Cytoskeletal crosstalk involving vimentin intermediate filaments and microtubules

Get Complete Project Material File(s) Now! »

Actin filaments regulate cell shape and movement

Discovered in the 1940s in muscle cells [Straub 1942], actin is the most studied subcomponent of the cytoskeleton so far. Actin forms filaments that are key players in mechanical support and in cell movement and that are implicated in a number of biological processes in eukaryotic cells. Providing an exhaustive list is not the aim here and we will only mention three of the most crucial biological processes involving actin in eukaryotic cells:
• Myosin motors bind actin and transport organelles along actin filaments. Myosin proteins – a superfamily of actin motor proteins – use cycles of Adenosine Triphosphate (ATP) hydrolysis to walk along actin filaments. In other words, myosins convert chemical energy (taken from ATP) into mechanical energy to generate forces and movements. In many eukaryotic cells, the actin network is referred to as the actomyosin network. Cells use myosin motors to transport organelles along actin filaments (see figure 1.3.C). For instance, myosin V walks from the pointed end to the barbed end, transporting vesicles and intracellular organelles.
• Contractile rings of actin filaments allow animal cells to perform cytokinesis. Other myosins, such as myosin II, are associated with contractile activity. During the last step of the cell cycle, the two daughter cells get separated in a process called cytokinesis. Several organisms, such as fungi, animals and amoebas, pinch their cells in two by using a contractile ring of actin filaments and myosin II. The process relies on myosin II polymerization into bipolar filaments able to produce a contraction by pulling actin filaments together (see figure 1.3.D). In animal cells, this contractile ring machinery is found in muscle cells, in which the actomyosin complex is very abundant.
• Cellular motility is achieved by actin filaments acting as a treadmill. The ability to migrate is a key feature of eukaryotic cells: embryonic morphogenesis mostly relies on cell migration; tissue integrity and regeneration is ensured by migrating cells; the motility of immune cells allows them to search and destroy pathogens; and many diseases – like cancer – take advantage of this cellular ability in order to move inside the organism. Cell polarization is essential to migration: the molecular processes at the front and at the back of a moving cell are different [Ridley 2003]. Actin plays a key part in the migration process which occurs in several steps. First, a complex linked to actin (called Arp2/3, see figure 1.3.A) initiates a protrusion at the front of the cell by growing branches of actin, making structures called lamellipodia and filopodia. These structures push the plasma membrane forward. During the extension of actin protrusions, the cell forms focal adhesions at the front of the cell to provide adhesion to the extra-cellular environment. Next, a forward force is generated by contractions of the actomyosin network. Finally, retraction fibres pull the rear of the cell in the direction of the leading edge [Mattila & Lappalainen 2008] (see figure 1.3.B). The treadmilling effect of actin is a meaningful description of this cycle of localized polymerizations and depolymerizations of actin which allows cells to move.

Microtubules provide a structural framework and orchestrate intracellular trafficking

Microtubules have become a resarch topic per se during the 1960s. Their association with several molecular motors and their dynamics make microtubules a key player in cell polarity, cell organization and intracellular transport. Here, we give three of the main functions of microtubules:
• Microtubules establish the global polarity of the cell. One of the most important properties of the microtubule network is that they are highly dynamic, constantly polymerizing and depolymerizing, as we will describe in 1.4.1. Microtubule half-time vary during the cell cycle, but it ranges from 10 s to 10 min. Interestingly, cells lacking a proper dynamic microtubule network – e.g. after using drugs affecting its dynamics – are often much less motile and poorly polarized. While actin structures are crucial in cell motility, it has been shown that microtubules induce cortical polarity and regulate actin dynamics [Siegrist & Doe 2007]. Microtubules, rather than establishing cell polarity, ensure its maintenance by mediating the distribution of inhibitory signals [Zhang et al. 2014]. Microtubules also impact the organization of the nucleus and of other organelles. For instance, in Xenopus, microtubule dynamics is balanced spatially and temporally for nuclear formation and its perturbation changes nuclear morphology [Xue et al. 2013].
• Molecular motors associate with microtubules to perform intracellular trafficking. As myosins bind actin filaments to transport vesicles along them, two types of molecular motors bind microtubules : kinesins and dynein. Discovered in 1985, kinesins are anterograde transport motors, which means that they transport vesicles, organelles and other cargoes towards the (+) ends of microtubules from the center of a cell to its periphery [Vale et al. 1985]. Fourteen families of human kinesins exist and all share a common amino acid sequence of the motor domain. Conversely, cytoplasmic dynein moves on microtubules towards their (-) ends: it is a retrograde transport motor. Dynein-induced retrograde transport is important to send endocytosis products to the center of the cell. As myosins, both kinesins and dynein convert the chemical energy stored in ATP into mechanical work.

