An Arp2/3 based nuclear squeezing allows dendritic cells to passage through micrometric constrictions

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Motiles cells in innate immunity

The macrophage, neutrophils, natural killer and dendritic cells are at the first line of the innate immune response. Indeed, upon invasion, those cells will move towards the site of infection to kill pathogens as well as infected cells. In vertebrate, the movement of those cells, particularly dendritic cells, is also essential to activate the adaptive immune response.

Macrophages: the long lived cells

In vertebrates, macrophages are the cells that first encounter invaders. They are indeed living in tissues all around the body with a particular concentration in sites where infection has a higher prob-ability to occur: connective tissues, liver, spleen. They are rapidly recruited to site of infection where they will display a set of machineries to phagocyte and kill pathogens. Their activation at sites of infection, leads to the production of cytokines that can activate other phagocytic cells such as neu-trophils but also triggers fever which by rising the body temperature inhibit the growth of pathogens. Macrophages often survive their first encounter with pathogens, they then keep patrolling the body waiting for the next invasion. They are thus the most long lived cells among the ones involved in innate immunity.(Adapted from [Alberts 2007])

Neutrophils: the short lived cells

Neutrophils are usually present in the blood stream and are rapidly recruited to sites of infection by activated macrophages, by molecules specific to the pathogens such as the formylmethionine-containing peptides or « by peptides fragments from the complement components » [Alberts 2007, p. 1528]. They are highly phagocytic cells which will usually die at the site of infection. As they are short lived cells, they can use their own DNA as nets to trap pathogens [Alberts 2007, p. 1532].

The natural killers cells

Natural killer cells target virus infected cells and trigger their apoptosis. They recognize infected cells through their levels of expression of a class of proteins called the Major Histocompatibility Complex (MHC) of class I. Indeed, many viral infections lead to the down regulation of the MHC class I as those proteins are used to present antigen to cytotoxic T lymphocytes during the adaptive immune response (see 2.2.3.1). By lowering the amount of MHC class I, viruses escape from the cytotoxic T cells but are then detected by natural killer cells. The apoptosis pathway will be activated in infected cells before viral replication [Alberts 2007, p. 1536].

Dendritic cells at the interface between innate and adaptive immunity

Dendritic cells are also phagocitic cells with a large variety of PRRs which allows them to detect a wide variety of pathogens. Once they encounter a pathogen, they will cleave it into peptide fragments which will be presented at their surface with MHC proteins. They will then carry such complexes toward the nearby lymphoid organ where they activate T cells. This will trigger the adaptive immune response. Dendritic cells are thus essential linkers between the innate and the adaptive immunity. (Adapted from [Alberts 2007])

Adaptive immunity

The adaptive immunity also called acquired immunity arose 500 millions year ago in vertebrates. While the innate immunity displays a common mechanism to fight against pathogens, the response gets more sophisticated and specific when the adaptive immune system comes to play. This highly specific response is carried out by Lymphocytes: the T and B lymphocytes.
Upon infection, activated dendritic cells migrate towards the nearest lymphoid organ where they ac-tivate T cells. T Cells activation occurs after the recognition of the antigen presented on the MHC proteins of the dendritic cells associated to some co-stimulatory molecules. This will lead to the on-set of the migration of some T cells towards the sites of infection where they will participate to the destruction of pathogens and infected cells.
Other dendritic cells activated T cells will remain in the lymphoid organ where they will keep den-dritic cells active in order to activate more T cells and help activating B lymphocytes which will produce antibodies against the pathogen. The adaptive immune response is then displayed in two phase: the humoral immunity based on B cells antibody production and the cell-mediated immunity based on T lymphocytes. In the following paragraph, I will describe the keys players of the adap-tive immune response: the T and B lymphocytes. Even though they derive from the same common lymphoid progenitor cells (see figure 2.1), they display specific feature in their fight against infection.

Antigen uptake in dendritic cells

Antigen uptake is a fundamental process in the accomplishment of dendritic cells task during immune response. Indeed, its role as an antigen presenting cell requires the ability to engulf pathogens and present antigens at its surface. Di erent modes of antigen uptake have been describe in dendritic cells. According to the size of the element to internalize, the cell will perform either phagocytosis, endocy-tosis or, macropynocytosis. Indeed, particles bigger than 0.5mm will be internalized by phagocytosis while smaller particles will be internalized by endocytosis through specific receptors/cargo interac-tion. Macropanocytosis is used to internalize fluid, it does not requires receptor/cargo interaction which makes it unspecific.
After internalization, two routes can be taken by the particle. In the first case, the particle will be released from endosomes to the cytoplasm where it will be cleaved into small peptides before being transported towards the endoplasmic reticulum by the ABC transporter where it will bind to the MHC class I molecules [Alberts 2007, p. 1583]. In the second case, the particle will be released from the early endosome to the late endosome where it will be joined by the complex MHC class II/Invarian chain. The release of the invarian chain from the MHC class II allows its binding to the particle. The MHC/particle complex is then targeted to the plasma membrane where it will be recognize by the T lymphocyte. Note that the MHC class I is recognized by the cytotoxic T cells while the MHC class II is recognized by the Helper T cells [Alberts 2007, p. 1584]. The dendritic cell , by activating di erent subset of e ector T cells will then define the type of adaptive immune response that will be displayed by the host.
The interaction between T cells and dendritic cells should lead to the activation of the T cell only if the dendritic cell is activated. Indeed, if the dendritic cell has not been activated, the activation of the T cell triggers apostosis. The activation of dendritic cells is thus an important process in the adaptive immune response.

From an immature to a mature state

Upon recognition of a so called « danger signal », the immature dendritic cell enters a di erentiation process to become a mature dendritic cells. A global set of particles including PAMPs, cytokines pro-duced by macrophages or neutrophiles can be recognized as danger signal [Reis e Sousa 2004]. The maturation of dendritic cells leads to the up-regulation of MHC-peptide complexes and co-stimulatory proteins as well as the production of immunomodulatory cytokines, all this has a major role in T cell Figure 2.3: « Pathways of entry into cells. Large particles can be taken up by phagocytosis, whereas fluid uptake occurs by macropinocytosis. Both processes appear to be triggered by and are depen-dent on actin-mediated remodeling of the plasma membrane at a large scale. Compared with the other endocytic pathways, the size of the vesicles formed by phagocytosis and macropinocytosis is much larger. Numerous cargoes can be endocytosed by mechanisms that are independent of the coat protein clathrin and the fission GTPase, dynamin. Most internalized cargoes are delivered to the early endosome via vesicular (clathrin- or caveolin-coated vesicles) or tubular intermediates (known as clathrin- and dynamin independent carriers (CLICs)) that are derived from the plasma membrane. Some pathways may first tra c to intermediate compartments, such as the caveosome or glycosyl phosphatidylinositol-anchored protein enriched early endosomal compartments (GEEC), en route to the early endosome. » Adapted from [Mayor 2007] activation and di erentiation. Another aspect of dendritic cells maturation is their upregulation of a chemokine receptor (CCR7) which through binding to the ligands CCL21, CCL19 directs mature dendritic cell migration towards the nearest lymphoid organs through the a erent lymphatic vessels [Weber 2013].

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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

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