thesis, I presented evidence that pCav1 localizes specifically to the podosome

ring component in microglia. Caveolin-1 has been shown to associate with invadopodia in

breast- and melanoma- cancer cell lines (Caldieri et al., 2009; Yamaguchi et al., 2009;

Yamaguchi and Oikawa, 2010). Tyrosine 14 phosphorylated caveolin-1 (pCav1), on the other

hand, localized to podosomes but required ACTH stimulation of Y1 adrenal cells (Colonna

and Podesta, 2005). Caveolin-1 tyrosine phosphorylation can occur in response to growth

factor signalling or oxidative stress, and is thought to be mediated by c-Src (Labrecque et al.,

2004; Parat and Fox, 2004). Subsequent to phosphorylation of caveolin-1, it can interact with

proteins that are involved in cell migration and Src signalling, and with other SFKs, which

suggests that pCav1 serves as an adaptor molecule to recruit other molecules (Labrecque et

al., 2004). In focal adhesions, the phosphorylation state of caveolin-1 determines the turnover

rate of these adhesion structures by regulating signalling cascades downstream of pCav1

52

(Nethe and Hordijk, 2011). In addition, caveolin-1 proteins are major constituents of

specialized structures in the plasma membrane, called caveolae. Caveolae are small flask-

shaped invaginations (50-100 nm) of the membrane that are rich in cholesterol. These

structures have been linked with many physiological functions, such as membrane

trafficking, clathrin-independent endocytosis and cholesterol transport (Fujimoto et al.,

2000). Many cells use caveolae to organize cell-signalling molecules, and even ion channels,

into signalling complexes. Podosomes are hotspots for many signalling molecules and as

such, microglial podosomes might exploit caveolae and its versatile constituent, caveolin-1,

to bring together regulatory molecules that affect podosome formation and expression.

Podosome formation is the result of complex interactions between signalling

pathways (see review Murphy and Courtneidge, 2011), that are themselves regulated by

second messenger molecules. Reactive oxygen species (ROS) can act as second messengers

to modulate signalling pathways. For example, ROS production can inhibit protein tyrosine

phosphatases (PTPs) (Wu et al., 2003; Goldstein et al., 2005; Kwon et al., 2005), activate

kinases (Griendling et al., 2000; Han et al., 2003; Torres and Forman, 2003; Touyz et al.,

2004; Mehdi et al., 2005), and can regulate ion channels involved in K+ permeation and Ca2+

signalling (Zimmerman et al., 2005; Lee et al., 2006; refer review Bedard and Krause, 2007).

Although ROS are also a by-product of the electron transport chain in mitochondria, a family

of NADPH oxidase (Nox) enzymes functions solely to generate ROS (Bedard and Krause,

2007). Nox1, the first identified member of the Nox family, was previously found in

invadopodia, and both the Nox-1 specific blocker, ML171, and the non-specific blocker,

diphenylene iodonium (DPI), disrupted invadopodia formation in human colon cancer cells

(Gianni et al., 2009; Gianni et al., 2010). The authors concluded that localization of

53

functional Nox1 is required for the formation of invadopodia; however, contribution of other

Nox isoforms is possible because the concentration of ML171 used (10 μM) was much

higher than their reported IC50 values: Nox-2 (5 μM), Nox-3 (3 μM) and Nox-4 (5 μM)

(Gianni et al., 2010; Wingler et al., 2011). Gianni and colleagues also stated that Tks

proteins, including Tks5, could serve as organizers of Nox proteins at invadopodia, and they

suggested that inhibiting Nox1-mediated ROS generation increases local tyrosine

phosphatase activity and reduces tyrosine kinase activity. My results show for the first time

that Nox1 localizes to the podosome ring in microglia. Because the adaptor protein, Tks5,

also localised to the microglial podosome ring, it might regulate Nox1 localization. Localized

production of ROS by Nox1 might then facilitate tyrosine kinase signalling in podosomes to

modulate their formation and/or stability. Conversely, ROS generation might be regulated by

podosome components, such as c-Src tyrosine kinase or ion channels, like SK3, for overall

modulation of podosome assembly/disassembly (Khanna et al., 2001; Gianni et al., 2008;

Vincent and Schlichter, 2010).

