Multicellular architecture

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Epithelial cells and their functions

To motivate the work done during this thesis, we will begin by introducing the biolog-ical system studied. In particular, in vitro cultures of epithelial cells were employed to model tissue organization and cell dynamics in vivo. In this section we will describe epithelial cells and their functions. Then we will present how they contribute to the architecture of an epithelium.
Epithelial cells form a major class of cells found in vivo, and are de ned by their di-rectional polarity and the specialized junctions they form to adhere to their neighbors. Epithelial tissues line organs and cavities within the body. They act as a protective barrier, but can also serve to absorb, secrete and transport uids and provide sensory responses. Epithelial cells are classi ed by their shape and the number of layers they form. As seen in Figure 2-1 an epithelium can be described as squamous, cuboidal, or columnar as well as simple, strati ed, and pseudostrati ed [3]. Simple epithelia are single layered and strati ed epithelial are multilayered. A pseudo-strati ed epithelia has nuclei arranged at more than one height from the basal plane while maintain-ing all cells in contact with the basement membrane. In some cases, for example the urethra, an epithelium can be made up of a combination of these organizations in close proximity in which case it is termed a transitional epithelium. The orga-nization of an epithelium inside a tissue isn’t xed and it can change dramatically during development. For example, the mammary gland is strati ed during embry-onic and postnatal branching stages but develops a simple columnar organization after branching morphogenesis [4]. This thesis describes work carried out primarily on Madin-Darby canine kidney epithelial (MDCK) cells a common model cell line prototypical of epithelial tissues.
Figure 2-1: The seven subcategories of epithelial cells divided by individual cell geometry and overall tissue organization []
Although the overarching aim of this work is to understand the dynamics and the collective organization of an epithelial tissue, in the following two sections character-istics of a single epithelial cell will be presented. Given that the epithelial cell is a building block of our multicellular system of interest, its internal properties closely inform the collective dynamics of the group.
Below the architecture of an epithelium at the scale of a single cell will be de-scribed and then the junctions and adhesions a cell forms with its environment will be detailed. Subsequently we return to our multicellular system of interest and discuss the supracellular architecture present in an epithelial tissue.

