EDUCACIÓN Y VALORES
2. L A NATURALEZA DE LA FORMACIÓN
The cytoskeleton serves three main roles in eukaryotic organisms: to physically separate contents within cells, to allow traffic of intra and intercellular cargoes, and to allow force generation needed for cell movement and morphogenesis (Fletcher and Mullins, 2010). The most studied groups of cytoskeleton polymers include actin filaments, microtubules and intermediate filaments that have largely been studied as separate entities. However, burgeoning evidence of dynamic crosstalk between these filamentous polymers is emerging to provide clues as to how the cytoskeletal network co-ordinate molecular signals, cell shape and mechanics.
Chapter 1: Introduction
The different cellular polymers differ in mechanical stiffness, assembly programmes, polarity and associated molecular motors (Fletcher and Mullins, 2010). Microtubules are most rigid with complex assembly and disassembly programmes. These are dynamically unstable as microtubules can dynamically grow and rapidly shrink. During mitosis, the dynamic microtubules form the mitotic spindle to align and separate chromosomes. The rapidity of microtubule growth and shrinkage allows these polymers to sense space quicker than other polymers that respond to either chemotactic gradients or activity of other regulatory proteins (Fletcher and Mullin, 2010).
Actin filaments are less rigid but are able to form stiff, organised networks that can be either bundled or branched (Fletcher and Mullins, 2010). Bundled actin filaments are commonly seen in filopodial protrusions that allow chemical sensing in the intercellular environment (Gupton and Gertler, 2007). Branched filaments are enriched especially at the leading edges of motile cells or cells forming initial contact with neighbouring cells (Ridley, 2011; Hoelzle and Svitkina, 2012). These branches of filaments generate forces that allow shape changes to accommodate cell movements. The presence and location of actin regulators: nucleation factors such as Arp2/3 complex and formins dictate the organisation of networks formed (Ridley, 2011; Section 1.3.3). Steady addition of nucleotide monomers to the growing end of actin filaments provide sustained mechanical forces to advance migrating cells (Fletcher and Mullins, 2010).
Many cell types form intermediate filaments to allow cells to resist mechanical stress such as those seen in keratinocytes and airway epithelial cells (Fletcher and Mullins, 2010). Actin and microtubules depend on nucleotide binding, hydrolysis and polarise to allow trafficking of molecular motors (Chang and Goldman, 2004). Intermediate filaments are not reported to have enzymatic activity and lack the ability to polarise compared to the actin and microtubule filaments (Fletcher and Mullins, 2010). Instead, these least stiff polymers resist tensile forces and can be cross-linked to each other, actin filaments and microtubules by plectins, which organises a subset of intermediate filaments in cells (Fletcher and Mullins, 2010). These networks extend in all directions, from the cell surface (to tether mature desmosomal plaques) to cage-like structures surrounding the nucleus that contains polymerised nuclear lamins (Chang and Goldman, 2004). When nuclear lamins are phosphorylated by cyclin-dependent kinase, this triggers the nuclear envelope initial breakdown in
Chapter 1: Introduction mitosis, suggesting that they provide mechanical integrity to the eukaryotic nucleus (Fletcher and Mullins, 2010).
1.3.2 Cell migration
Cell migration requires changes in cell shape mediated by the actin cytoskeleton in response to extracellular stimuli (Schmidt and Hall, 1998). Movement of cells has been studied extensively in animal development and cell cultures (Friedl and Gilmour, 2009; Ridley, 2011). To move across substrates, cells at the front, ‘leading edge cells’ have to extend their membranes (Ridley, 2011). Currently, four types of membrane protrusions coordinated by actin polymerisation have been identified. Leading edge protrusions include lamellipodia, filopodia, invadopodia and membrane blebs. Each type of structure aids cell migration depending on the biological circumstance. For example, lamellipodia protrusions propel cells through
extracellular matrix (ECM) in vivo (Friedl and Gilmour, 2009). Filopodia act as
sensors for chemo-attractants and are involved in relaying signals between cells (Gupton and Gertler, 2007). Invadopodia are protrusions that enable the degradation of the ECM to allow cell invasion (Mader et al., 2011). In development, membrane blebbing has been reported to direct cell migration (Charras and Paluch, 2008). These different types of protrusions can act separately or synergistically at the leading edge. For example, lamellipodia and filopodia coexist dynamically during the initial cell-cell junctions assembly in endothelial cells (Hoelzle and Svitkina, 2012).