I have shown that microglial podosome/podonut formation requires Ca2+ entry. I then

hypothesized that podosomes are signalling foci that contain Ca2+ entry channels and other

Ca2+ responders, which might allow formation of Ca2+ nanodomains and local signalling. My

observation of Orai1 in the podosome core supports this hypothesis. The CRAC channel is a

multimeric protein, with Orai1 being the critical pore forming subunit (Smyth et al., 2010).

Our lab showed that store operated Ca2+ entry (SOCE) in rat microglia occurs through the

CRAC channel (Ohana et al., 2009). Its important biophysical properties include inward

rectification, high selectivity for Ca2+, and a very small single-channel conductance (<0.2 pS)

(Zweifach and Lewis, 1993; Ohana et al., 2009). CRAC currents (sometimes referred to as

54

ICRAC) have been observed in several immune cell types, including microglia (Norenberg et

al., 1997; Ohana et al., 2009), mast cells (Hoth and Penner, 1992), the Jurkat T cell line

(Zweifach and Lewis, 1993), dendritic cells (Hsu et al., 2001); as well as in some non-

immune cells (Fasolato et al., 1993; Delles et al., 1995; Parekh and Penner, 1995;

Somasundaram et al., 1995; Rychkov et al., 2001). ICRAC is generated when intracellular Ca2+

stores in the endoplasmic reticulum (ER) are depleted (Hoth and Penner, 1992; see reviews

Parekh and Putney, 2005; Parekh, 2006; Smyth et al., 2010). Although many different

models have been postulated for the CRAC activation mechanism, the most widely accepted

model involves STIM1-Orai1 interaction after store depletion, as follows. Depletion of ER

Ca2+ is sensed by STIM1, an ER-resident Ca2+ sensor, which then undergoes a

conformational change that allows oligomerization with other STIM1 molecules. This

increases the affinity of STIM1 oligomers for Orai1 in the plasma membrane, and binding of

STIM1 to Orai1 opens the CRAC channel allowing influx of Ca2+. In general, influx through

Ca2+-permeable channels increases the Ca2+ concentration immediately adjacent to the

channel pore; free Ca2+ declines sharply with distance as Ca2+ diffuses away and is buffered

(Allbritton et al., 1992; Baimbridge et al., 1992; Smith et al., 1993; Kasai and Petersen, 1994;

Weber et al., 2010). In future, we want to determine whether Ca2+ microdomains form in

podosomes, and will begin by immunolabeling for STIM1 and podosome markers to see if

STIM1 and Orai1 co-localize in the podosome core.

Calmodulin is another Ca2+-dependent molecule that was present in microglial

podosomes (and throughout the microglia cytoplasm), providing a potential mechanism

whereby Ca2+ entry through CRAC could influence podosome-associated regulatory proteins.

Ca2+-CaM regulates many molecules, including CaM kinases and the gating of SK1–SK4

55

channels (Chin and Means, 2000). In addition, trafficking of SK3 and SK4 to the plasma

membrane depends on CaM-channel interactions, as shown by our lab and others (Khanna et

al., 1999; Joiner et al., 2001; Maylie et al., 2004). Because CaM co-assembles with SK

channels during their biogenesis, it was surprising to find CaM mainly in the microglia

podosome ring and SK3 mainly in the core.

I discovered a third novel Ca2+-binding protein in the microglial podosome core: the

‘ionized calcium-binding adapter-1’ protein (Iba1). Iba1 is a specific marker used to identify

microglia in the CNS. It is involved in binding and cross-linking filamentous actin, and is

regulated by Ca2+ (Sasaki et al., 2001), but has never been identified in podosomes or

invadipodia. Localization of Iba1 to podosomes might facilitate growth of the actin core by

cross-linking filamentous actin and stabilising the core structure. In future, it would be

interesting to determine whether Ca2+-mediated regulation of Iba1 contributes to podosome

expression and microglia migration. In contrast to my results on Iba1, another actin cross-

linking protein, α-actinin, is found in both the podosome ring and core in other cells

(Maruyama and Ebashi, 1965; Mimura and Asano, 1978; Marchisio et al., 1987; Stickel and

Wang, 1987; Imai et al., 1996; Gimona et al., 2003).

