Cell Motility. Junfeng Ji ( 纪俊峰 ), PhD

Transcription

1 Cell Motility Junfeng Ji ( 纪俊峰 ), PhD Professor Laboratory of Somatic Reprogramming and Stem Cell Aging Center for Stem Cell and Tissue Engineering School of Medicine Zhejiang University jijunfeng@zju.edu.cnedu

2 Cytoskeleton 1. The cytoskeleton provides the structure and shape of the cell. 2. Eukaryotic cells contain three main kinds of cytoskeletal filaments: microfilaments, intermediate filaments, and microtubules. 3. The cytoskeleton undergoes continual turnover and rearrangement which underlies the changes in cell morphology and migration that shape the organism.

3 Cytoskeleton

4 Actin Microtubule Intermediate filament

5 Cell Movement in vitro

6 1. The crawling movement of cells is driven by the continuous reorganization and turnover of the actin cytoskeleton. 2. Actin polymerization drives the extension of sheet-like and rod-like protrusions at the cell front, termed lamellipodia ( 板状伪足 ) and filopodia ( 丝状伪足 ), respectively. 3. Behind the protruding front, actin interacts with myosin to form contractile arrays that drive the translocation of the trailing cell body.

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8 Treadmill The breadth of the lamellipodium network at the cell front remains constant as the cell moves forward. This feature is explained by a so-called treadmilling of actin filaments, whereby the addition of actin monomer at the front of the lamellipodium is balanced by the release of monomer at the rear.

9 Adhesion and traction 1. For a cell to move, it must adhere to a substrate and exert traction. 2. Adhesion occurs at specific foci at which the actin cytoskeleton on the inside of the cell is linked via transmembrane receptors (integrins) to the extracellular matrix on the outside.

10 Actin cytoskeleton focal adhesion interplay Step 1: Actin polymerization and myosin IIdependent contractility generate forces that affect specific mechanosensitive proteins in the actin-linking module (perhaps talin and vinculin), the receptor module (represented by integrins, such as α5β1 integrin and αvβ3 integrin), the associated actinpolymerizing module (for example, zyxin and formins) and the signalling module (represented by, for example, focal adhesion kinase and p130cas). Step 2: Acting in concert, these interacting modules, with their particular mechanosensitive components, form a mechanoresponsive network. The effect on the actin cytoskeleton depends on the integrated response of the entire system to interactions with the matrix (FIG. 1) and to applied mechanical forces. Geiger et al Nat Rev Cell Mol Cell Bio 2009

11 Actin cytoskeleton focal adhesion interplay Step 3: Stimulation of the signalling module eventually leads to the activation of guanine nucleotide-exchange factors and GTPase-activating proteins, leading to activation or inactivation of small G proteins, such as Rho and Rac. Step 4: These G proteins affect actin polymerization and actomyosin contractility through cytoskeleton-regulating proteins. Step 5: The cytoskeleton regulating protein can then modulate the force-generating machinery. Geiger et al Nat Rev Cell Mol Cell Bio 2009

12 Focal adhesion formation and maturation A diagram that t summarizes the stages of FA formation and maturation, ti and the simultaneous advancement of the boundary between fast (lamellipodium) and slow (lamella) actin flow zones. Nascent and mature FAs are shown as red ellipses of different sizes; and stress fibres are shown as purple lines of different thicknesses. Note that the process by which the boundary advances between the flows in lamellipodium (fast) and lamella (slow), and that of FA maturation, are presented in different panels, for clarity. In fact, these two processes proceed simultaneously. Geiger et al Nat Rev Cell Mol Cell Bio 2009

13 1. The extracellular matrix, integrins and the cell cytoskeleton interact at sites called focal contacts. Molecular architecture of focal contact 2. Focal contacts are dynamic groups of structural and regulatory proteins that transduce external signals to the cell interior and can also relay intracellular signals to generate an activated integrin state at the cell surface. 3. The integrin-binding proteins paxillin and talin recruit focal adhesion kinase (FAK) and vinculin to focal contacts. 4. α-actinin is a cytoskeletal protein that is phosphorylated by FAK, binds to vinculin and crosslinks actomyosin stress fibres and tethers them to focal contacts. 5. The composition of a focal contact is therefore constantly varying depending on external cues and cellular responses. Mitra et al Nat Rev Cell Mol Cell Biol 2005

14 Focal adhesion kinase integrates signals to promote cell migration 1. Focal adhesion kinase (FAK) is activated by growth factors and integrins during migration, and functions as a receptor-proximal regulator of cell motility. 2. At contacts between cells and the extracellular matrix, FAK functions as an adaptor protein to recruit other focal contact proteins or their regulators, which affects the assembly or disassembly of focal contacts. Mitra et al Nat Rev Cell Mol Cell Biol 2005