Single cell architecture: the cytoskeleton

The internal structure of an epithelial cell is comprised of a set of bers which attach together to form a sca old or cytoskeleton inside the cytoplasm that dynamically grows and rearranges through polymerization and depolymerization. The cytoskele-ton attaches to its environment through substrate adhesions and cell junctions. Fi-nally, molecular motors interact with the cytoskeleton to apply forces and transport cargo.
The cell cytoskeleton can be divided into three types of laments: actin (or ’mi-cro laments’ of diameter 5-9nm), intermediate laments (diameter 10nm), and mi-crotubules (diameter 25nm), each of which help build a rigid structure inside the cell. In the case of microtubules and actin laments, the bers have molecular motors associated with them which can apply force inside the cell.
In the following subsections, the three types of lament will be presented with a particular focus on the actin laments and their associated molecular motors, myosins, as they will be studied in depth during a large part of this thesis.
Actin laments and their associated molecular motors: myosins
Actin filaments
Actin laments (micro laments) are approximately 7 nm in diameter and are formed through the polymerization of actin monomers, or G actin (aka globular actin) into a polar helical lamentous structure. The lamentous actin (F actin) has a po-larity with monomers being added faster to their \+ plus end » than their \ { minus end » or \barbed end » ( g 2-2A). Each monomer is added through the hydrolysis of an adenosine triphosphate or ATP, attached to the monomer, to adenosine diphos-phate or ADP [3]. Actin laments are dynamic and can also shrink in size at their end preventing other monomers to be added.
Figure 2-2: Actin lament polymerization. During actin lament assembly monomers as-semble through dimerization and trimerization, steps which are energetically unfavorable. The subsequent monomer, however, is more stable and subsequent additions are much more favorable. An actin lament has two ends the barbed (B) and the plus end (P). Actin monomers are added to the plus end and removed from the barbed end. B) Arp2/3 (orange) is an actin nucleator that binds to the side of a lament and creates a new branch at a 70 angle. Repeated Arp2/3 branching forms a dendritic actin network. C) Formin (purple) nucleates a lament and can either move progressively with the elongating barbed end or bundle laments together. [5]
The polymerization of actin can be stimulated with the help of other proteins. Formins are a family of proteins that regulates linear actin assembly. Formins move with the barbed end of the lament during elongation aiding in successive nucleation events. Formin can also bind multiple bers together forming an actin bundle (Fig 2-2C). The Arp2/3 complex, binds to the side of an actin lament or competes with capping proteins to bind to a barbed end. Once bound, Arp2/3 creates a new daughter branch at a 70 angle from the original one. Repeated actin branching is possible on both the mother and the new daughter branch resulting in a dense dendritic network of actin (Fig 2-2B) [5] .
Actin laments can also take shape into larger structures with the help of cross-linking proteins which further organize actin into gel-like networks and bundles. Actin bundles are commonly cross-linked together by mbrin or -actinin. Filamin cross-links actin at large angles in a gel-like formation.
The di erent forms of actin work together during cell migration. As illustrated in Fig 2-3 a migrating cell on a rigid at surface often forms an elongated fan-like protrusion. The branched organization of actin created from actin polymerization catalyzed by the Arp2/3 complex is present in the protruding lamellipodium. The tight parallel actin bundles, protrude further forward in a crawling cell in the form of lopodium.
Figure 2-3: Structure of a migrating cell. Actin in the lamellipodium is arranged in a branched network while actin in protruding lopodium is organized into parallel bundles. The arrow indicates the direction of migration. [6]
Myosin II is a molecular motor that functions with actin to produce force. Myosin
II is composed of two heavy chains and two light chains which can be divided into a head, neck and tail domain (Fig 2-4). The heavy chains have head regions and long -helical tails, which wrap around one another to form dimers. Myosin’s head domain exerts force by binding to an actin ber through ATP hydrolysis and it \walks » towards the ‘+’ end of an actin lament through successive movements: the head domain binds, undergoes a conformational change, releases from the lament and nally undergoes a conformational reversal to prepare for another step cycle.
For each step, myosin can cause a displacement of 5-10nm and generate 1-5pN of force[3]. Myosin II can assemble together with the tail of one myosin II interacting with the tails of adjacent motors. Together the group of myosins can form a \thick lament » with many heads available for actin binding on both ends. [7]
Figure 2-4: A) Myosin II: Myosin II is comprised of three domains, the head, neck and tail regions [3]. B) Actin stress ber: Myosin II (blue) head domains bind to actin laments (red) in a bipolar manner. ATPase activity results in the movement of actin bers in an anti-parallel manner [7].
Stress Fibers
The actin stress ber is a contractile structure composed of actin laments and myosin II motors. The stress ber is formed from bundles of actin laments of alter-nating polarity cross linked by -actinin (Fig 2-4B)[8]. Thick laments of myosin bind to actin in a bipolar manner pulling bers together in an antiparallel con guration. The bundles, or stress bers are commonly anchored to the cell plasma membrane through substrate adhesions or ‘focal adhesions’ (discussed further in section2.2.2) but their morphology and position in the cell can vary. Stress bers inside a single cell are divided into four main categories: dorsal, ventral, transverse arcs and perinu-clear actin cap. The dorsal stress bers are connected to the substrate at a single end, ventral bers possess two anchors while the transverse arcs lack a direct attachment (Fig 2-5). The perinuclear actin cap forms a dome-like actin cap on top of the nucleus and has been shown to regulate nuclear shape, for example under cell con nement[9] by providing a lateral compressive force[10].
Stress bers assemble or rearrange when the cell encounters a mechanical force and their thickness and orientation inside a cell has been found to depend on the rigidity of the substrate a cell is plated on[11]. Generally stress bers are well de ned in cells plated on sti substrates and weakly de ned or absent from cells grown on soft substrates[12].
Figure 2-5: Actin stress ber structure: A) U2OS osteosarcoma cell stained for F-actin. Three types of stress bers are highlighted in this cell, dorsal stress bers (red), arcs (yellow), and ventral stress bers (green). Scale bar 10 m B) Ventral stress bers are present on the bottom of the cell below the nucleus. Dorsal stress bers connect the focal contacts along the cell edge up to the transverse arcs stress bers present at the cell surface. [13]

