MCB L19-20
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MCB L19-20 - Leaderboard
MCB L19-20 - Details
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| Connecting Cell-Cell and Cell-ECM junctions to the cytoskeleton | In multi-cellular organisms, such as humans, Cell-Cell junctions are crucial for the organization and function of tissues by maintaining adherence to neighbouring cells and preserving both cell shape and integrity. Non-adhered and some adhered cells also contain transmembrane receptor complexes that mediate interaction with the Extracellular matrix (ECM). The ECM not only provides structural support to cells and tissues but also controls cell migration, proliferation and differentiation. Both Cell-Cell junctions and Cell-ECM transmembrane receptor complexes are frequently linked to the intracellular cytoskeleton to enable cells to both resist and react to physical forces exerted by their environment |
| Tight junctions link to the actin cytoskeleton | Also known as an occluding junctions, or zonulae occludens (ZO) are multiprotein junctional complexes whose general function is to prevent leakage of transported solutes and water and seals the paracellular pathway. They also help to maintain the polarity of cells by preventing the lateral diffusion of integral membrane proteins. TJs are composed of the tetraspan membrane proteins, Claudin and Occludin, and TJ-associated proteins including a family of Zona Occludens proteins (1,-2, and -3) which associate not only to the intracellular domains of Claudin and Occludin but also bind directly to F-actin and other actin-regulatory proteins including cortactin, α-actinin and Type 1 Myosin |
| Adherence junctions link to the actin cytoskeleton | Adherens junctions (AJs) or zonula adherens are cell-cell adhesion complexes that are continuously assembled and disassembled, allowing cells within a tissue to respond to forces, biochemical signals and structural changes in their micro-environment. AJs are usually found closer to the basal membrane than TJs and are composed of E-Cadherins, a family of transmembrane proteins that form homodimers with other E-Cadherin molecules on adjacent cells and other proteins including b-catenin and a-catenin which links the inner face of E-Cadherin to actin filaments. |
| The application of tension across the Adherence junction | It causes signalling molecules to be recruited to the intracellular domain of the E-cadherin receptor. In particular, biophysical experiments indicate that a-catenin undergoes a tension-dependent conformational change which triggers recruitment and activation of the RhoA - Rho kinase (ROCK) - Myosin II pathway leading to contraction of the stress fibres of the adherence belt and, in doing so, resists tension across the AJ. Notably, b-catenin can also bind a transcription factor (YAP1) in the nucleus to cause changes in gene expression when cell adhesion is lost. |
| Desmosomes link to intermediate filaments | Desmosomes, also known as macula adherens, is a cell structure specialized junctional complex, that are localised randomly arranged on the lateral sides of plasma membranes. Desmosomes are one of the stronger cell-to-cell adhesion types and are found in tissue that experience intense mechanical stress, such as cardiac muscle tissue, bladder tissue, gastrointestinal mucosa and epithelia. Desmosomes are composed of a network of the transmembrane cadherins, Desmoglein and Desmocolin, and adaptor proteins, Plakoglobin and Plakophilin, which help secure Desmoplakin to Keratin (Type I and II intermediate filaments) to the desmosome structure. |
| Type I and II intermediate filaments: keratins | Intermediate filaments comprised of heteropolymers of type I and II keratins attach to the plasma-membrane through desmosomes and the extracellular matrix (basal lamina) through hemi-desmosomes. This provides mechanical strength and integrity to epithelial cells and their derivatives to resist sheer and pressure forces. |
| Differential expression of keratins in layers of the skin | Histology reveals that skin is composed of distinct layers. Keratinocytes in the basal layer express K5/K14 heterodimers. As they move up through the skin to the spinous layer, they then express K1/K10 heterodimers. Cells in the stratum corneum are dead but nevertheless provide protection to the granular and layers below. Mutations in the K5/14 keratins heterodimer produce human blistering disease |
| Where is the Extracellular Matrix found? | The extracellular matrix (ECM) is the non-cellular component present within all tissues and organs and provides not only essential physical scaffolding for the cellular constituents but also initiates crucial biochemical and biomechanical cues that are required for tissue morphogenesis, differentiation and homeostasis. Components of the ECM are secreted by the cells of connective tissue. |
