Breaking the Lamina network to allow nuclear passage through narrow pores 

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The cytoskeleton as a load bearing structure

The cytoskeleton is a three dimensions network made of bio-polymers. Mechanical loads applied on the cell are supposed to be held by the cytoskeleton. As for other networks of bio-polymer, any deformation leads to a certain loss of energy in the form of heat [Starodubtseva 2011]. Therefore, the cytoskeleton is often described as a viscoelastic material meaning that it exhibits both elastic as well as viscous properties when it undergoes deformations.
Elasticity is a measure of the capacity of an object to come back to its original shape after deformation.
The main elastic modulii used for the definition of cellular mechanical properties are the tensile modulus (Young’s modulus E), the shear modulus (G), the bulk modulus (K) and the bending modulus (). The tensile modulus defines the response of an object under uniaxial tension while the bulk modulus describes the object deformation under compression. The bending modulus defines the object resistance to bending along its length and the shear modulus sets the response of an object to a shear stress (illustrated in figure 3.1). Until now, most of the mechanical measurements of the cytoskeleton have focused on the shear elastic and on the shear viscous modulii. The shear elastic modulus measures how mechanical energy is store while the shear viscous modulus measures the dissipation of mechanical energy in a system. They are usually determined by the relation between the applied stress and the resulting strain. The cell contains three types of bio-polymers: actin filaments, microtubules and intermediate filaments. Their organization, dynamics and interaction plays an important role in the mechanical properties of a cell. In the following paragraph, I will describe the mechanical properties of those bio-polymers with a main focus on the actin cytoskeleton which has been at the heart of my PhD work.

The actin cytoskeleton

Actin is the most abundant protein in a cell where it can exist at as high concentration as 300 M [Blanchoin 2014]. In most eukaryotic cells, globular monomeric actin (G-actin) assembles into dimers before polymerizing with a helical arrangement of the monomers [Pollard 2009] to form 5nm diameter filaments (F-actin) [Gardel 2008]. F-actin is a polar filament as actin subunits in the filament are oriented towards the same direction. It also has a fast growing end, the barbed end in opposition to the pointed end which elongates much slower [Pollard 2009]. ATP-G-actin dimers are added at the barbed end, ATP is then hydrolyzed and releases a phosphate. ADP-G-actin is removed from the pointed end of the filament leading to depolymerization of the F-actin. ADP in the ADP-G-actin is exchanged for ATP making the G-actin available for another round of polymerization [Baum 2005].
F-actin length is regulated by capping proteins which through their binding to either the barbed or pointed end, prevent the adding or removal of G-actin. Severing proteins such as ADF/Cofilin, cleave actin filaments thus stopping the filament growth.
Because actin plays a central role in many cellular processes, it has been the subject of many studies. In vitro studies on single actin filaments measured a mean persistence length of 8-17 m [Gittes 1993], a young modulus of 109 pascal ([Kojima 1994], [Gardel 2008]) and a buckling force (minimal force to apply for bending anactin filament) of 0.4 pN to 1.6 pN for a micrometer long filament [Berro 2007]. With such a persistence length of the order of magnitude of the cell diameter, actin filaments should behave like rigid rods inside the cell.

Formins based actin networks

Formins form a large family of several proteins whose role in actin polymerization was described in 2002 [Breitsprecher 2013]. They are characterized by their formin homology 1 and 2 domains at their C-terminus (FH1 and FH2 respectively). The  H2 domain has been shown to promote the nucleation of actin filaments in vitro ([Pruyne 2002], [Sagot 2002]). The proposed mechanism of actin nucleation by formins relies on the high binding anity of the donut-shaped FH2 domain to the barbed end of F-actin [Chesarone 2009]. Because the FH2 domain lacks detectable binding anity to G-actin, it has thus been proposed that formins promote actin polymerization by stabilizing spontaneously formed actin dimers or trimers. After polymerization, the formin FH2 domain stays at the barbed end of F-actin and moves progressively with the barbed end allowing rapid G-actin insertion ([Chesarone 2009], [Breitsprecher 2013]).
This current model of FH2 based actin nucleation is under controversy. Indeed the FH2 domain based actin nucleation was shown to be very inecient with profilin bound G-actin which is the most available actin substrate in vivo [Chesarone 2010]. However, one hypothesis which would support the FH2 based actin nucleation theory is the recruitment of G-actin by sequences at the C-terminus of the FH2 side [Chen 2010] or by FH2 partners (in the same way than class I NPFs bind G-actin and Arp2/3). If there are some evidences of the existence of formins regulators which bind to G-actin (bud 6 a Saccharomyces cerevisiae polarity factor [Moseley 2004]) a direct proof of such a mechanism is still lacking. Therefore, the mechanism of Formin based actin nucleation is still far from being understood.

