The LINC complex connects the cytoskeleton to the nucleoplasm

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IFs in cancer: focus on Glioblastoma

A glance at what happens in healthy astrocytes is therefore important to better understand how malignancies such as glioblastoma rise and can thrive and spread in the brain. Understanding how the cell interacts with the many different extracellular signalling cues seems to be key in finding remedies, and personalised therapies. Intermediate filaments in astrocytes seem to be at the centre of the cellular response to its environment and it is therefore important to study IFs, with higher magnification, in astrocytes.
Nestin in brain cancers. Nestin being a marker for poorly differentiated cells, and expressed when cells acquire the necessity to migrate and proliferate, it is used as a marker for a more defined grading of the tumour stages, especially tumour rising from progenitor cells. Logically, this protein is involved in cancer, especially in the different sorts of brain cancers, the CNS being one of the two regions expressing nestin. Intermediate filaments especially nestin co-expressed with vimentin are thought to control cell shape, morphology in GBM (Matsuda et al., 2013).
Nestin expression is tightly correlated with invasive phenotypes in astrocytoma cell lines (Rutka et al., 1999). The depletion of nestin in glioblastoma, the highest grade of gliomas, impairs their extension within the subventricular zone, acting through Notch and Kras signalling (Shih & Holland, 2006). Notch is a receptor in a simple signalling pathway indispensable for development and often implicated in the transformation of malignancy. Notch and its ligand are transmembrane proteins with an extracellular domain constituting epidermal growth factor (Bray, 2006). Notch in gliomas can directly act on nestin’s regulatory region to activate its expression (Shih & Holland, 2006).
GFAP in brain tumours. GFAP expression levels have been debated to be an indicator of malignancy level for gliomas. However, the serum level of GFAP could well serve as a diagnostic tool in glioblastomas. In a study driven by Jung et al, GFAP was found in the serum of glioblastoma multiform (GBM) patients before tumour resection at a median of 0.18μg/L, compared to the absence in non-GBM patients and healthy controls. Levels of GFAP in the serum also correlate with tumour size and necrosis, making GFAP a good potential biomarker for GBM (Jung et al., 2007; Tichy et al., 2016).
Vimentin in brain tumours. As for Nestin and GFAP, an increase in vimentin expression is observed in gliomas and GBM. High vimentin expression in glioblastoma is associated with a lower survival rate and lower progression-free survival (J. Zhao et al., 2018). Vimentin therefore can be used as a prognostic outcome for the patients. Interestingly, glioblastoma patients expressing low vimentin are more susceptible to the therapy temozolomide, commonly used in glioblastoma treatment (L. Lin et al., 2016). Vimentin is also involved in migration as the downregulation of vimentin in GBM cells, significantly impairs their migration (Nowicki et al., 2019).

Interactions between cytoskeletal networks

The cytoskeleton forms within the cytoplasm an intertwined and highly dynamic network of filaments. They are interacting in several ways, indirectly, thanks to cytoskeleton cross-linker, and directly. There is indeed a dynamic and active crosstalk between the different components of the cytoskeleton. This has been well studied for the crosstalk between actin and MT. This crosstalk is essential for a lot of cell processes such as cytokinesis and migration. As they share regulators and crosslinking proteins such as EB1 protein interacting with the plus tip of MT, they can act as a guidance system for MT growth and give dynamic links between the two networks (Alberico et al., 2016). More interactions between MT and actin are thoroughly explained in this review: (Dogterom & Koenderink, 2019).
IFs exert as well a regulatory role through intricate crosstalk between actin and MTs. IFs interact with the other cytoskeleton components mainly through crosslinkers such as adenomatous polyposis coli (APC) or plectin. The effect of that interaction is yet to be clearly understood. Vimentin, for example, controls the formation of actin stress fibres by inhibiting RhoA through interaction with guanine exchange factor GEF-H1 (Jiu et al., 2017). Hence, an increase of traction forces is observed in the U2OS osteosarcoma line in absence of vimentin. Vimentin associate with MT and are aligned in polarised cells (Sakamoto et al., 2013). MTs that are directly associated with vimentin are more resistant to MT depolymerization drugs nocodazole. After treatment, MTs grow along the vimentin filament as a template for its network (Gan et al., 2016). This provides evidence that crosstalk between IFs and the other two networks is of utmost importance for cellular homeostasis.