Intermediate laments

Intermediate laments are the most diverse cytoskeletal component and are comprised of numerous polypeptides which can vary across cell type. Some common building blocks of intermediate laments are lamins, vimentin, and keratins. In neurons and epidermal cells these are 10 times more abundant than actin laments. Unlike other cytoskeletal laments, intermediate laments do not have a polarity. Intermediate lament associated proteins crosslink individual laments into a staggered array which bundles into a larger ber(Fig. 2-6). Intermediate laments can link to microtubules, actin laments, the plasma membrane and the nuclear surface interconnecting parts of the cytoskeleton thus playing an integral role in the stability of the cytoskeleton[14].
Figure 2-6: Intermediate laments are formed of smaller laments that align to form a staggered array and twist into a rope-like structure. [ laments-14706444]


Microtubules are hollow cylinders formed from 13 pro lament strands (in mammalian cells) which contain alpha and beta forms of the globular protein tubulin. Similar to actin laments, microtubules are polar and dynamic polymerizing and depolymerizing at both ends to adjust their size (Fig. 2-7). The tubules are typically nucleated from and attached to one end to the microtubule organizing center, or centrosome and while they grow to an average of 25 m in length their size varies between cell type[15]. The tubules support the migration of kinesines and dyneines two molecular motors which can move organelles and vesicles through the cell. Microtubules also play a signi cant role in cell division as they form a major structural component of the mitotic spindle which segregates a cell’s chromosomes.
Figure 2-7: A schematic of microtubules and their growth mechanism. Micro-tubules are composed of – and – tubulin subunits that are assembled into pro-laments. A single microtubule in a mammalian cell contains 13 pro laments that wind together to form a cylinder of 24nm in diameter. Microtubules are dynamic in structure and can grow through polymerization and shrink via depolymerization. [ laments-14052932]

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Cell Junctions and Adhesions

After discussing the properties of a cell’s cytoskeleton we now consider the manner in which the cytoskeleton and the cell in general connect to the surrounding environment. These connections can appear as either adhesions to neighboring cells or as adhesions to a substrate.

Cell-cell Junctions

Epithelial cells are joined to their neighbors in a manner that can be classi ed ac-cording to nature and function into three categories: anchoring junctions, occluding junctions and communicating junctions.
Communicating junctions (gap junctions for epithelial cells) enable the electric and chemical cell-cell communication.
Occluding junctions (tight junctions for vertebrates) seal the cells together, selectively inhibiting molecules from penetrating the epithelium or membrane proteins and lipids from mixing between di erent domains of the cell membrane. Tight junctions divide di erent membrane regions of an epithelial cell and their role in polarizing epithelial tissue is discussed further in section (2.2.3) on cell polarity.
Anchoring junctions mechanically link the cytoskeleton of two neighboring cells. Anchoring junctions are further subdivided into two categories according to the cytoskeleton lament they bind to. These are termed adherens junctions, that link to actin laments and microtubules and desmosomes that link to intermediate laments.
Here, focus will be placed speci cally on the anchoring junctions as they are attached to the cytoskeleton and the thesis also focuses on unraveling the complexity of the multicellular cytoskeletal organization.

Adherens junctions

Adherens junctions (Fig. 2-8) contribute to the strength of the mechanical link between neighboring cells in the epithelium and aid establishing apical-basal polar-ity across a cell sheet. Adherens junctions are more basal than tight junctions and in some epitheliums adherens junctions completely enclose the circumference of the cell and are thus associated with an actin belt of F-actin that also surrounds the cell. An important component of adherens junctions is a family of cadherin trans-membrane, calcium dependent proteins. In epithelial cells E-cadherin together with the cytoplasmic molecule catenin forms the basis of mature adherens junctions. The cadherin- -catenin complex binds to -catenin which subsequently binds either di-rectly to actin or to actin associated proteins [16].
In addition to connecting with actin, an adherens junction protein also associates with microtubules[17]. The plus end of radially extending microtubules can therefore be targeted to adherens junctions and the presence of microtubules at the junctions aids in the further accumulation of junctional E-cadherin [18].
Figure 2-8: A schematic of adherens junctions between adjacent cells. Cadherins link neighboring cells as the extracellular domain of one cadherin dimer links to the extracellular domain of the cadherin in a neighboring cell. As well, rings of lamentous actin form a belt around the junctions inside the cell. [3].