Actin networks and their mechanical properties

Actin networks are usually crosslinked in migrating cells. Indeed, the lammelipodium at the leading edge is made of branched actin networks, the contractile fibers (stress fibers and transverse arcs), are based on bundled actin filaments. The combination of branched and bundled filaments is found in the cortex and to some extent at the leading edge, between the lammelipodia and the filopodia (see figure 3.3).
The global eect of crosslinkers in an actin network is to link actin filaments together. Some crosslinkers such as Myosin II or Fascin will keep the filaments aligned creating a bundled network while others such as 􀀀actinin, Filamin and Fimbrin will entangle the filaments leading to a branched actin network. It is also interesting to underline the role of Arp2/3 in network generation.

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Table of contents :

0.1 Abstract
0.2 Foreword
1 Migration: a multiscale process 
1.1 Migration: a multiscales process
1.2 Cell migration
1.2.1 Cell migration in health and disease
1.2.1.1 Cell migration during embryogenesis
1.2.1.1.1 Neural crest cells migration:
1.2.1.2 Cell migration to maintain tissue homeostasis
1.2.1.2.1 Cell migration during wound healing
1.2.1.3 Metastasis formation: when cell migration goes out of control
1.2.1.3.1 Some history:
1.2.1.3.2 Cancer cell migration:
2 Cell migration in the context of the immune response 
2.1 Introduction to the immune system
2.1.0.4 Some history:
2.2 The immune system
2.2.1 Innate immunity
2.2.2 Motiles cells in innate immunity
2.2.2.1 Macrophages: the long lived cells
2.2.2.2 Neutrophils: the short lived cells
2.2.2.3 The natural killers cells
2.2.2.4 Dendritic cells at the interface between innate and adaptive immunity
2.2.3 Adaptive immunity
2.2.3.1 The T lymphocytes
2.2.3.2 The B lymphocytes
2.2.3.3 Generation of memory
2.3 Dendritic cells
2.3.1 Antigen uptake in dendritic cells
2.3.2 From an immature to a mature state
2.3.3 Dendritic cells migratory routes
3 The physics of cell migration 
3.1 Cell mechanics in migration
3.1.1 The cytoskeleton as a load bearing structure
3.1.1.1 The actin cytoskeleton
3.1.1.1.1 Actin polymerization:
3.1.1.1.1.1 Arp2/3 based actin networks:
3.1.1.1.1.2 Formins based actin networks
3.1.1.2 Actin networks and their mechanical properties
3.1.1.2.1 Actin networks mechanical properties
3.1.1.2.2 Mechanical properties of dendritic actin network
3.1.1.2.3 Mechanical properties of unbranched actin network
3.1.1.2.4 Force production by actin networks
3.1.1.2.4.1 Contractile forces
3.1.1.2.4.2 Protrusive forces
3.1.1.2.4.3 An exemple of actin based motility: actin comet tails
3.1.1.3 The microtubule network
3.1.1.3.1 Crosstalk between microtubules and actin:
3.1.1.4 The intermediate filaments
3.1.2 Proposed model of the cytoplasm
3.1.3 Contribution of the plasma membrane to the cell mechanics
3.1.4 The nucleus in cell mechanics
3.2 Physical properties of the extracellular environment
3.2.1 in vivo environments
3.2.2 In vitro assays for mimicking in vivo environments
3.2.2.1 The 2D migration assay
3.2.2.2 3D collagen gels
3.2.2.3 1D/3D migration assays
3.3 Mechanotransduction in cell migration
3.3.1 Ions channels in mechanotransduction
3.3.1.0.1 Role of membrane potential in cell migration
3.3.1.0.2 Role of cell volume in cell migration
3.3.1.0.3 Calcium signalling in cell migration
3.3.1.0.4 pH in cell migration
3.4 Mechanism of cell migration
3.4.1 2D/planar migration
3.4.1.1 Adhesion dynamics
3.4.1.2 The molecular clutch model
3.4.2 3D cell migration
3.4.2.1 3D mesenchymal migration
3.4.2.2 3D amoeboid migration
3.4.2.2.1 Mechanism of blebbing migration
3.4.2.2.1.1 Myosin II as force generator
3.4.2.2.1.2 Models for force transmission in blebbing motility
3.4.2.2.2 Polymerization driven amoeboid migration
3.4.2.3 Switching migration mode as function of the ECM physical and chemical properties