15 Focal adhesion kinase integrates signals to promote cell migration 3. FAK activity and downstream signalling can promote changes in actin and microtubule structures. t 4. FAK signalling can affect the formation and disassembly of cell cell (cadherin-based) contacts. 5. The Rho-family GTPases (RhoA, Rac and Cdc42) direct local actin assembly into stress fibres, lamellipodia and filopodia, respectively. FAK can influence the activity of Rho-family GTPases through a direct interaction with, or phosphorylation of, protein activators or inhibitors of Rho GTPases. RhoA can also influence the stability of microtubules through its effector Diaphanous (mdia). Mitra et al Nat Rev Cell Mol Cell Biol 2005

16 Focal adhesion kinase domain structure and phosphorylation sites 1. Focal adhesion kinase (FAK) contains a FERM (protein 4.1, ezrin, radixin and moesin homology) domain, a kinase domain and a focal adhesion targeting (FAT) domain. 2. The FERM domain mediates interactions of FAK with the epidermal growth factor (EGF) receptor, platelet-derived growth factor (PDGF) receptor, the ETK tyrosine kinase and ezrin. 3. The FAT domain recruits FAK to focal contacts by associating with integrinassociated proteins such as talin and paxillin. It also links FAK to the activation of Rho GTPases by binding to guanine nucleotide-exchange factors (GEFs) such as p190 RhoGEF. Mitra et al Nat Rev Cell Mol Cell Biol 2005

17 Focal adhesion kinase domain structure and phosphorylation sites 4. FAK contains three proline-rich regions (PRR1 3), which bind Srchomology-3 (SH3) domain-containing proteins such as p130cas, the GTPase regulatorassociated with FAK (GRAF) and the Arf-GTPase-activating protein ASAP1. 5. FAK is phosphorylated (P) on several tyrosine residues. Tyrosine phosphorylation on Tyr397 creates a Src-homology-2 (SH2) binding site for Src, phospholipase p p Cγ γ (PLCγ), suppressor of cytokine signalling g (SOCS), growth- factor-receptorbound protein 7 (GRB7), the Shc adaptor protein, p120 RasGAP and the p85 subunit of phosphatidylinositol 3-kinase (PI3K). Phosphorylation of Tyr576 and Tyr577 within the kinase domain is required for maximal FAK catalytic activity, whereas the binding of FAK-family interacting protein of 200 kda (FIP200) to the kinase region inhibits FAK catalytic activity. FAK phosphorylation at Tyr925 creates a binding site for GRB2.

18 Focal adhesion kinase (FAK) Src signals regulate cell motility and focal contact localization 1. Integrin clustering promotes FAK autophosphorylation (P) at Tyr397, which creates a binding site for the Src-homology (SH)2 domain of Src. 2. Src-mediated phosphorylation of FAK at Tyr576 and Tyr577promotes maximal FAK catalytic activity. 3. Active FAK Src facilitates SH3- mediated binding of p130cas to FAK and its subsequent phosphorylation. 4. Crk binding to phosphorylated p130cas facilitates Rac activation, lamellipodia formation and cell migration. Mitra et al Nat Rev Cell Mol Cell Biol 2005

19 Focal adhesion kinase (FAK) Src signals regulate cell motility and focal contact localization 5. Paxillin binding to the FAK focal adhesion targeting (FAT) domain is important for FAK focal contact localization. 6. Src-mediated phosphorylation of FAK at Tyr925 creates an SH2 binding site for the growth-factor-receptor-bound protein 2 (GRB2) adaptor protein, which leads to the activation of Ras and the extracellular signal-regulated kinase-2 (ERK2) cascade. 7. ERK2 activation promotes FAK phosphorylation at Ser910, which is also associated with decreased paxillin binding to FAK. Within focal contacts, FAK Src-mediated phosphorylation of paxillin at Tyr118 promotes ERK2 binding. ERK2-mediated phosphorylation of paxillin can facilitate FAK binding to paxillin and enhances FAK activation. So, there might be a cycle whereby Src- and ERK2-mediated phosphorylation of FAK promotes its release from focal contacts and ERK2-mediated phosphorylation of paxillin promotes the association of unphosphorylated FAK with paxillin at new or growing focal contact sites. Mitra et al Nat Rev Cell Mol Cell Biol 2005

20 Focal adhesion kinase promotes cytoskeletal fluidity 1. Stress fibres and cortical actin are continuously destabilized/stabilized by focal adhesion kinase (FAK)-regulated processes. 2. Normally, the actin cytoskeleton exists in a semi-solid state, owing to a high degree of α-actinin-mediated crosslinking of stress fibres, which are tethered and exert tension at focal contacts (left panel). Mitra et al Nat Rev Cell Mol Cell Biol 2005

21 Focal adhesion kinase promotes cytoskeletal fluidity 3. Conversion to a more soluble state (right panel) is promoted by FAK phosphorylation (P) on Tyr12 of α-actinin, which results in reduced crosslinking and the release of actin stress fibres from focal contacts.. 4. Cytoskeletal fluidity is also regulated by the effects of FAK on Rho-family GTPases and on the neuronal Wiskott Aldrich syndrome protein (N-WASP). Mitra et al Nat Rev Cell Mol Cell Biol 2005