Transport of IFs in the cell: interaction with cellular protein motors

There have been two different kinds of transport of IFs within the cell identified. Both kinds rely on the interaction with the other cytoskeleton proteins: actin and MT.
MT dependent transport of IF proteins. The first type depicts a rapid, discontinuous and bidirectional movement. As MT depolymerisation drug nocodazole disrupts this type of transport, it was characterised as MT dependant (Francis et al., 2005; C. L. Ho et al., 1998).
IFs proteins or ULFs are seen as cargoes by the motor proteins for transportation along actin or MTs (Figure 20). Determination of the interaction between the MT molecular motor kinesin at the plus tip of the MT was pivotal to understand this type of movement in the cell (Gyoeva & Gelfand, 1991). The interaction with IFs and the kinesin is specific to the tail region of the heavy and light chain of the kinesin. The interaction also requires the detyrosination of MTs, acting as a signal for the recruitment of IFs to the MT, via the binding of kinesin (Kreitzer et al., 1999; Liao & Gundersen, 1998). It was shown a few years later that IFs also bind the second MT-based motor protein dynein at the minus end. This was demonstrated for vimentin, neurofilaments and peripherin. The medium subunit of the neurofilament bind directly to the dynein intermediate chain (Helfand et al., 2002, 2003; Shah et al., 2000). Binding to either dynein or kinesin is determining the direction of movement of IFs subunits (Figure 20). This explains the bidirectional movement observed early on. The IFs cargo can be attached to both dynein and kinesin at the same time. They will then start a “tug-of-war” pulling on the cargo. Mathematical modelling seems to predict that the elasticity of the cargo and the number of motor binding sites will affect the directionality of the IF cargo (Dallon et al., 2019; Ikuta et al., 2014). A study about peripherin IF motility showed that peripherin can be co-translated locally Figure 20 Transportation of cargoes along microtubule. Here cargoes can represent IF subunits being transported to be incorporated within the filament. Commonly, cargoes are moving from the end to the +end attaching to the light chains of kinesins and on the oppos ite direction with the dynein complex composed of dynactin with spectrins and anchored thanks to ankyrin (Shah et al, 2020).
for a fast local reorganisation of the network. Indeed, peripherin mRNA containing ribonucleoproteins have been observed moving along MTs. Upon the end of the movement, the translation is induced in a process of dynamic co-translation (Chang et al., 2006). Actin dependent transport of IFs. The second kind of transportation of IFs in the cell is described as slow, continuous and unidirectional. It is linked to actin flow. Where extensive researches have been done on the transport of IFs by MT and interaction with MT motor protein, very little research on this second type of transport have been done. This type of transport starts at the periphery of the cell and is directed towards the cell centre. It has been studied for keratin network organisation. After disruption of the actin network, keratin subunits polymerise at the plasma membrane but remains there. Keratin starts being generated at FAs and assembles along actin filament to be integrated into the network (Figure 21) (Kölsch et al., 2009; Leube et al., 2017).

A fully formed network of IFs interacts with actin and MT

The structural and mechanical functions of IFs are reinforced by their tight connection and crosstalk with the actin and MT networks. This is perfectly illustrated in muscle fibres, where desmin filaments connect the Z-discs to the plasma membrane, mitochondria and nuclei to increase the mechanical resilience of muscle cells (Conover & Gregorio, 2011; Mermelstein et al., 2006; Milner et al., 2000). In addition to a possible direct interaction with actin microfilaments, a wide variety of cytoskeletal crosslinkers connect IFs to actin and MTs: these include plectins, alphaB-crystallin, fimbrin, filamin A, adenomatous polyposis coli (APC), as well as several cytoskeletal motors (Djabali et al., 1997; Karashima et al., 2012; Leduc & Etienne-Manneville, 2017; Sakamoto et al., 2013). The impact of IFs not only is a result of their physical interactions with actin and MTs but also involves their function in key signalling pathways controlling, for instance, RhoA activity and cell contractility (Jiu et al., 2017). IFs influence the dynamics and organization of the MT and actin networks. Accumulating evidence points to IFs as major regulators of actin stress fibres and actomyosin-mediated forces, although the effects vary with cell type, possibly as a consequence of differences in IF composition (De Pascalis et al., 2018; Jiu et al., 2017). Although no major change in the global organization of the MT network has been observed in IF-depleted cells, IFs can modulate the dynamics of MTs. Vimentin IFs interact with MTs and serve as a template to subtly direct MT growth (Gan et al., 2016). MTs that are directly associated with vimentin are also more resistant to MT-depolymerizing drugs like nocodazole (Gan et al., 2016). Whether all types of IF have the same effect on the other cytoskeletal networks awaits further investigation. However, it is already clear that keratin and vimentin networks behave very differently concerning MTs, as MT depolymerization induces the perinuclear collapse of the vimentin IF network without affecting keratin IF organization (Kölsch et al., 2009). Overall, it is likely that IF composition or post-translational modifications influence cell contractility and motile behaviour and may be key to the adaptation of various cell types to the specific, and possibly evolving, mechanical properties of their environment (Dutour-Provenzano & Etienne-Manneville, 2021; van Bodegraven & Etienne-Manneville, 2021). Interactions with adhesive structures. The scaffolding structure formed by cytoplasmic IFs, together with the other cytoskeletal networks, is connected to the extracellular microenvironment. IFs interact with, are regulated by and also influence cell-cell and cell-matrix adhesive structures (Jones et al., 2017). This was initially illustrated by the association of keratin filaments with desmosomes via desmoplakin and with hemidesmosomes via bullous pemphigoid antigens 1 and 2 (BPAG1 and 2) (Fuchs & Wiche, 2013). Keratin IFs stabilize hemidesmosomes and desmosomes, which are essential for the cohesion of epithelial tissues. The stabilizing role of IFs on cell adhesions is not limited to epithelial cells. IFs are involved in the stability of gap junctions in cardiomyocytes and of adherens junctions in endothelial cells. IFs also associate with FAs in many cell types, including fibroblasts, endothelial cells and astrocytes (Leube et al., 2015). The organization of IFs at FAs and the exact molecular link that connects IFs to integrins are not entirely clear and likely vary depending on the IF composition and on the integrin involved. One specific IF protein, synemin, directly interacts with several FA proteins, such as talin, vinculin and zyxin (Russell, 2020; Sun, 2008). In the absence of synemin, plectin (more specifically plectin 1f) is involved in the association of vimentin and desmin with integrins (Sun et al., 2008a). IFs tend to stabilize or reinforce FAs in immobile cells. However, the molecular mechanisms physically and functionally bridging IFs to FAs need to be further investigated, focusing on the specific relationship of each IF protein with various integrins and FA proteins (Dutour-Provenzano & Etienne-Manneville, 2021).