3.4.3 1D-3D cell migration
3.4.3.1 Actin slab at the leading edge of neutrophils undergoing interstitial migration
3.4.3.2 Pushing on the walls to move forward
3.4.3.3 Interstitial migration independent of the actomyosin system: the osmotic engine model
4 Nuclear mechanics in cell migration 
4.1 Nuclear mechanics
4.1.1 The nucleoplasm define the nuclear rheological properties
4.1.2 Mechanical properties of the lamina network
4.1.2.1 Structure of the lamina network
4.1.2.2 Lamina mechanics
4.2 Linking the nucleus to the cytoplasm
4.2.1 The LINC Complex
4.2.1.1 The SUN-domain proteins:
4.2.1.2 The Kash-domain proteins
4.3 Nuclear mechanics in health and disease
4.3.1 Laminopathies
4.3.1.1 The structural hypothesis
4.3.1.2 The genome regulation hypothesis
4.4 The nucleus during cell migration
4.4.1 Nuclear positioning
4.4.1.1 Microtubules based nuclear positioning
4.4.1.2 Actin based nuclear positioning
4.4.1.2.1 The Transmembrane Actin-associated Nuclear (TAN) lines
4.4.2 The nucleus in mechanotransduction
4.4.2.1 Can the nucleus feel the force?
4.4.2.1.1 The actin caps
4.4.2.2 How could the nucleus react to forces?
4.4.3 The nucleus as limiting factor for cell migration
5 Objectives 
6 Methods 
6.1 Cellular models for cell migration
6.1.1 Dendritic cells as model system
6.1.1.0.1 Dendritic cells maturation
6.1.1.0.2 Genetic manipulation of dendritic cells
6.1.2 Neutrophils as a second model
6.2 Microchannels as an original setup for transmigration
6.2.1 The common setup: transwells
6.2.2 The microfabrication based setup: microchannels with constrictions .
6.2.2.1 From soft photolithography to PDMS chambers
6.2.2.2 3D visualization of the channels geometry
6.2.2.2.1 Optical profilometer
6.2.2.2.2 Confocal imaging based channels measurement
6.3 Live cell imaging of cells migrating through constrictions
6.3.1 Channels preparation
6.3.2 Putting cells in channels
6.3.3 Video microscopy of cells crawling through constrictions
6.4 Immunostaining in microchannels
7 Results 
7.1 An Arp2/3 based nuclear squeezing allows dendritic cells to passage through micrometric constrictions
7.1.1 Summary
7.2 Manuscript in preparation for submission
7.2.1 Main text
7.2.2 Remarks
7.2.2.1 Analysis to perform
7.2.2.2 Experiments planed before submission
7.2.2.3 More general remarks
8 Discussion 
8.1 An Arp2/3 based nuclear squeezing mechanism allows immature dendritic cells to pass through narrow gaps
8.2 The nucleus as a limiting factor for dendritic cells migration
8.2.1 A microchannel based set up
8.2.2 The nucleus limits migration below 12 m2 constrictions
8.3 Mechanisms of perinuclear actin meshwork formation
8.3.1 A retrograde flow based actin increase
8.3.2 Adhesion based actin formation
8.3.3 Microtubules based F-actin recruitment
8.3.4 Arp2/3 based nucleation
8.3.4.1 Arp2/3 recruitment at the nucleus
8.3.4.2 NPFs confinement induced Arp2/3 activation
8.3.5 Conclusion
8.4 Role of the actin accumulation in nuclear passage through constrictions
8.4.1 Breaking the Lamina network to allow nuclear passage through narrow pores
8.4.2 Phosphorilation based nuclear passage through constrictions
8.4.3 How are forces transmitted to the nucleus
8.4.4 Conclusion on the role of the perinuclear actin meshwork in constrictions
8.5 Role of myosin II in nuclear squeezing
8.6 Squeezing the nucleus through a small pore
8.6.1 Our model of nuclear squeezing during cell migration
8.6.2 Limits of this model
8.7 Perspectives
8.7.1 An intriguing Arp2/3 based nuclear squeezing
8.7.2 Cell survival during migration: role of the LINC complex
8.7.3 Importance of nuclear squeezing for in vivo cell migration
9 Conclusion 
Bibliography 
A Appendix 1 : Research article 
A.1 A computational mechanics approach to assess the link between cell morphology and forces during confined migration

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