Collective cell migration, tissue dynamics, ngers and complex ow patterns

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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 scaold or cytoskeleton inside the cytoplasm that dynamically grows and rearranges through polymerization and depolymerization. The cytoskeleton attaches to its environment through substrate adhesions and cell junctions. Finally, 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 ‘micro laments’ of diameter 5-9nm), intermediate laments (diameter 10nm), and microtubules (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.

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.
Epithelial cells are joined to their neighbors in a manner that can be classied according 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 dierent domains of the cell membrane.
Tight junctions divide dierent 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 specically 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.

In vitro models of epithelia

Epithelial tissue formation is a complex collective process of cell growth, rearrangement and migration, as presented brie y above (sec. 2.2.4) and further in the following chapter (2.4). Much work has been dedicated to understanding the mechanics of how groups of epithelial cells interact and move together collectively. However, given the many challenges associated with monitoring and quantifying epithelial tissues in vivo, in vitro systems have been extensively employed.
The following discussion consequently focuses on in vitro studies of two dimensional at epithelial tissues. The goals of this presentation are twofold. First, to demonstrate important information can be extracted from 2d in vitro models which mimic many characteristics of in vivo tissues and secondly, to present the limitations these systems have on describing some aspects of three dimensional tissue, for example its out-of-plane curvature.
Examples of two dimensional assays that expose some properties of epithelial tissue are discussed below. These assays applied on MDCK cells reveal spatial correlations during migration, the dependency of wound geometry on the formation and function of actin cables during re-epithelization, and eects of population connement on epithelial tissues.

Collective cell migration, tissue dynamics, ngers and complex ow patterns

Collective cell migration has been widely studied in 2d epithelial tissue since the presence of strong junctions between cells help form complex correlated movements. The scratch wound assay is the traditional technique employed to study collective migration. Cells are grown to con uence and then a portion of the monolayer is scraped away using a pipette tip or another sharp object[30]. Cells migrate to close the wound and migration parameters can be measured[31]. Scratching, however, does not yield reproducible wound geometries and damaged border cells not mechanically removed introduce changes in the local signaling environment [32].
To isolate the role of wound geometry in wound healing, MDCK cells were grown to con uence inside a PDMS micro-stencil. The stencil was removed and cells migrated outward into the newly created free space (Fig. 2-13). Clusters of cells migrated together creating ows inside the monolayer, with a correlation length of typically 10 cell sizes [33, 34]. In addition to velocity correlations inside the tissue, multicellular digitations or ngers form at the leading edge guided by a wide \leader » cell (Fig. 2- 14) moving almost two times as fast as the mean border speed[34]. The cells inside a nger have a preferential orientation and polarity compared to cells inside the monolayer[35]. Finally, collective patterns were also observed in the mechanics of the migrating tissue. Traction force microscopy on a migrating monolayer revealed mechanical waves driven by tissue expansion. These waves demonstrated that (at least for substrates of rigidity 3kPa) traction forces involved in migration originate across large distances in the tissue and not just at the leading ngers (Fig. 2-15) [36].

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Actin cables during re-epithelialization

Actomyosin cables, present in vivo as discussed above (section 2.2.4), can be recapitulated in vitro. Cells were seeded around PDMS pillars of radius 25-100m pressed onto a glass coverslip, conning their growth. Upon removal of the pillars at con uence, an actomyosin cable assembled in minutes around the wound edge[2, 1]. The actomyosin cable was present during the entire closure and ablation of the cable conrmed that it was exerting a centripetal force towards the center of the wound. However despite the elastic tension stored in the circular cable, it does not pull the wound closed using an in-vivo-like purse string mechanism described above. Inhibition experiments and analysis of the dynamics of the closure revealed that for wounds of diameter larger than a cell size the cable plays predominantly a regularization role at the front edge and active crawling of protrusive cells at the boundary is the principle propeller of closure.

Assembling epithelial tissues in vivo: tubulogenesis

Tissues exhibit an intermediary level of organizational complexity between a cell and an organ. Epithelial cells organize into tissues of tubes or cysts which function inside larger organs. Some organs, for example the kidney, possess a tubule network of varying diameters in dierent regions. Other epithelial tissues, for example the breast, bronchioles, pancreas, salivary glands, are formed from shorter tubular networks that nish in spherical acini as seen in gure 2-22c.Since this thesis focuses on work carried out on a cell line derived from kidney cells, below we will focus on the mechanisms of in vivo tube formation and acinus formation will not be discussed. As well, even though some mechanisms of tubulogenesis apply to both epithelial tissue as well as tubular endothelial vessels found throughout the body, focus will be placed on epithelia as endothelia are beyond the scope of this thesis.

Two case studies of in vivo curvature

The discussion of tubulogenesis above emphasized the wide variety of curved epithelial tissue present in vivo. In this thesis, out-of plane curvature is studied in a controlled manner by growing epithelial cells on curved substrates. Before describing such in vitro studies, two in vivo tissues will be presented: the drosophila trachea and the drosophila egg chamber, both examples of epithelia that develop on curved rigid extracellular matrix whose presence guides the alignment of actin across the tissue.
Although the polarity and localization of the actin is dierent in the two cases, each generates aligned actin bers on their surface facing the ECM. In the trachea, bers align on the the apical surface which is in contact with a luminal chitinous ECM while in the egg chamber bers orient on the basal side that contacts the layer of collagen IV ECM.

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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