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Physical and mechanical properties of IFs

Due to their unique mechanical properties, IFs can take on the load as mechanical support of the cells. They are structuring the cells creating a scaffold anchored to the nucleus and throughout the cytoplasm (Patteson, Vahabikashi, Pogoda, Adam, Goldman, et al., 2019). Different ways are used to study the mechanical properties of IFs (Lowery et al., 2015). They can be reconstructed in vitro, studied within the cell or in tissues.
IFs are grown in solution or coverslips where their mechanical properties can be closely assessed. IFs display a flexible structure, assembled by 45 nm long coiled-coil dimers with a persistence length ranging from 0.2μm for Neurofilaments to 2.1μm for Vimentin (Block et al., 2015a). The flexibility of the filament is depending on their specific type of assembly involving an axial sliding upon force application allowing the filament to be stretched (Mücke et al., 2004). This ability to stretch and deform under tension comes from their coiled-coil alpha-helical rod domains that can unfold and form ß-sheets. IFs proteins come in contact through different 22 Stretching and elastic properties of vimentin Vimentin filament stretched to increasing distances with each cycle), (c) Sketch of the experimental protocol for stretching cycles, including waiting time (twait). (d f) Examples for force strain data from experiments with different waiting times ((d) twait = 0, ( twait = 30 min, and (f) twait = 60 min). IFs are sensitive to repeated stretching. They do not recover completely by themselves and need introduction of crosslinker (Forsting et al, 2019).
points of assembly along the rod domain to form a filament (C.-H. Lee et al., 2020). Recent studies have shown that the stretching of the filament is only partially reversible, meaning that when subjected to the second cycle of stretching, the filament displays lower extendibility (Figure 22). The filament once stretched in ß-sheet does not refold when relaxed, thus exerting lower forces. The reversibility can be achieved with the introduction of a crosslinker, preventing the filament to enter the ß-sheet state (Forsting et al., 2019). Inter-filament crosslinking also gives the filaments their astonishing elasticity. In order for the filament to stay in a stretched condition, energy is required. This energy is given by the hydrophobic and electrostatic inter-filament bounds. IFs are extremely elastic in vitro giving them great importance in cell integrity.
The remarkable mechanical properties of IFs are also essential within the cells. The cells are constantly afflicted by mechanical forces with, for example, membrane tension allowing the cells to change shape under external forces (Pontes et al., 2017). Therefore, they require a scaffolding structure to maintain their integrity. In keratinocytes, a strain stiffening is observed upon AFM (Atomic Force Microscopy) indentation measurement on junctions and lamellae in comparison with Keratin KO, suggesting that Keratin protects cell structures and may play a role in transducing forces from the junction to the cell body (Ahrens et al., 2019). In the same cell line, using AFM and magnetic tweezers, a significant softening of the cell is observed when the entire Keratin gene cluster is missing. This reduces cell viscosity and thus increases the cell deformability, a phenotype that can be rescued upon reintroduction of Keratin K5 and K14 (Ramms et al., 2013). In a study using unanchored MSCs, the authors stipulate, that cells with higher Vimentin expression exhibit a higher capability to deform upon strain application on different substrate rigidities (Sharma et al., 2017). This indicates the role of Vimentin as a cell shaper under external tension, especially during mesenchymal stem cell transition when the cell has to squeeze through the epithelium cells. The role of Vimentin as a protector of the cell is also described in MEFs cells where Vimentin protects the nucleus and regulate its shape, avoiding nucleus rupture when the cell is migrating through confined spaces. The nucleus of Vim KO MEFs is more susceptible to leakage, as seen using transwell migration assays (Patteson, Vahabikashi, Pogoda, Adam, Goldman, et al., 2019). Lamins also participate in the upholding of nucleus integrity. Their elastic capabilities are crucial during cell compression. The rod domain of Lamins can vary from 40 nm to the originally measured 52 nm. This size reduction might be due to the sliding of dimer creating an increase in rod overlap or a simple shortening of the rod by 15 to 20% (Makarov et al., 2019). Lamins are the only IFs that are positively charged right after the rod domain, creating an electrostatic interaction facilitating the assembly of dimers. These interactions and the presence of cross-linkers allow the lamin to get stretched upon force application and to go back to their initial shape once the force is removed (Makarov et al., 2019).