Some data that were omitted from this thesis include several molecules that showed

no evidence of localization to podosomes in microglia; i.e., SK4 and TRPM7 channels, and

the matrix metalloproteinases, MMP-9, MMP-12 and MMP-14. Figure 20 is an example

showing punctate TRPM7 staining that was evenly distributed throughout the cytoplasm,

with no podosome-like staining or co-localization with the podosome core marker, F-actin.

This finding is interesting in light of my finding that blocking TRPM7 with spermine did not

affect podonut formation in microglia. The lack of MMP localization to podosomes suggests

56

that the fibronectin degradation by microglial podosomes observed by our lab (Vincent and

Schlichter, 2010) depends on other ECM-degrading proteins. Other proteins with ECM-

degrading capacity (ADAMs, cathepsins) have been reported in invadosomes (Linder, 2007;

Murphy and Courtneidge, 2011), and in future, we plan to assess whether they are present in

microglia podosomes.

In conclusion, this thesis presents evidence that microglial podosomes contain several

components that are common to invadosomes in other cell types: F-actin, talin, Arp2,

tyrosine phosphorylated proteins, Tks5, Tyr-14-phosphorylated caveolin-1 and Nox1. These

findings suggest that signalling pathways that regulate podosomes in other cell types might

also function in microglia. Novel findings in this thesis include the association of the

following molecules with microglial podosomes: SK3, Orai1, CaM, and Iba1. The presence

of a Ca2+ permeable channel (Orai1/CRAC) and several Ca2+ responsive proteins (CaM,

SK3, Iba1) suggests that podosomes might be a hub for local signalling within a Ca2+

microdomain, and is supported by my finding that podonuts/podosomes in microglia depend

on Ca2+ entry through CRAC channels. If so, this would provide migrating cells with

additional regulatory mechanisms for the cell-substrate adhesion and matrix degradation that

is necessary for traversing through tissue. While it will be important to demonstrate that

microglia migration depends on Ca2+, this is very likely the case, and is well established for

many migrating cells.

57

F‐Actin  TRPM7 F‐Actin TRPM7

Figure 20. Immunolabeling of TRPM7 channels and F-actin in microglia. Colour-separated

images of a cell stained for the podosome core marker, F-actin (phalloidin; green) and

TRPM7 (red); merged image at right. Scale bars = 5 µm

58

WORKING MODEL

Based on the literature regarding podosomes and the above findings from the

Schlichter lab and in this thesis, I propose a model for the podosome structure, assembly,

disassembly and function (Figure 21). This is by no means complete, but is a culmination of

the literature and our novel findings in microglia.

It is thought that podosome assembly begins when the protruding lamellipodium of a

migrating cell finds, and adheres to a suitable ECM substrate. Upon binding matrix ligands,

integrins undergo a conformational change that leads to changes in cytosolic factors,

including activation of Src kinases, recruitment of phosphoinositide-3-kinase (PI3K), and

interaction with cytoskeletal rearrangement proteins such as talin, vinculin and paxillin (van

der Flier and Sonnenberg, 2001; Tvorogov et al., 2005; Alberts et al., 2008), which modulate

other signalling cascades (Hynes, 2002). This ‘outside-in’ signalling translates integrin

binding to extracellular matrix into effects on cytosolic factors (Hynes, 1992; Alberts et al.,

2008). In addition, integrins mediate ‘inside-out’ signalling, in which the cytoplasmic tail

interacts with proteins (e.g., talin) that can induce a conformational change, and increase

integrin affinity for matrix proteins (Ginsberg et al., 1992; Calderwood et al., 1999; Hynes,

2002; Alberts et al., 2008). Recruitment of adhesion-associated molecules then activates actin

nucleators, such as Arp2/3 complex, WASP and/or N-WASP, to produce filamentous actin

from G-actin monomers in the podosome core (Alberts et al., 2008). Integrin clustering at the

cell-matrix contact site strengthens the adhesion (Alberts et al., 2008). Clustered integrins

might localize to cholesterol-rich domains along with their ligands, such as ECM

components, and bring along adhesion-associated machinery in the cytosol to these domains

(del Pozo et al., 2005). Conversely, integrin clustering at adhesion contact sites might recruit

59

caveolin-1 (Nethe and Hordijk, 2011), a structural component of cholesterol-rich membrane

domains (caveolae) that might serve as the initial organizing center for integrin signalling

(del Pozo et al., 2005; Nethe and Hordijk, 2011).

