flUÍJI SCI Extracellular matrix and cell-cell interactions RNDr. Jan Škoda, Ph.D. Department of Experimental Biology Outline - Supportive extracellular structures -Cell wall in prokaryotes -Cell wall in plants and fungi - Extracellular matrix (ECM) in animals -Cell-ECM interactions -Cell-cell interactions 2 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Supportive extracellular structures -Cell wall (Bacteria, Archea, plants, fungi) -Glycocalyx (animals) Multicellular eukaryotic organisms - Interactions among cells: tissues = cells + extracellular matrix (ECM) - Interconnection of cells - direct contact or via ECM - Cell growth, migration, proliferation, differentiation 3 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell wall in prokaryotes - ECM and cell-cell interactions (4 May 2022) Gram-positive bacterial cell wall Gram-negative bacterial cell wall Lipoteichoic acid Cell wall (ITA) Phospholipid (fatty acid D-glycerol esters) Peptidoglycan Cytoplasmic membrane protein Cytoplasmic membrane Outer membrane Pilus protein Lipopolysaccharide (LPS) Do„tw„r,i„„„ r'~1~L" H 1^ H HH H I- Periplasmic space Inner membrane I Cytoplasmic membrane protein Archaeal cell wall (Methanobacterium, Methanosphaera, Methanobrevibacter) Pseudopeptidoglycan (pseudomurein) Phospholipid -h (isopranoid alcohol L-glycerol ethers) Heteropolysaccharide Cytoplasmic membrane protein Archaeal cell wall {Methanococcus, Holobacterium) - . . Phospholipid Cytoplasmic ,. . , isopranoid alcohol membrane , , ... L-glycerol ethers) '—\ *—\ ,— MM öcmicl Cytoplasmic membrane protein 5 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Gram staining Hans Christian Gram -G+: thick peptidoglycan walls retain crystal violet Microscopic View Iodine (potassium iodine) forms complexes with crystal violet - Ethanol (acetone) dehydrates peptidoglycan layer ■ Crystal violet trapped and insoluble in water ■ Safranin counterstain all bacteria ■ G+ purple; G- pink/red 6 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Gram staining result Purple: G+ Staphylococcus Pink: G- Escherichia coli 7 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell wall in plants and fungi 9 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell wall in plants - Primary cell wall - Thin, flexible, extensible - Cellulose, hemicelluloses, pectin - Secondary cell wall - Additional layers underneath the primary cell wall (always produced by the cell itself) - Thick layer, strengthens and/or waterproofs - Additional components: lignin (xylem), suberin (cork) - Middle lamella - Between cell walls of neighboring cells - Calcium and magnesium pectates Plasma 10 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) fl - Arabidopsis thaliana stem cross section Cellulose, pectin 20 Mm 2 fim 11 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Plasmodesmata - Plasmodesma - A channel interconnecting cytosol of two cells; cell-cell communication - Primary - Forms during cell division, around ER trapped between the dividing cells = desmotubule - Secondary - Between non-dividing cells, unknown mechanism Plant cell wall layers Middle lamella Primary wall Secondary wall Simple Complex Primary wall CELL 1 Three layers of secondary wall Middle lamella 1 (im — Membrane Cytosol Cell wall □ Endoplasmic reticulum (desmotubule) 12 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Plasmodesmata Intercellular Junction Plasma Membrane — Cross-Linking Glycan Pectin Cellulose Microfibrils Cell Walls of Adjacent Plant Cells Smooth Endoplasmic Reticulum Middle Lamella — Primary Cell Wall Desmotubule Figure 1 13 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell wall composition and synthesis -Cellulose microfibrils cross-linked with other polysaccharides, hemicellulose and pectin -Specificcomponents: lignin (xylem - wood), cutin (epidermis), suberin (cork), silica crystals (in grasses) Middle J— Lamella I_ Primary Cell Wall * Plasma J Membrane!