toskeleton rearrangement
Migration is a process which requires the communication between two opposing ends of the cell. Not only coordination of protrusions in the leading front and retraction at the rear are important, also shape, organisation and polarity that are driven by the cytoskeleton. In respect of locomotion three distinct activities are involved to move a cell forward: 1) Protrusions in the front of cell structures, rich in actin, are pushed out. 2) Attachment, in which the cell membrane is connected to the substratum by the actin cytoskeleton. 3) Traction in the rear. The trailing cytoplasm is drawn forward. These cytoskeletal rearrangements are governed by the Rho protein family: Rho, Ras-related C3 botulinum toxin substrate (Rac) and cell division control protein 42 homologue (Cdc42). The function of Rho is to regulate actin stress fibre assembly, whereas Rac regulates membrane ruffles and lamellipodia formation at the cell periphery, while Cdc42 is responsible for filopodia formation389.
Figure 1.23: Illustration of Rho-GTPase activation. Rho-GDIs release inactive Rho-GTPases in the cytoplasm. Sequestered Rho-GTPases are targeted to the cell membrane. GEFs mediate their activation by binding to GTP. GAPs promote their inactivation by binding to GDP. The image is adapted from Huveneers and Danen, Adhesion signaling - crosstalk between integrins, Src and Rho, Journal of Cell Science, 2009390.
CHAPTER 1. GENERAL INTRODUCTION 79
Within the cytoplasm GTPases are bound by Rho-GDP dissociation inhibitors (Rho-GDI) as shown in figure 1.23. Sequestered Rho-GTPase from Rho-GDIs are targeted to the plasma membrane. GTPase switches between an active GTP-bound and an inactive GDP-bound state. Guanine nucleotide exchange factors (GEFs) promote activation of GTPase through the catalytic removal of GDP and enabling GTP binding whereas GTPase-activating proteins (GAPs) show the opposite effect, causing down regulation of GTPase activity.
The initiation of cell adhesion and spreading occurs in parallel with inhibition of RhoA and activation of Rac1 and Cdc42. This increases actin-mediated protrusion formation and suppresses actomyosin contractility. Later, the activities of Rac1 and Cdc42 decline while RhoA activity increases which leads to FA and stress fibre for- mation. The reason of this mutually exclusive behaviour is that RhoA suppresses the activity of Rac1 and vice versa. Rac1 inhibits low-molecular-weight protein tyro- sine phosphatases by induction of reactive oxygen species (ROS) production. This leads to an increase in tyrosine phosphorylation and activation of p190RhoGAP which inhibits RhoA activity391. In contrast, RhoA inactivates Rac1 by promoting Rho-associated coiled-coil containing kinases (ROCK)-mediated phosphorylation of FilGAP392. FilGAP is a filamin A-binding RhoGTPase-activating protein. It func- tions as a GAP for Rac1, localising to sites of membrane protrusions.
Integrins are the major regulators of RhoGTPases largely via the FAK-Src com- plex. A simplified illustration of integrin-mediated RhoGTPse activation is shown in figure 1.24. Upon binding of an integrin to a ligand the cytoplasmic domain un- folds and various FA initiation proteins are recruited to the the membrane such as talin and FAK. Recruitment of FAK causes its autophosphorylation at Y397 which serves as binding site for the SH2 domain of Src. This leads to transphosphoryla- tion of FAK within the kinase domain activation loop at Y576 and Y577 as well as C-terminal domain at Y861 and Y925393. This phosphorylation events serve two purposes: They maximise the kinase activity of FAK and generate new binding sites for other proteins. The active FAK/Src complex recruits and phosphorylates the scaffolding protein p130Cas394. Active p130Cas associates with the adaptor protein v-crk sarcoma virus CT10 oncogene homologue (Crk) thereby recruiting en- gulfment and motility 1 (ELMO1) and dedicator of cytokinesis-180 kDa (Dock180). The Dock180-ELMO1 complex promotes the formation of membrane protrusions by functioning as an unconventional GEF for Rac1395. Apart from activating p130Cas the FAK/Src complex also phosphorylates paxillin that subsequently recruits the Ar- fGAP paxillin-kinase linker (PKL) as well as the mutual GEF for Rac1 and Cdc42, Pak-interacting exchange factor-β (β-PIX). Rac1 is recruited and activated by β-PIX via direct interaction396. A proline-rich region of Rac1 binds to the SH3 domain of β-PIX396.
Figure 1.24: Simplified illustration of integrin induced regulation of Rho-GTPases. At the site of integrin activation Src and FAK are recruited and form a complex. This complex modulates Cdc42, Rac1 and RhoA activity via several pathways. The image is adapted from Huveneers and Danen390 and Dr´aber, Sulimenko and Dr´aberov´a397.
CHAPTER 1. GENERAL INTRODUCTION 81
It has been demonstrated that PKL can be directly phosphorylated by Src and/or FAK398 while β-PIX is activated by Src399. Furthermore, Src mediates the transient inhibition of RhoA through regulation of p190RhoGAP400.
There are multiple downstream targets of RhoGTPases with direct effects on actin cytoskeleton rearrangements. This includes formins, kinases and members of the Wiskott-Aldrich syndrome protein (WASP) family and other scaffolding proteins. The most prominent contributors are the mammalian Diaphanous formin (mDia), proteins of WASP and Wiskott-Aldrich syndrome protein-family verprolin homol- ogous protein (WAVE), ROCK and p21-activated kinase (PAK). Cdc42 and Rac1 activate PAK which promotes LIM-motif containing kinase (LIMK) activity. Both, Cdc42 and Rac1, activate the Arp2/3 complex, Cdc42 through WASP while Rac1 stimulates it via WAVE401. Cdc42-mediated Arp2/3 activation leads to filopodia formation while Rac1-regulated Arp2/3 stimulation leads to membrane ruffling and lamelipodia formation. Interestingly, the ADAM15 adaptor protein Grb2 is an al- ternative activator of WASP, stimulating actin polymerisation. Cdc42 and Grb2 elicit a synergistic effect by simultaneous binding to N-WASP which increases its activity402.
RhoA promotes stress fibre formation via activation of mDia and ROCK. Besides, ROCK also mediates actomyosin contractility via phosphorylation of myosin II light chain (MLC) and myosin light chain phosphatase (MLCP). Activation of LIMK by ROCK leads to phosphorylation of cofilin. This leads to inhibition of cofilin which is associated with actin-filament turnover403.
RhoGTPase-mediated mDia activation has a promoting effect on cytoskeleton rear- rangement by stabilising microtubules404.
In addition to integrins, GPCRs and RTKs have the ability to activate RhoGT- Pases405,406. Furthermore, GPCRs can also activate G-protein subunits such as G 12/13 regulating RhoGEFs407. These subunits modulate the turnover of membrane ruffles upon growth factor induction408. The Swiss 3T3 cell line has shown stress fibre formation and FA assembly prior to G 12/13 stimulation via the RhoGTPase pathway. Overexpression of β1-integrin enhances Rac activity and lamellipodia formation whereas the overexpression of β3-integrins showed an increase in Rho activity and stress fibre formation409. More than half of the 58 known RTKs activate at least one Rho family member. Several RTKs are able to activate the same Rho GEFs, but it is also possible that a single RTK activates different Rho GEFs (16 in total) which increases the complexity of RTK signalling.