Once integrins are activated, Src tyrosine kinase activation is thought to promote

phosphorylation and activation of other substrates, including caveolin-1 at Tyr-14 (Lee et al.,

2000; Cao et al., 2002), Tks5 (Lock et al., 1998; Abram et al., 2003), and PLCγ (Kanner et

al., 1993; Clark and Brugge, 1995). Activated PI3K can phosphorylate the membrane lipid,

phosphatidylinositol, to produce PIP2 and PIP3, and PIP2 binds to phosphorylated Tks5,

where it presumably serves as an organizer, bringing other proteins, such as Nox1, to the

maturing podosome (Sechi and Wehland, 2000; Gianni et al., 2010). Nox1 generates ROS,

which can inhibit protein tyrosine phosphatases (PTPs) (Wu et al., 2003; Goldstein et al.,

2005; Kwon et al., 2005), and possibly enhance Src tyrosine kinase signalling in a positive

feedback loop.

Phosphorylation of PLCγ activates it to cleave PIP2, which releases IP3 and DAG

(Nishibe et al., 1990; Tvorogov et al., 2005). IP3, in turn, activates IP3 receptors in the ER

membrane, depletes intracellular Ca2+ stores, and activates Ca2+ influx through CRAC

channels, which might then form a Ca2+ nanodomain around a podosome. Influx of Ca2+

potentially activates other molecules, including the tyrosine kinase, Pyk2 (Lev et al., 1995;

Yu et al., 1996); Iba1, which cross-links actin filaments along with α-actinin (Maruyama and

Ebashi, 1965; Mimura and Asano, 1978; Sasaki et al., 2001); and CaM, which can further

interact with other molecules (Chin and Means, 2000). A relevant Ca2+-CaM interaction

occurs at the SK3 channel, which is opened by Ca2+ binding to CaM and then mediates K+

efflux (Xia et al., 1998). The best known role of SK channels is to hyper-polarize cells,

60

which maintains the large driving force required for Ca2+ influx through CRAC channels

when the ER lumen is depleted of Ca2+ (Lewis and Cahalan, 1995; Fanger et al., 2001;

Ahmmed and Malik, 2005). Once assembly of podosome structure is well underway,

microtubules can traffic molecules, such as matrix-degrading proteins to this structure

(Poincloux et al., 2009; Wiesner et al., 2010).

There is some information regarding disassembly of podosomes. This might begin

with pCav1, if it initially serves to recruit adhesion associated molecules. Tyr-14

phosphorylation of caveolin-1 is required for internalization of cholesterol-enriched

membranes, including caveolae (del Pozo et al., 2005). Along with internalization of

caveolae and podosomes, integrins located in these structures would detach from the matrix,

thereby stopping the integrin-mediated signal required to maintain Src kinase activity. Other

intrinsic factors could also inactivate integrins by reducing their affinity for matrix ligands or

preventing them from binding matrix proteins (Millon-Fremillon et al., 2008; Harburger and

Calderwood, 2009). For example, the intracellular protease, calpain, can break down talin

(Franco et al., 2004), and together with proteolysis of the integrin cytosolic tail (Flevaris et

al., 2007), can interfere with integrin signalling. Without integrin signalling, there would be

dissociation of actin nucleators (e.g. Arp2/3, WASP), actin-association proteins (e.g. α-

actinin), and adhesion-associated proteins (e.g. vinculin, paxillin, talin). PI3K activity in

podosomes would cease, preventing production of PIP2 at the membrane, which would

prevent Tks5 anchoring to the podosome membrane, allowing it to diffuse away. In turn,

Tks5 loss would diminish Nox1-mediated ROS generation in podosomes, and increase PTP

activity. When ER Ca2+ is replenished, the CRAC channel would close, and Ca2+ would be

removed from the nanodomain by buffering (by cytosolic proteins), uptake into organelles, or

61

pumping out due to plasma membrane Ca2+ pumps. The reduction in Ca2+ would close SK3

channels as apoCaM forms. Loss of the Ca2+ signal could influence other molecules; e.g.,

inactivating Pyk2 tyrosine kinase and other Ca2+ regulated molecules, such as Iba1.