1.3.3 Regulation of the actin cytoskeleton organisation
Membrane associated complexes such as adherens junctions have been reported to act as nucleation sites of actin (Yamada and Geiger, 1997). Actin polymerisation is important to generate mechanical force for the cells to move forward. Numerous actin binding regulators mediate actin polymerisation, nucleation and cross linking to allow the dynamic assembly of actin monomers into polymers and eventually filamentous networks (Schmidt and Hall, 1998). One of the main drivers of new actin filament formation near membranes is the Arp2/3 complex (Actin related protein 2/3 complex) (Ridley, 2011). WASP (Wiskott-Aldrich syndrome protein) acts as a nucleation-promoting factor to stimulate the activity of the Arp 2/3 complex (Stevenson et al., 2012). The Arp2/3 complex binds the side branches of actin filaments to nucleate new branched actin filaments via actin polymerisation. Dysregulated expression of these proteins has been implicated in diseases. For
Chapter 1: Introduction
example, N-WASP (Neural WASP) has been found to be overexpressed in human invasive ductal carcinoma (Yu et al., 2012).
1.3.4 Regulation of the actin cytoskeleton orchestrated by GTPases signalling The network of actin cytoskeleton network also acts as a signalling hub in different cell structures (Schmidt and Hall, 1998; Yu et al., 2012). Ras homologous (Rho) family small GTPases are key signal regulators that integrate extracellular or intracellular stimuli with the assembly and organisation of actin cytoskeleton (Ridley, 2011; Schmidt and Hall, 1998). The most conserved Rho GTPases associated with actin-specific structures and migration include the Rac, CDC42 and RhoA that drive the formation of lamellipodia, filopodia and stress fibres initially discovered in fibroblasts (Abercrombie et al., 1970; Boureux et al., 2007; Ridley, 2011; Ridley and Hall, 1992; 2004). These Rho GTPases function as binary switches that cycle between active GTP-bound form and inactive GDP-bound forms (Figure 1.6). These molecular switches are regulated by guanine nucleotide exchange factors (GEF), GTPase activating proteins (GAP) or guanine nucleotide dissociation inhibitors (GDI) (Cook et al., 2014). RhoGEFs favour the formation of Rho-GTP to Rho-GDP by accelerating the intrinsic exchange activity (Ridley, 2011). RhoGAPs stimulate the intrinsic GTP hydrolysis activity of Rho GTPases to form the inactive GDP- bound state. RhoGDI binds Rho-GDP and sequesters the RhoGTPases from the cell membrane where RhoGTPases can be activated (Cook et al., 2014). The first RhoGEF discovered, Dbl from genomic DNA of the MCF7 human breast carcinoma cell line caused tumorigenic growth when transfected into NIH 3T3 fibroblasts (Fasano et al., 1984). Aberrant expression of GTPases and their regulators have since been implicated in cancer such as upregulation of Rac-GEF PRex1 in human breast cancer that leads to cancer metastasis (Sosa et al., 2010; Wertheimer et al., 2012). In non-tumorigenic mammary epithelial cells, Rac1 activation via RacGEF Vav2 on cell membranes was implicated in the disassembly of adherens junctions implying the possibility of cell migration (Duan et al., 2011).
1.3.5 EGF regulation of cell shape
Upstream of the cytoskeletal signalling modulators, EGF has been shown separately to induce cell shape change and is implicated in cell motility. Often, this has been studied in oncogenic cell systems. For example, metastatic rat mammary adenoma cells, MTLn3 extend lamellipodia towards EGF upon stimulation (Bailly et al., 1998; Bailly et al., 2000). Invasive bladder cancer cells displayed higher levels of EGFR at the tumour regions than the sites further from the tumours (Rao et al.,