| What does the Extracellular Matrix (ECM) do? | Compartmentalizes tissues, Serves as a highway for cell migration, Presents signaling molecules, Provides structural support |
| What is the Extracellular Matrix made of? | 1. Fibrous proteins: such as collagen and elastin, which make fibers that provide strength and flexibility 2. Hydrated gel-like matrix containing protein-polysaccharide complexes called proteoglycans, which are glycoproteins with glycosaminoglycan linkages. 3. Adhesive glycoproteins such as fibronectin and laminin, that link components of the matrix to one another and to cells via cell surface receptors |
| ECM cell surface receptors: Integrins | Integrins connects proteins in the basal lamina with hemi-desmosomes on the inner basal membrane of epithelial cells. Hemi-desmosomes provide an attachment site for type I and II intermediate filaments and thus promote promote adhesion of epithelial cells to the underlying basement membrane. In skin epithelial cells loss of hemi-desmosomes or intermediate filaments causes blistering diseases |
| ECM cell surface receptors: Integrin (Pt. 2) | Integrins are crucially important because they are the main receptor proteins that cells use to both bind to and respond to the ECM. Integrins differ from cell-surface receptors for other extracellular soluble signal molecules in that they usually bind their ligand with lower affinity and are present 10-100 higher concentration on the cell surface. If the binding were too tight, cells would become irreversibly glued to the matrix and would be unable to move. In some way integrin binding resembles “Velcro”. Integrins also activate intracellular signalling pathways that communicate to the cell the character of the ECM that is bound. There are multiple types of α and β subunits, resulting in many types of integrin heterodimers. Many integrins recognize the RGD sequences in the adhesive glycoproteins such as Laminin and Fibronectin. Integrin tails interact with cytosolic proteins that link integrins to intracellular cytoskeleton. |
| Adhesive glycoproteins: Laminin | Laminin is an adhesive glycoprotein that is found mainly in the basal lamina, a thin sheet of specialized loose connective tissue which underlies epithelial cells, separating them from connective tissues. The basal lamina is a structural support and a permeability barrier. All forms of basal lamina contain type IV collagen, proteoglycans, laminins, and other accessory glycoproteins. Cells can alter the properties of the basal lamina by secreting enzymes that catalyze changes in the lamina. |
| Laminin structure | Laminin consists of three long polypeptides α, β, and γ; several types of each can combine to form many types of laminin. Disulfide bonds hold the polypeptides together in the shape of a cross. Laminins have several domains, including binding sites for components of the ECM proteins including type IV collagen and other accessory proteins and a distinct domain which enables binding to integrin receptors on cell surfaces. Since integrins are transmembrane receptors this association tethers the basal lamina to the cell membrane. |
| Focal adhesion sites | Migratory and non-epithelial cells such as lymphocytes, macrophages and fibroblasts attach to the Extracellular matrix via focal adhesion sites. These sites contain clustered integrins that interact with bundles of actin filaments via the adaptor protein, Talin, and the actin binding protein, Vinculin, on the inside of cells and Fibronectin (a component of the extracellular matrix) on the outside of the cells. These dynamic linkages allow cells to migrate to where they are needed. |
| Adhesive glycoproteins: Fibronectin | Fibronectins are a family of closely related glycoproteins in the ECM that help guide cell movement. Fibronectin has two large subunits linked near the C-terminals by two disulfide bonds. Each of the two Fibronectin subunits is folded into a series of rod-like domains which bind one or more ECM macromolecules, including several types of Collagen. Other domains of Fibronectin recognize cell surface receptors (Integrins) via a RGD (arg-gly-asp) sequence. This enables Fibronectin to act as a bridging molecule between cells and the ECM. |
| Integrin signalling | Integrin signalling can be bi-directional namely either Inside-out signalling or Outside-in signalling. In the former situation interaction of molecules to the Integrin tails on the inside of the cell can trigger a conformational change in the exterior portion of the dimer such that it is able to bind to ligand. Conversely, the application of tension from association of the ECM to the outside portion of the Integrin complex can cause changes in the activity of protein within the focal adhesion site to stimulate actin nucleation and bunding and other signalling pathways that alter gene transcription in the nucleus. |
