CELL SIGNALING AND MOTILITY (BIOL 3373) )

1. CELL SIGNALING AND MOTILITY (BIOL 3373)) Lecture 8

2. Definition and Classification of cell junction • Cell junction (also called membrane junction) is the connection between the neighboring cells or the contact between the cell and extracellular matrix. Cell junction are classified into three types: • Anchoring junction • Occluding junction • Communicating junction can be either channel forming junction orsignal releasing junction

3. Types of cell junction in animal tissue Communicating junction

4. Cell Adhesion Molecules (CAMs) • Cell junctions are constitutes by Cell Adhesion Molecules (CAMS): • Important cell surface proteins molecules promoting cell–cell and cell–matrix interactions. • Important for many normal biological processes -embryonic cell migration, immune system functions, wound healing. • Involved in intracellular signaling pathways (primarily for cell death/survival, secretion etc.)

5. STRUCTURES of Cell Adhesion Molecules (CAMs) • Express 3 major domains: • The extracellular domain allows one CAM to bind to another on an adjacent cell. • The transmembrane domain links the CAM to the plasma membrane through hydrophobic forces. • The cytoplasmic domain is directly connected to the cytoskeleton by linker proteins.

6. Binding of an adhesion molecule on one cell to the same adhesion molecule on a second cell Cadherin - cadherin An adhesion molecule on one cell type binds to a different type of cell adhesion molecule on a second cell Selectins – mucins The linker molecule in most cases is Laminin, a family of large cross shaped molecules with multiple receptor domains. Interactions between CAMs can be mediated by :

7. Cell Adhesion Molecules (CAMs) • Cell adhesion molecules can be divided into 4 major families: • The cadherin superfamily • The selectins • The immunoglobulin superfamily • The integrins

8. The Cadherin superfamily • Cadherins are the most prevalent CAMs in vertebrate: • 125 kD transmembrane glycoproteins - mediate intercellular adhesion in epithelial and endothelial cells by Ca2+ dependent homophilic adhesion. • Primarily link epithelial and muscle cells to their neighbors: Form desmosomes and adherens junctions • Play critical role during development (cell sorting). • Do not interact with extracellular matrix.

9. The Cadherin superfamily EC1 EC2 EC3EC4 Contain a short transmembrane domain and a relatively long extracellular domain containing four cadherin repeats (EC1-EC4), each of which contains calcium binding sites. Cadherins interact with specific cytoplasmic proteins, e.g., catenins (α, β and γ), through which they link the actin microfilament. The binding ofcadherinsto the catenins is crucial for cadherin function.

10. The Selectins Selectins are cell-cell adhesion receptors expressed exclusively on cells in the vascular system. Selectins function to arrest circulating leukocytes in blood vessels so that they can crawl out into the surrounding tissue. Three forms of selectin have been identified: L-selectin P-selectin E-selectin • Structure of Selectins: • NH2-terminal C-type Ca2+ dependent lectin like binding domain, which determines the ability of each selectin to bind to specific carbohydrate ligands. • an epidermal growth factor-like region. • a number of repeat sequences. • a membrane-spanning region • a short cytoplasmic region

12. Immunoglobulin Superfamily Molecules • Consists of more than 25 molecules. • Important ones being: • Intracellular adhesion molecule 1(ICAM1; CD54) • Intercellular adhesion molecule 2 (ICAM2), • Vascular cell adhesion molecule1 (VCAM1; CD106), • Platelet endothelial cell adhesion molecule 1 (PECAM 1; CD31)

13. Theintegrins Integrins are transmembrane proteins Integrins couple ECM to cytoskeleton through cytosolic proteins, talin, a-atinin, paxillin, filamin, vinculin

15. Theintegrins • Formed by 1 αand 1 β subunit: • 50 different α and 8 different β subunits give rise to over 20 different heterodimeric combinations at cell surfaces. • Bind epithelial and muscle cells to allow platelets to stick to exposed collagen in a damaged blood vessel • Allow fibroblasts and white blood cells to adhere to fibronectin and collagen as they move

16. Integrin ACTIVATION SIGNALS Sanford J. Shattil, Chungho Kim & Mark H. Ginsberg The final step of integrin activation: the end of the game Nature Reviews Molecular Cell Biology 11, 288-300 (April 2010) doi:10.1038/nrm2871 There are two directions of integrin signaling, which have different biological consequences: 1) In 'outside–in' signaling, Integrins behave like signaling receptors. Binding of Ligandschanges the conformation of the integrin and contributes to integrin clustering. This leads to intracellular signals that control cell polarity, cytoskeletal structure, gene expression and cell survival and proliferation. 2) In 'inside–out' signaling, an intracellular activator, such as talin, binds to the β-integrin tail, leading to conformational changes that result in integrin activation. Inside–out signalling controls adhesion strength and enables sufficiently strong interactions between integrins and extracellular matrix (ECM) proteins to allow integrins to transmit the forces required for cell migration and ECM remodelling and assembly. Although we conceptually separate the two processes, they are often closely linked; for example, integrin activation can increase ligand binding, resulting in outside–in signaling. Conversely, ligand binding can generate signals that cause inside–out signalling