Desmosomes link intermediate laments to the plasma membrane and are crucial in maintaining the mechanical integrity of an epithelium. Desmosomes assist cells in relieving mechanical stress as they support \hyperadhesive » junctions, namely junc-tions that remain intact during calcium depletion. The strength of these junctions, however, can be modulated through intracellular signaling and desmosomes strength is often varied over the course of wound healing and some parts of development. In MDCK cells for example, calcium independent desmosomes are never present before con uence, while after con uence nearly all the cells develop such junctions over a period of days [19].

Cell-substrate adhesion

In order to move in vivo, a cell must form adhesions with a surrounding sca old called the extra-cellular matrix (ECM). The main components of the ECM are collagen I and IV, laminin and bronectin. When a tissue is grown on a virgin substrate, the cells must rst produce ECM on the substrate and then build adhesion between it and its basal membrane. These cell-substrate adhesions can be classi ed into two categories: focal adhesions and hemidesmodomes each of which link actin laments and intermediate laments to ECM. Properties of focal adhesions will be presented here as these adhesions are studied during the thesis.

Focal adhesions

Focal adhesions tie a cell to a substrate by linking actin stress bers to the ex-tra cellular matrix via transmembrane integrin receptors. In a migrating cell, focal adhesions are preceded by focal complexes. Focal complexes are formed toward the leading edge of the migrating cell and are smaller protein complexes comprised mainly of vinculin and talin which aggregate near the membrane on the cytoplasm side and link the actin lament to an integrin. The integrin spans the membrane and attaches to the extracellular matrix outside the cell (Fig. 2-9). Focal adhesions are matured focal complexes and are more stable structures comprised of vinculin, and talin but also other proteins such as paxillin, lamin, alpha-actinin, and zyxin.
Similarly to stress bers, focal adhesions react to externally applied force and respond by growing in size[20]. Focal adhesions are clearly de ned on 2d cultures and their size is correlated with cell migration speed[21]. Cells migrating in 3d envi-ronments, such as gels, have been observed to possess a di use distribution of focal adhesion proteins unlike in 2d where they self-assemble. The presence of these pro-teins, however, has been found to regulate migration dynamics by controlling cell protrusions and the ability of a cell to deform the surrounding matrix[22].

Cell Polarity


Most epithelial cells exist in cohesive sheets or monolayers with an asymmetric struc-tural organization, or polarity. Polarized epithelial cells possess apical and basolateral domains each with a distinguished by domain-speci c proteins that form an apical-basal axis. The development of apical-basal cell polarity is a complex mechanism resulting in separated specialized domains on the plasma membrane and in the cyto-plasm. Generally, an extrinsic cue from a cell’s environment, for example an adhesion either to extra cellular matrix, or neighboring cells, initiates the development of cell polarity.
A polarity axis inside a cell leads to directed behaviors such as uid secretion and transport. In vivo the apical domain commonly faces the lumen of internal cavities or the outside surface of the body. The apical surface facing the cavity therefore possesses a higher positive curvature with respect to the external basal surface ( g 2-10A). When present, cilia, and microvilli localize on the apical surface and the basal surface binds to a basement membrane through focal adhesions. Cells can contribute to an existing basement membrane by secreting their own layer of extra cellular matrix entitled ’basal lamina’ a layer 20-300nm thick comprised predominantly of laminin and type IV collagen[23].
In vitro, MDCK cells embedded inside gels develop polarized cysts which can form tubular branches when stimulated with HGF[24]. These three dimensional structures have an apical-basal polarity similar to that of in vivo tubes with their apical side facing the lumen. The polarity of MDCK cysts in vitro, however, is reversed when they are grown in suspension. In this condition their basal surface faces the lumen, secreting basal lamina into the cavity Fig.(2-10B) [?].
Figure 2-10: A) An epithelial tube with the polarity found either in vivo or in tubes and cysts grown in gels in vitro. The apical membrane faces the lumen of the tube and the basolateral membrane faces the exterior of the tube. In this geometry, the apical membrane is more curved than the basolateral surface. B) An example of inverted polarity seen in suspended epithelial MDCK cysts. Cells secrete ECM inside the cavity thus developing a basolateral membrane inside and an apical side on the outside of the cyst facing the medium. Here, the basolateral surface of is at higher curvature than its apical membrane. [25]