22 Focal adhesion kinase promotes cytoskeletal fluidity 3. FAK phosphorylates Cdc42-activated N-WASP at Tyr256, thereby retaining phosphorylated N-WASP in the cytoplasm where it can affect ARP2/3-mediated actin polymerization. 4. Through associations with Rho GTPase-activating proteins (GAPs) and Rho guanine nucleotide-exchange factors (GEFs), FAK can regulate actomyosin stress fibre polymerization. Mitra et al Nat Rev Cell Mol Cell Biol 2005

23 Focal adhesion kinase promotes cytoskeletal fluidity 5. Reduced tension can be attributed in part to increased RhoGAP activity of GTPase regulator associated with FAK (GRAF). Conversely, FAK can promote cytoskeletal tension through phosphorylation and activation of p190 RhoGEF. 6. Subsequent Rho activation indirectly regulates myosin light chain (MLC) phosphorylation h through h Rho-associated kinase (ROCK) phosphorylation h of MLC phosphatase, which leads to increased MLC kinase (MLCK) activity through the downregulation of MLC phosphatase activity. Mitra et al Nat Rev Cell Mol Cell Biol 2005

24 Focal adhesion kinase influences phospholipid and microtubule structures 1. The phospholipid kinases that have a role in the modification of phosphatidylinositol (PtdIns) cooperate with focal adhesion kinase (FAK) at several levels. 2. Integrin and FAK signalling also promote the translocation of specific components of lipid rafts to membranes. The stabilization of lipid rafts through integrin signalling facilitates the coupling of Rac to target proteins. Mitra et al Nat Rev Cell Mol Cell Biol 2005

25 Focal adhesion kinase influences phospholipid and microtubule structures FAK-mediated translocation of the lipid ganglioside GM1 to the membrane, which is mediated through the activation of the Rho GTPase effector Diaphanous (mdia), regulates microtubule polarity at the leading edge of motile cells. Microtubule polarization and Rac activation contribute to the formation of membrane ruffles and stable lamellipodia. Mitra et al Nat Rev Cell Mol Cell Biol 2005

26 Rho GTPases 1. GTPases are molecular switches within cells, which control the formation and disassembly of actin cytoskeletal structures (STRESS FIBRES, LAMELLIPODIA and filopodia). 2. It functions to provide the molecular framework that supports directed cell motility.

27 The Rho GTPases cycle Manneville SE and Hall A. Nature They cycle between an active (GTP-bound) and an inactive (GDP-bound) conformation. In the active state, they interact with one of over 60 target proteins (effectors). 2. The cycle is highly regulated by three classes of protein: in mammalian cells, around 60 guanine nucleotide exchange factors (GEFs) catalyse nucleotide exchange and mediate activation; more than 70 GTPase- activating proteins (GAPs) stimulate t GTP hydrolysis, leading to inactivation; and four guanine nucleotide exchange inhibitors (GDIs) extract the inactive GTPase from membranes.

28 GDPase regulate cytoskeletal changes ERM proteins are required for GTPase-mediated cytoskeletal changes. It has been proposed that ERM proteins exist in a closed (inactive) conformation and an open (active) conformation. There is evidence to suggest that this transition can be regulated by Rho, perhaps through the activation of a Ser-Thr kinase or a lipid kinase (through PIP2). In the active conformation, the NH2-terminus of ERM proteins (pink) can interact with transmembrane proteins, such as CD44, whereas the COOH-terminus (blue) interacts with F-actin. This membrane ERM F-actin unit is an essential prerequisite for the Rho GTPases to induce cytoskeletal changes. In the case of Rho, this is likely to be mediated by the bundling and reorganization of preexisting actin-myosin filaments to generate stress fibers, whereas in the caseof Rac and Cdc42 it is likely that preexisting filaments are uncapped to allow actin polymerization and filament growth leading to the formation of lamellipodia and filopodia. Manneville SE and Hall A. Nature 2002

29 Hall A. Science 1998

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31 Manneville SE and Hall A. Nature 2002

32 Manneville SE and Hall A. Nature 2002

33 Manneville SE and Hall A. Nature 2002

34 Cell Polarity The loss of polarity of fibroblasts by disassembly of microtubules

35 Importance of cell movement 1. Wound healing: Vascular endothelial cells in the epidermis, and fibroblasts in the surrounding connective tissue close and remodel wounds. 2. Cell movement in embryogenesis. 3. Cell movement in host s defense against infection 4. Uncontrolled cell migration of cancer cells

36 Uncontrolled cell movement: Cancer Metastasis

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38 Epithelial mesenchymal transition (EMT) Invasion Anoikis Angiogenesis Transport through vessels Outgrowth of secondary tumours

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40 Epithelial Mesenchymal Transition (EMT) From Wikipedia

41 EMT

42 Polarized actin cytoskeleton Non-polarized actin cytoskeleton t cwp.embo.org

43 Signaling events during EMT Lee JM JCB (7):

44 Invasion and Cell Migration Lee JM JCB (7):

45 Quiz What are the main three Rho GTPases? How are Rho GTPases regulated? What is EMT? 45