READ  An Algorithm for Non-Redundant Sampling of RNA Secondary Structures 

Structure and assembly of intermediate filaments

• The tripartite monomer structure of intermediate filament proteins. Like all proteins, intermediate filament proteins have two ends: an amine group, called the N-terminus and a carboxylic group, called the C-terminus. As they are translated from messenger Ribonucleic Acid (RNA), they are created from N-terminus to C-terminus. The domain next to the N-terminus of intermediate filament proteins is called the head and the one next to the C-terminus is called the tail. Between these two domains, we can find several -helical structures: they form the rod (see figure 1.5.A). All intermediate filaments share this tripartite monomer structure. Under stress, the rod domain can stretch and some parts of the -helical domain can be uncoiled and form sheets instead [Qin et al. 2009]. Nuclear intermediate filaments – lamins – also have this structure. Their structural singularity relies on the much longer C-terminal domain in which can be found a Nuclear Localization Signal (NLS), required for transport into the nucleus (see figure 1.5.B-C that compares human lamin and human vimentin structures).
• Assembly of intermediate filament proteins depends on their type. All intermediate filaments are assembled from fibrous proteins that exhibit a central -helical rod domain which facilitates the formation of dimeric coiledcoil complexes. However, their assembly vary a lot depending on the type of intermediate filament protein involved. The first step common to all interme.

Cytoskeletal mechanics measurements in vitro

The cytoskeletal subcomponents come in many forms. They can be soluble proteins in the cytoplasm, form individual filaments, bundles1 or networks. Studies carried out in vitro reflect and explore all this hierarchical organization, from individual filaments to crosslinked networks. Experimental techniques are accordingly selected for each biological object which is being probed, and we will detail here the main methods that have been developed to study the mechanics of the cytoskeleton in vitro. We can distinguish between two different types of measurements of mechanical properties: active measurements, where a force is applied on the cytoskeletal filament and passive measurements, where spontaneous fluctuations give access to the mechanical properties.

Table of contents :

List of Acronyms
1 The cytoskeleton 
1.1 A network of crosslinked filamentous biopolymers
1.2 Functions of the cytoskeleton in eukaryotic cells
1.2.1 Actin filaments regulate cell shape and movement
1.2.2 Intermediate filaments maintain cellular integrity
1.2.3 Microtubules provide a structural framework and orchestrate intracellular trafficking
1.3 Intermediate filaments
1.3.1 Diversity of intermediate filaments in eukaryotic cells
1.3.2 Structure and assembly of intermediate filaments
1.3.3 Vimentin, an intermediate filament protein present in many cells
1.4 Microtubules
1.4.1 Dynamics of microtubules
1.4.2 Microtubules and post-translational modifications
2 Mechanical properties of the cytoskeleton 
2.1 Measuring mechanical properties in cell biology
2.1.1 Cytoskeletal mechanics measurements in vitro
2.1.2 Whole-cell-scale, cortical and intracellular force measurements
2.1.3 Our approach: combining optical tweezers-based intracellular rheology with live cell imaging
2.2 Mechanics of microtubules in vitro
2.2.1 Anisotropic stiffness of microtubules
2.2.2 Variability in the measurements of microtubule flexural rigidity
2.2.3 Microtubule response to repeated mechanical stress
2.3 Mechanics of intermediate filaments in vitro
2.3.1 Networks of intermediate filaments: highly deformable and almost unbreakable
2.3.2 Individual intermediate filaments exhibit nonlinear strain-stiffening
2.4 Microtubules and intermediate filaments in cellulo
2.4.1 Measuring mechanics of cytoskeletal filaments in cellulo
2.4.2 Mechanical contribution of microtubules in cells
2.4.3 Mechanical contribution of intermediate filaments in cells
3 Mechanical coupling within the cytoskeleton 
3.1 Cytoskeletal crosstalk involving vimentin intermediate filaments and microtubules
3.1.1 Crosstalk through molecular motors
3.1.2 Crosstalk through crosslinking proteins
3.2 Impact of cytoskeletal crosstalk on network organization and cell mechanics
3.2.1 Synergistic organization of cytoskeletal networks
3.2.2 Mechanical reinforcement mediated by cytoskeletal interactions
4 Aims of the PhD project 
5 Materials and Methods 
5.1 Cell culture
5.2 Immunofluorescence staining and fixed cell imaging
5.3 Live cell imaging of vimentin and tubulin in cellulo
5.4 Drugs targeting microtubules
5.5 ATP depletion
5.6 Optical tweezer-based microrheology
5.7 Data analysis: from the movies to the effective stiffness
5.8 Statistical tests
6 Results and Discussion 
6.1 Mechanics of vimentin bundles and microtubules
6.1.1 In cellulo, vimentin bundles are stiffer than microtubules
6.1.2 Sequential deflections make vimentin bundles more rigid
6.2 Mechanical coupling between microtubules and vimentin intermediate filaments
6.2.1 The vimentin network does not play a key role in the mechanical properties of microtubules
6.2.2 Modifying microtubule stability affects vimentin mechanical behaviour
6.3 Study of a post-translational modification: acetylation
6.3.1 Acetylation leads to microtubule softening
6.3.2 Acetylated microtubules impact vimentin bundle mechanics .
6.4 Preliminary results: role of ATP in cytoskeletal mechanics
7 Conclusion and Perspectives 
A Protocols 
A.1 Cell culture
A.2 Immunofluorescence staining
A.3 Live cell imaging of vimentin and tubulin in cellulo
A.4 Post-treatment of images before creating kymographs
B MATLAB codes 
C Power law analysis 


Related Posts