17. Anchoringjunction Anchoring junction are the junction ,which provides strength to the cell by acting like mechanical attachment. These junction provide firm structural attachment between two cells or between a cell and extracellular matrix Anchoring junction are responsible for structural integrity of the tissue.

18. Anchoring Junctions • Desmosomes (cell – cell junction) • Hemidesmosomes (cell- Matrix Junction)

19. Desmosomes Also known as macula adherens is a cell structure specialized for cell-to-cell adhesion. Are molecular complexes of cell adhesion proteins and linking proteins that attach the cell surface adhesion proteins to intracellular intermediate filaments keratins. The cell adhesion proteins of the desmosome, desmoglein and desmocollin, are members of the cadherin family.

20. Hemidesmosomes Hemidesmosomes look like half-desmosomes that attach cells to the underlying basal lamina. Hemidesmosomes use Integrins cell, instead than cadherin proteins The integrin molecule attach to extracellular matrix protein such as laminin, forming adhesions between cell and matrix.

21. Summary of AnchoringJunctions FEATURES

22. Occluding Junction Function of occluding Junction • Strength and stability • Selective permeable for ions. • Fencing function • Maintenance of cell polarity • Blood-brain barrier • Choroid plexus A cell-cell junction that closures cells together in an epithelium in a way that prevents leaking from one side of the sheet to the other. membranes join together forming a virtually impermeable barrier to fluid Also known as Tight Junction

23. Occluding Junction Three main transmembrane protein families are found in tight junctions:  occludin, claudins, and junctional adhesion molecules (JAMs). 1)Occludin proteinsconsist of 4 domains- 2 intracellular and 2 extracellular and are involved with the regulation of signaling event. 2) Claudins proteins compose the major structural and functional elements of tight junctions. They also consist of 4 domains- 2 intracellular and 2 extracellular and mediate calcium-independent cell-cell adhesion. 3) JAMs have only a single transmembrane domain, unlike occludin and claudins. While JAM proteins are believed to regulate the paracellular barrier, direct evidence is lacking to support this.

24. Communicating Junction Cell junction which permit called communicating junction, these junction permitthe intercellular exchange of substance (e.g ions and molecules from one cell to another cell). Two Types of Communicating Junction: Gap junction Chemical synapse

25. Gap Junction Gap Junction consist of intercellular channels in the plasma membrane of adjacent cells. So small molecules can diffuse across the channel and into the cytoplasm of the other cell. Gap junctions were first discovered in myocardium and nerve because of their properties of electrical transmission between adjacent cells (Weidmann 1952; Furshpan and Potter 1957). Gap Junction are present in heart, basal part of epithelial cell of intestinal mucosa, nerve cells, etc

26. Gap Junction STRUCTURE The structural unit of gap junction is called Connexon- The connexon of cell A is alligned with the connexon in cell B (fig). A Connexon is composed by 6 connexins, a transmembrane protein, which make the connexon having an hexamer structure.

27. Chemical synapse The space between the pre- and postsynaptic membrane of two nerve fiber is called the synaptic cleft. The structure of synaptic cleft is stabilized by adhesion proteins (i.e Cadherin, Ig-superfamily members). The key feature of all chemical synapses is the presence of small, membrane-bounded organelles called synaptic vesicles within the presynaptic terminal. The synaptic vesicles contain neurotransmitters, chemical signals that act as messengers between neuron-neuron or neuron- muscle fiber cell. (Remember! In case of neuromuscular junction the neurotransmitter is Acetylcholine) Chemical synapse is the junction between two nerve fibre or between a nerve (motor) fibre and a muscle fiber (Remember neuromuscular junction!!!). which signals transmitted by the release of chemical transmitter.

28. Overview of various cell junctions in epithelial cells

29. The extracellular matrix (ECM)

30. The extracellular matrix (ECM) • The extracellular matrix is a dense network of proteins that: • lies between cells • is made by the cells within the network • The extracellular matrix together with cell junctions control: • the three-dimensional organization of cells in tissues • the growth, movement, shape, and differentiation of these cells

31. The extracellular matrix (ECM) Electron Micrograph of cells surrounded by ECM Embryonic chick limb bud

32. Cells communicate through ECM to affect Cell shape

33. The extracellular matrix ECM consists of two fundamental components: • proteoglycans composed by proteins covalently linked to Glycosaminoglycans (GAGs) • Fibrous proteins: collagen, fibronectin,elastin, Laminin.