IFs involvement in signalling

IFs not only serve as a structural scaffold but also form a molecular scaffold that connects with signalling pathways to influence cell behaviour in physiological and pathological situations (Pallari & Eriksson, 2006). The connection of IFs with intracellular structures parallels the ability of these filaments to influence cellular functions. While focal-adhesion-mediated signalling influences the organization of the IF network, IFs can in turn influence the dynamics of FAs. They participate in focal-adhesion-associated signalling, as shown for vimentin, which regulates the expression level and the localization of FA kinase (FAK) and the Rac1 guanine nucleotide exchange factor Vav2 (Havel et al., 2015). In addition to their role in controlling actomyosin contractility, vimentin and more generally type III IFs facilitate cell migration and invasion of mesenchymal cells by controlling the dynamics and distribution of FAs (De Pascalis et al., 2018; Mendez et al., 2010; Menko et al., 2014). Depending on the cell type, IFs have different effects on cell migration that may be explained by the difference in IF proteins or integrin expression patterns. Mirroring their protective role against mechanical stresses, the signalling functions of IFs are also involved in cellular survival by promoting cell-cycle progression, maintaining organelle homeostasis and protecting against apoptosis. Most IF proteins, including keratins, GFAP, vimentin and neurofilament proteins, interact with 14-3-3 proteins.

Table of contents :