Podosome association with the microtubule plus-end associated molecule, KF1C, might also

lead to disassembly of podosomes (Kopp et al., 2006).

62

Figure 21. Updated schematic showing podosome-associated molecules (modified from

(Linder and Aepfelbacher, 2003), with the molecules found in microglia in this thesis shown

in red boxes.

Adapted and reprinted from The Lancet, Vol. 13, Linder S, Aepfelbacher M, Podosomes: adhesion hot-spots of invasive cells, 376-385, Copyright (2003), with permission from Elsevier

63

FUTURE STUDIES

In this thesis, I conducted extensive immunostaining on primary cultures of rat

microglia, and identified both known and novel molecules in podosomes. Knowledge about

podosome components provides us with the foundation to explore some of the signalling

pathway(s) that might be involved in podosome formation and functions in microglia.

Continued characterization of the components of podosomes will help identify potential

pharmacological targets (e.g. SK3, Orai1, CaM, Iba1) for promoting or inhibiting podosome

formation. Selective drugs could then be used with immunocytochemistry and functional

assays to study podosome formation and roles. Both 2-D migration- and 3-D invasion assays

using brain-relevant ECM substrates would shed light on whether active podosomes are

needed for microglial migration or invasion. Because Ca2+ removal disrupted lamellipodia

formation, a structure required for microglia migration (Vincent and Schlichter, 2010), we

expect that microglia invasion through ECM-coated filters in Transwell™ chambers would

be inhibited in the absence of extracellular Ca2+, buffering of intracellular Ca2+, or blocking

specific Ca2+-entry and responding pathways (e.g., Orai1/CRAC and SK3 channels).

Unfortunately, primary microglia are extremely resistant to gene transfection, siRNA-

mediated knockdown or viral-mediated infection (Ohana et al., 2009); therefore, we have to

mainly rely on pharmacological approaches for these proposed studies.

I suggested in this thesis that microglial podosomes represent a nexus for Ca2+- and

ROS signalling pathways. Therefore, I would like to: (i) use Fura2-FF, an analogue of the

fluorescent Ca2+ indicator, Fura-2, to monitor and measure any rapid changes in local Ca2+ in

and around the tiny podosome structures; and (ii) monitor ROS levels with Rhodamine 123

in podonuts, and (iii) determine whether these two signals interact and are interdependent.

64

This would address the possible roles of Ca2+ and ROS as second messengers for podosome

regulation and function.

Our discovery of podosomes in microglia in vitro raises the possibility that migrating

microglia in the brain use these structures to locally degrade ECM and to navigate through

CNS tissue to reach the site of injury. Only a handful of studies have identified podosomes in

vivo, and never in microglia or the CNS. At this point, our in vitro studies have been limited

by using microglia grown in an artificial 2-dimensional environment. Ultimately, to study

podosomes in a 3-dimensional in vivo environment, we would like to use adult rats and our

ischemic and hemorrhagic stroke models, in which massive microglia migration is observed.

This would allow analysis with the naturally occurring complement of ECM molecules, and

factor in changes that occur after acute damage. For instance, Iba1, a microglia/macrophage-

specific marker that has been extensively used in our lab, is strongly up-regulated in

microglia/macrophages in rat brain after stroke. Thus, Iba1 could be used to identify these

cells and simultaneously label podosomes to assess their formation in the CNS in vivo.

In summary, there is evidence that microglia are involved in most neuropathological

processes (Tambuyzer et al., 2009), and microglial migration to the site of injury is a crucial

early response to acute CNS injury. Hence, determining the role of podosomes, and

mechanisms regulating their specific functions, will bring us an important step closer to

developing therapeutic measures that target microglia and brain inflammation.

65

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