| The Dystroglycan Complex stabilizes attachments of Muscle Cells to the ECM | The Costamere is an attachment structure at the surface of striated muscle which links the actomyosin cytoskeleton to the ECM and is important for maintenance of muscle integrity. Costameres contain many of the same proteins found at focal adhesion sites in addition to the protein Dystrophin, a very large skeletal muscle protein which makes up 5% of membrane-associated cytoskeleton in skeletal muscle cells. Dystrophin links cortical cytoskeleton actin filaments to the extracellular matrix (ECM) via Laminin and a transmembrane glycoprotein, called the Dystroglycan complex. |
| Types of PCD in animals | Necrosis: External signal, no genetic program, no signal transduction, cytoplasmic swelling, expansion of organelles, inflammation response. PCD; apoptosis: external or internal signal, involvement of signal transduction, defined catabolism of cellular components, chromatin condensation, fragmentation of the nucleus, almost no morphological modifications of organelles, |
| What triggers apoptosis? (Intrinsic vs extrinsic pathway) | The main differences between in intrinsic and extrinsic pathway: Apoptosome and Death-Inducing Signalling Complex (DISC) |
| Extrinsic apoptotic pathway | DISC formation and activation of the caspase cascade are the two most crucial events in apoptotic signalling. Death ligands: Bind specifically to one or multiple receptors, of TNF family. Death receptors: Tumour Necrosis Factor Receptor (TNFR) superfamily. 1. DISC is signalling platform of the extrinsic pathway and forms upon ligand binding to death receptor. 2. DISC activates caspase 8 by dimerization 3. FLIPs can inhibit DISC formation. 4. Initiator caspase 8: links to intrinsic pathway via BID, and Activates effector caspase 3, caspase 7 and others |
| Caspase activation | Proteolytic enzymes that cleave their substrates at defined positions, Highly specific. Intrinsic pathway: Apoptosome activates caspase 9, Initiator caspase 9, Effector caspase 3, caspase 7 and other caspases 1. Initiation: Caspase-9 2. Execution: caspase-7 |
| Intrinsic pathway | Cytochrome c originates from mitochondria, Activating formation of apoptosome. Apoptosome assembly and activation by cytochrome c: -CARD – caspase recruitment domain -NB-ARC – nucleotide oligomerisation domain -> hydrolyses ATP -Winged helix domain – interacts with (d)ATP / (d)ADP -> ATPase – hydrolyses bound ATP. -WD40 repeat – repeat sequence of ~40 aa; repeats form circular β-propeller structures -> involved in cytochrome c binding |
| Mitochondrial Membrane Permeabilization (MMP) | MMP -> cytochrome c release. This is point of no return in apoptotic pathway. Mitochondrial contents flow to cytosol to activate apoptotic signalling. Intrinsic pathway can be activated independently by intracellular stress. Chemotherapeutic agents, irradiation or growth factor withdrawal activate BAD -> MMP. Severe ER stress (Ca2+ ion release) cause MMP. Cytochrome c: is critical to PCD signalling, also essential for oxidative phosphorylation, Normally found in associated with inner membrane of mitochondria. Released from the inner mitochondrial membrane by oxidation of cardiolipin. |
| TWO mechanisms for MMP | 1.MOMP - Mitochondrial outer membrane permeabilization 2. Mitochondrial permeability transition (MPT) opens the Permeability Transition Pore Complex (PTPC) |
| MOMP - Mitochondrial outer membrane permeabilization | Proteins of BH3 family activate BAK/BAX to form channels in outer membrane cause MMP BH3 proteins can also inhibit antiapoptotic factors (BCL-2 proteins), liberating BAK/BAX activation. BH3-only proteins include: BID (activated by extrinsic) and BAD (activated by intrinsic) |
| Mitochondrial permeability transition (MPT) opens the Permeability Transition Pore Complex (PTPC) | Immediate loss of membrane potential cause swelling and rupture. Activated by Ca2+ accumulation and ROS at the inner membrane (IM). Or activated by BAK/BAX. PTPC sensitised by inhibition of BCL-2 antiapoptotic proteins by BH3 family (BID/BAD). BAK/BAX and BH3s can link the 2 MMP mechanisms. |
| Regulation of apoptosis | Bcl-2 family: Act in the intrinsic pathway (NOTE: Pro-apoptotic BAX and BAK are also Bcl-2 family members). Inhibitor of Apoptosis (IAP) family: Act in the intrinsic and extrinsic pathway. |
| Anti-apoptotic proteins | Act at different levels. AP family proteins act as E3 ligases: RING domain is essential E3 ligase activity, Ubiquitinates specific pro-apoptotic proteins, Degradation of ubiquitinated caspases can prevent PCD |
| Plant do not have caspases | Distantly related Metacaspases (MCs) and Vacuolar Processing Enzymes (VPEs) which lead the cascade have been ID’d in plants. MCs and VPEs have caspase activities and participate in plant PCD. Indicates convergent evolution of (similar) PCD pathways in plants and mammals |