Preface
Summary
Résumé
Abbreviations
List of figures
I. The cytoskeleton
I-1. Actin
I-1-1. Form and assembly
I-1-2. Different structures of actin
I-1-3. Post-translational modifications
I-2. Microtubule
I-2-1. Form and assembly
I-2-2. Post-translational modifications
I-3. Intermediate filaments:
I-3-1. Structure of IF proteins
I-3-2. Assembly into filament
I-3-3. Classification of IF proteins
I-3-3-1. Differences between nuclear and cytoplasmic IFs
I-3-3-2. Different categories of IF proteins
I-3-3-3. Alteration and mutations
I-3-4. Post-translational modifications
I-3-4-1. Phosphorylation
I-3-4-2. Farnesylation, glycosylation, transglutamination
I-4. IFs in Astrocytes
I-4-1. Description of the astrocyte model
I-4-2. Different IFs present in Astrocytes:
I-4-2-1. GFAP
I-4-2-2. Synemin
I-4-2-3. Nestin
I-4-2-4. Vimentin
I-4-3. IFs in cancer: focus on Glioblastoma
I-5. Cytoskeletal interactions:
I-5-1. Interactions between cytoskeletal networks
I-5-2. Transport of IFs in the cell: interaction with cellular protein motors
I-5-3. A fully formed network of IFs interacts with actin and MT
I-6. Key messages
II. The mechanical functions of the cytoskeletal network
II-1. Actin and actomyosin
II-2. Microtubules
II-3. Intermediate filaments
II-3-1. Physical and mechanical properties of IFs
II-3-2. Structural role of IFs
II-3-2-1. At the tissue level
II-3-2-2. At the cell level
II-3-3. IFs involvement in signalling
II-4. Key messages
III. Mechanotransduction
III-1. Mechanotransduction definition
III-2. Substrate Rigidity
III-3. Mechanosensing at adhesive structures on different substrate rigidity
III-3-1. Mechanosensing at FAs
III-3-2. Mechanosensing at adherens junctions
III-4. Cytoskeletal network in mechanotransduction
III-4-1. Actin
III-4-1-1. Role of actin/actomyosin in mechanosensing
III-4-1-2. Impact of mechanotransduction on actin and forces.
III-4-2. Microtubules
III-4-3. Intermediate filaments
III-5. Key messages
IV. Mechanotransduction at the nucleus
IV-1. The LINC complex connects the cytoskeleton to the nucleoplasm
IV-2. The cytoplasmic and nuclear cytoskeleton protect the nucleus from mechanical stress
IV-2-1. Lamins
IV-2-2. Actin cap
IV-2-3. Cytoplasmic Intermediate filaments
IV-3. Cytoskeleton and nucleus positioning
IV-4. Control of nuclear morphology
IV-5. Mechanotransduction induces changes in gene expression
IV-5-1. Effects of mechanical signals on chromatin
IV-5-2. Control of gene expression in response to mechanical tension
IV-6. Key messages
V. Objectives
V-1. What are the effects of mechanical cues on nuclear morphology and structure?
V-1-1. Does substrate rigidity affect the nucleus size and morphology?
V-1-2. Does substrate rigidity affect the chromatin status?
V-1-3. Does substrate rigidity affect the nuclear recruitment of transcription factor YAP?
V-1-4. Does geometrical constrain affect the nucleus morphology?
V-2. Is the IF network reorganised in response to mechanical cues?
V-2-1. Does IF organisation change with the substrate rigidity?
V-3. Do IFs mediate the effect of substrate rigidity on the nucleus?
V-4. Looking for effectors of IFs new partners
VI. Material and methods
VI-1. Cell culture:
VI-2. Transfection:
VI-3. Hydrogel substrates of different rigidities.
VI-4. Micropatterns
VI-5. Immunofluorescence
VI-6. Immunoprecipitation:
VI-7. Mass spectrometry
VI-8. Western Blotting
VI-9. Image analysis
VI-10. Quantification and statistical analyses
VII. Results
VII-1. Substrate rigidity affects the nucleus of astrocytes.
VII-1-1. Substrate rigidity affects the size and shape of the nucleus but not its position within the cell
VII-1-2. YAP nuclear localisation in astrocytes
VII-1-3. Substrate rigidity affects histone post translational modifications
VII-2. IFs organisation is changing with the rigidity of the substrate
VII-2-1. IFs form a cage-like structure around the nucleus on stiff substrates.
VII-2-2. Substrate rigidity affects the phosphorylation status of vimentin
VII-3. Decreased IF protein expression by siRNA
VII-4. IFs mediate some effect of the substrate rigidity on the nucleus
VII-4-1. Specific IFs affect the size and morphology of the nucleus on soft stiffness .
VII-4-2. IFs protect the nucleus against nuclear blebbing.
VII-4-3. IFs are responsible for the positioning within the cell on different rigidities
VII-4-4. Vimentin increases YAP localisation on soft substrate
VII-5. IFs affect the rigidity dependent changes in histone PTM.
VII-6. Discovery of new interactors of IFs
VII-6-1. Mass spectrometry analyses of potential interactors
VII-6-2. Confirmation of interaction between IFs and HDAC6
VII-7. Key messages
VIII. Discussion
VIII-1. Substrate rigidity affects the nucleus.
VIII-1-1. Substrate rigidity affects the size and the shape of the nucleus.
VIII-1-2. Substrate rigidity affect the tension on the nucleus
VIII-1-3. Substrate rigidity affect the structure of the chromatin
VIII-2. The organisation of IFs is changing with rigidity.
VIII-2-1. IFs organisation around the nucleus.
VIII-2-2. Phosphorylation of vimentin makes the network more soluble.
VIII-3. IFs mediate the effects of substrate rigidity on the nucleus.
VIII-3-1. Lack of IFs changes the morphology of the nucleus depending on substrate rigidity.
VIII-3-2. IFs protect the nucleus from blebbing
VIII-3-3. IFs keep the nucleus centred in the different rigidity
VIII-3-4. Lack of IFs affect the acetylation of histone H3 depending on the substrate rigidity
VIII-4. Geometrical constrain and the nucleus
VIII-5. HDAC6 interaction with IFs.
IX. Perspective
Appendix 1
Appendix 2
Appendix 3
Appendix 4
Appendix 5
Publication: “Intermediate filaments”
X. Bibliography

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