34. Proteoglycans • Proteoglycans consist of a central protein “core” to which long chains of disaccharides, called glycosaminoglycans (GAGs), are covalently attached. GAG chains on proteoglycans are negatively charged due to the presence or both carboxyl ( COO-) ANDsulfate group (SO3-).

35. The assembly of proteoglycan • The protein core is produced by membrane-bound ribosomes. • The linkage of GAG to a protein occurs in the Golgi apparatus. • The assembly of proteoglycan starts from a serine into the protein core: this serine binds GAG through a tetrasaccharide link. • Inside the Golgi GAG can be modified (e.g. epimerization which alters the structure of the sugar) promoting the heterogeneity among proteoglycan • After the assembly proteoglycan are secreted to the exterior by exocytosis

36. Proteoglycans FUNCTIOn • Proteoglycans attract water to form gels that: • keep cells hydrated • cushion tissues against hydrostatic pressure • The GAG molecules act as filters to limit the diffusion of viruses and bacteria in tissues.

37. Proteoglycans • Proteoglycans can bind to a variety of extracellular matrix components, including: • growth factors • structural proteins • cell surface receptors • Expression of proteoglycans is: • cell type specific • developmentally regulated

38. Proteoglycans types • Different combination between core proteins and GAG makes proteoglycans highly heterogeneous (e. g. DECORIN, AGGREGAN RIBONUCLEASE). • Proteoglycans differs for dimension:

39. Proteoglycans: Aggrecan Aggrecan is the major component of cartilage. Into the cartilage matrix aggrecans bind non-covalently to hyaluronan molecules.

40. Proteoglycans:HEPARAN SULFATE • Heparan sulfates are composed of distinct combinations of GAG (more than 30 different sugar subunits). • Heparan sulfate proteoglycans are a cell surface co-receptors of : • soluble proteins such as growth factors • insoluble proteins such as extracellular matrix proteins

41. Proteoglycans: HYALURONAN • Hyaluronan is a glycosaminoglycan. • It forms enormous complexes (MW 8 x 10^6) with proteoglycans in the extracellular matrix • Hyaluronan is highly negatively charged therefore It binds to cations and water in the extracellular space. • This increases the stiffness of the extracellular matrix . • This provides a water cushion between cells that absorbs compressive forces .

42. HYALURONAN StRUCTURE Hyaluronan consists of repeating disaccharides ( glucuronic acid and N-acetylglucosamine) linked into long chains Hyaluronan does not contain sulfate group as the rest of proteoglycan

43. Proteoglycans: HYALURONAN • Unlike other glycosaminoglycans, hyaluronans chains are: • synthesized on the cytosolic surface of the plasma membrane • translocated out of the cell

44. Fibrous proteins: Collagen • Collagens provide structural support to tissues. • Collagen family comprises over 20 different extracellular matrix proteins. (they are the most abundant proteins in the animal kingdom).

45. Electron micrograph of Fibroblasts surrounded by collagen fibrils in embryonic chick skin Collagen fibrils Fibroblast

46. Fibrous proteins: LAMININ • Laminins are heterotrimers comprising three different polypeptides a, b, g wrapped together in a coiled-coil configuration. Laminin heterotrimers do not form fibers. • They bind to linker proteins that enable them to form complex webs in the extracellular matrix. A large number of proteins bind to laminins, including more than 20 different cell surface receptors.

47. SPECIALIZATION OF ECM: The basal lamina • The basal lamina,also known as basement membrane, is a thin sheet of extracellular matrix: It is found at: • the basal surface of epithelial sheets • Muscle ( neuromuscular junction) • Kidney glomerulus

48. The basal lamina SEM micrograph of Basal Lamina in the cornea of a chick embryo

49. A molecular structure Model of basal Laminae • The components of the basal lamina vary in different tissue types. • Most Basal Lamina share four principal extracellular matrix components: • sheets of collagen IV and lamininheld together by perlecanproteoglycans and by the linker protein nidogen (A). In B the arrows indicates molecules that can bind directly to each other

50. The basal lamina The basal lamina separates cells and ephitelia from the underlying or surrounding connective tissue The cells of connective tissue are embedded in the extracellular matrix, that consists of protein fibers embedded in an amorphous mixture of proteoglycas