_ 14 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell wall composition and synthesis Cellulose synthesis: proteins embedded in the plasma membrane Cellulose synthase (CESA) ■ Oligomers form complexes - rosettes trimer Transport of saccharide monomers across the plasma membrane Microfibril orientation - microtubules 15 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) CM U accessory proteins? Apoplast (extracellular space) Trends in Plant Science Cell wall composition and synthesis Cellulose synthesis - Probable model: cellulose microfibrils synthesized as the complexes move along the microtubules Cellulose microfibril (right-handed twist) Other cell wall components -Synthesized inside the cell and secreted by exocytosis EXTRACELLULAR SPACE distal ends of cellulose microfibrils being integrated into preexisting wall plasma membrane CYTOSOL cellulose synthase complex, or rosette 0.1 urn microtubule attached to plasma membrane 16 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Fungal cell wall Cell membrane Mannoproteins ■Chiti Membrane proteins Candida albicans Aspergillus fumigatus Cryptococcus neoformans CalXM GXM Capsule -Chitin, chitosan -Glucans (mostly ß-glucans) - Differ between species - Mannoproteins - Melanin Melanin Rodlet GAG Galactomannan a-1.3-glucan Candida yeasts b Pneumocystis spp. c Aspergillus conidium d Aspergillus e Cryptococcus f Histoplasma capsutaturn fumigatus and Blastomyces hyphae dermatitidis 18 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Extracellular matrix in animals 19 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Glycocalyx Pericellular matrix (vs. extracellular matrix!) Dense glycoprotein and glycolipid cover Plasma membrane components Physical barrier, involved in regulation of cell-cell interactions e.g., zona pellucida in oocytes, endothelial cells Healthy status <3 Syndecan-1 Endothelial cell Sepsis ± Sepsis-induced release of sheddases (Heparanase/MMP) Dissipated hydration 9 Sc Uli k FLUID LOSS 20 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Extracellular matrix (ECM) - Organization and composition based on the tissue type - Connective tissue (bone, tendon, cartilage, vitreous body): predominance of ECM (mechanical strength and support) - Muscle and epithelial tissue: minimal ECM - basal lamina (mechanical support provided by cytoskeleton) Roles of ECM: 1. Mechanical support 2. Tissue-specific functions and cell adhesion signaling 3. Regulation of developmental processes 21 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) ECM composition -Components produced by cells and secreted by exocytosis 1. Collagen fibers (provide tensile strength; collagens) 2. Elastic fibers (provide elasticity; elastin and fibrillin) 3. Space filling components (prevents compression; glycosaminoglycans: hyaluronan and proteoglycans) 4. Adhesive molecules (glycoproteins) Different types of ECM in different tissues 22 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Collagen fibers -Collagens - in mammals -40 genes, 25% of the total protein mass -Triple-stranded helical structure of three collagen a chains - Rich in proline and glycine: superhelix -> collagen microfibril -> collagen fibril (10-300 nm in diameter) -> collagen fiber - Organization of fibrils - tissue- and function-dependent: ■ Skin - plywood-like arrangement: different layers laid at nearly right angles; withstand tension stress in multiple directions - Tendons - fibrils organized in parallel; withstand tension in the major axis -Synthesis by fibroblasts and epithelial cells 23 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) fl a. Fibril 24 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Collagen fibrils produced by a fibroblast 25 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) I_I 5 |xm Collagen fibrils in the skin Elastic fibers - Elastin and fibrillin -Synthesis limited to embryonal and juvenile fibroblasts -Skin, blood vessels, lungs - Network of elastin molecules (core) and glycoprotein microfibrils (sheath; fibrillin) - Elastin network recoils after stretch: elasticity 26 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Space filling components ff Glycosaminoglycans (GAG) ) 1 nu -Hyaluronan: glucuronic acid & A/-acetylglucosamine; up to 50 K disaccharide units in one molecule - Proteoglycans: core protein and covalently bound GAG chains - Highly polar = attract water - form viscous hydrogels - Attain large volumes - lubricants or shock absorbers -Hyaluronan surface receptors (e.g., CD44): activation promote cell proliferation, migration, and invasion 28 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) fl Hyaluronan Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Proteoglycan Proteoglycan Integrin Microfilaments of cytoskeleton 30 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Adhesive glycoproteins - Mediate interactions in the ECM, important for cell-ECM interactions 4 major types: - Laminins: basal lamina glycoprotein - Fibronectins: binds to integrin, cell-ECM attachment -Tenascins: antiadhesive effects, often overexpressed in tumor stroma (metastasis), connective tissue, brain, bone, smooth muscles - Fibrinogen: thrombocyte aggregation (coagulation factor) 31 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) fl