Tight junctions

Apical and basolateral domains are separated by ring of tight junctions located towards the upper part of the lateral cell surface. Tight junctions constitute a physical boundary between the two parts of the membrane preventing the di usion of speci c proteins and lipids.
Figure 2-11: a) Morphology of a polarized epithelial monolayer. Vertical epithelial polar-ization is divided into distinct apical, basal, and lateral domains of the plasma membrane. Apical surfaces often contain microvilli which are used for absorption and excretion. The basal surface binds to a brous extracellular matrix (ECM) through transmembrane integrin which is part of focal adhesions. Lateral membranes are linked to adjacent cells through junctional complexes (tight junctions, adherens junctions, and desmosomes) and provide di usion barriers. b) Horizontal epithelial polarization of planar cell polarity (PCP) pro-teins forms the proximal-distal axis in the plane of the epithelium. PCP can cause di erent proteins to localize to the proximal and distal cortical domains; here the di erential protein localizations are illustrated in green and red. [26]

Table of contents :

1 French abstract 
2 Introduction 
2.1 Forward
2.2 Epithelial cells and their functions
2.2.1 Single cell architecture: the cytoskeleton
2.2.2 Cell Junctions and Adhesions
2.2.3 Cell Polarity
2.2.4 Multicellular architecture
2.3 In vitro models of epithelia
2.3.1 Collective cell migration, tissue dynamics, ngers and complex ow patterns
2.3.2 Actin cables during re-epithelialization
2.3.3 Population connement
2.3.4 Limitations on 2D models
2.4 Assembling epithelial tissues in vivo: tubulogenesis
2.4.1 Tube formation
2.4.2 Tube elongation
2.4.3 Tube diameter regulation
2.5 Two case studies of in vivo curvature
2.5.1 Drosophila trachea
2.5.2 Drosophila egg chamber
2.6 In vitro studies of curvature
2.6.1 Denition of terms: curvature
2.6.2 Two dimensional in-plane curvature assays
2.6.3 Three dimensional curvature assays
2.6.4 Out-of-plane curvature assays
3 Materials and Methods 
3.1 Surface fabrication
3.1.1 Pillar assays
3.1.2 Glass wires
3.1.3 Polystyrene (PS) wires
3.2 Surface coating
3.2.1 Fibronectin
3.2.2 Pll-Peg
3.2.3 Micropatterning
3.3 Cell culture
3.3.1 Cell lines
3.3.2 Culture protocols
3.4 Microscopy
3.4.1 Video microscopy
3.4.2 Confocal spinning disk microscopy
3.4.3 Two photon laser ablation
3.5 Immuno uorescence
3.6 Drug Inhibitions
3.7 Image processing
3.7.1 Image projections
3.7.2 Image feature orientation
4 Results & Discussion 
4.1 Pillar vs. wire assays
4.2 Static properties of the monolayer
4.2.1 Polarity
4.2.2 Cell morphology
4.2.3 Density proles
4.3 Curvature-induced EMT
4.3.1 Connement vs. curvature
4.4 Monolayer molecular architecture
4.4.1 Actin cytoskeleton alignment
4.4.2 Photoablation of stress bers and cables
4.5 Collective migration
4.5.1 Front velocity vs. radius
4.5.2 Connement vs. curvature
4.5.3 Mechanisms governing collective migration
4.5.4 Theoretical models of migration
4.6 Extreme curvatures: cone tips
5 Conclusion


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