Adhesive glycoproteins interact with other ECM components Laminin - Three polypeptide chains - Crosslinked by disulfide bonds to cross-like structure self-assembly 20 nm self-assembly integrins dystroglycan perlecan Fibronectin - Dimeric structure, disulfide bonds - Cell binding: trough integrins m (A) 100 nm (b) domain self-association collagen binding cell binding heparin binding \s* n \ module 32 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Human lung fibroblasts Rat pulmonary artery smooth muscle cell Nuclei /fibronectin-GFP (ECM) Nuclei / tenascin (ECM) / F-actin 33 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) ECM remodeling - Mechanisms of degradation and re-organization - Important during development, wound healing, inflammation etc. - Exploited by cancer cells (metastasis) -Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinase (TIMPs) 34 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Basal lamina - basement membrane - Underlies epithelial cells, surrounds muscle, fat and Schwann cells -Cell polarity, mechanical connection with connective tissue, filter (kidney) - Relatively thin ECM layer: 50-200 nm -Collagen type IV, laminins, nidogens (linker proteins; formerly entactin), perlecan muscle epithelium basal lamina connective tissue uaaai lamina lumen or external surface •-WW. , muscle cell plasma membrane connective tissue basal lamina kidney glomerulus blood endothelial cell s T-^rinTV^ epithelial cell basal lamina 35 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) laminin integrin epithelium basal lamina — macrophage fibroblast hyaluronan, proteoglycans, and glycoproteins 50 (im epithelial cells basal lamina collaqen_*: .&&'7iS^ - -" , k 'f^O^m-^uH Chick embryo cornea 10 (xm 36 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell interactions 37 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell interactions - Expression of specific receptors: cell adhesion molecules (CAM) - CAM interactions: cell-cell interactions & cell-ECM interactions - Intracellular^ linked with cytoskeleton (stability and signaling) Regulation by translocation (membrane vs. vesicles; secretory vs. endocytic pathway) 4 major CAM classes: - Integrins (cell-ECM) -Cadherins (cell-cell) -Selectins -Immunoglobulin-like CAMs (ICAMs) 38 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Focal adhesion Hemi-desmosome Integrin Membrane proteoglycan 39 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell-ECM interactions 40 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Focal adhesions Connecting actin filaments to ECM through integrins Important for cell signaling (FAK/Src) cell survival, migration, proliferation... r i.ii.iu'iini,ir m.niii: 41 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Vinculin (focal adhesions) / F-actin / nuclei 42 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Hemidesmosomes -Connecting intermediary filaments to ECM through integrins - Anchor epithelial cells _ Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Cell-cell interactions 45 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Types of cell-cell interactions Cell adhesions -Adherens junctions (adhesion belt) - Desmosomes Tight junctions Channel-forming junctions -Gap junctions - Plasmodesmata (plants) 46 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Adherens junctions Vinculins - Connect actin filaments in neighboring a-actinin cells via Cadherins \ Actin filaments 47 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Desmosomes -Connect intermediary filaments in neighboring cells via desmosomal Cadherins 48 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Tight junctions -Seal gaps between epithelial cells close to apical membrane - Impermeable (selectively permeable) barrier -Claudin, occludin, JAMs (junction adhesion molecules) • V lumen • «• ••••••• •1 • • • cell 1 ■ H II i (A) _ tracer molecule cell 2 _ tight ce|| Junction 3 0.5 |xm 0.5 |xm 49 Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Bi1700en Cell Biology / 09 - ECM and cell-cell interactions (4 May 2022) Gap junctions (parallel: plasmodesmata in plant cells) - Directly link cytoplasms of adjacent cells; animal cells - Diffusion of water, ions, small molecules: cell-cell communication - Hexamer of connexin proteins = connexons (1.5 nm wide channels) Tight junction :; Claudin !! > *_■ 11j lOccludin:: Adherens Junction Actin p120 cateniif;; Desmosome xfc \0 \g> ICS.......HiODPMDPi -»—DM Gap Junction Ca 2+ Proteins CAMP IP3 IConnexins ,Ca2+ Proteins 5 cAMP IP3 52 Bi1700en oen cioiogy / u» - toivi ana cen-ceii inieracuons ^ iviay