FCP-1: Cell Biology

1. FCP-1: Cell Biology 1st contact session: cell membranes, cytoplasmic organelles, the cytoskeleton, intercellular connections, cell adhesion molecules, transport across cell membranes, ATP production

2. Part 1: intracellular structures and organelles

3. Simplified depiction of a cell

4. Cell membrane components • Main component: phospholipids • (hydrophilic outside, hydrophobic • inside, spontaneous bi-layer) • Selectively permeable • Inner membranes have similar • structure • Proteins: integral vs peripheral • Modifications • Anchors Cell adhesion molecules, pumps, channels, receptors, enzymes

5. Mitochondria (1) Main function: energy production through oxidative phosphorylation

6. Mitochondria (2) • Used to be free-living bacteria • Contains the components of the electron transport chain (energy production) in the inner membrane • Contains own genome (smaller than nucleus) and ribosomes (protein synthesis machinery) • Zygote mitochondria come from the ovum: maternal inheritance of mtDNA • Very ineffective DNA repair leads to mistakes: results in a large number of rare diseases associated with defects in energy metabolism

7. Mitochondria (3) Electron transport chain (oxidative phosphorylation, generation of ATP/energy): Later…

8. Lysosomes: rubbish bins • Large, irregular structures in the cytoplasm • Acidic interior, digest endocytosed bacteria and discarded cell components • Filled with acid hydrolases, cannot function at normal cellular pH, will not destroy other cell components • Lysosomal storage diseases result from absence of enzyme, accumulation/engorgement of lysosomes

9. Peroxisomes: detox and more • Catalyse various anabolic and catabolic reactions, e.g. breakdown of very long chain fatty acids, production of plasmalogen (myelin), production of bile acids • Enzymes oxidize substrates, generating toxic H2O2, used to oxidize other substrates, neutralizing H2O2 • NB for the detox of ethanol • PXR gene product is outer pxome receptor, PEX gene products import proteins into pxome, and enzymes are targeted into pxome by PTS signal • Errors in pxome assembly result in Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease (lethal in infants)

10. Nucleus: command HQ • Contains all of the DNA (nuclear genome) required for gene expression, in the form of chromatin • Site of gene expression (DNA → mRNA)

11. Nucleus • DNA (chromosomes) normally unravelled, disorganized: chromatin • Individual chromosomes condense before cell division • Nucleolus contains RNA, proteins: ribosome assembly • Nuclear envelope a double-layer membrane • Contains pore complexes for shuttling of proteins, ribosomes and RNA: ribosomes and RNA produced in nucleus, must shuttle to cytoplasm for protein synthesis, some proteins (i.e. transcription factors) must shuttle back to nucleus

12. Ribosomes: protein assembly lines

13. Endoplasmic reticulum: processing • Complex series of tubules in the cytoplasm • Contiguous to the nuclear membrane • Smooth ER: steroid synthesis • Rough ER: covered with ribosomes, protein synthesis, folding and modification

14. Golgi apparatus: add some sugar • Stacked membrane-enclosed sacs • Proper glycosylation (sticking on carbohydrate/sugar chains) of lipids and proteins • Directional (cis→trans) • Vesicles shuttle from the ER, through the Golgi, out for secretion

15. Cytoskeleton: intracellular highways • Maintains structure, helps to move and change shape • Also moves proteins and organelles around

16. Molecular motors to move cargo Kinesin, dynein, myosin: all use ATP (energy)

17. Part 2: Intercellular connections

18. Holding cells together: Tight junctions • Surround the outer layer of epithelial cells (intestinal mucosa, renal tubules, choroid plexus in brain) • Also contribute to endothelial barrier function • Totally obliterates the gap between cells, prevents protein • leakage between cells

19. Holding cells together: zonula adherens

20. Holding cells together: desmosomes • - Adhesion protein = cadherin, helps to withstand shear stress in epithelium, • particularly in epidermis • - Defining feature: dense plaques on cytoplasmic side, attached to • cytoskeletal filaments • Blistering diseases (Pemphigus) are auto-immune, attack desmogleins • (cadherins), cause layers of skin to pull apart

21. Attaching cells to the basal lamina: hemidesmosomes and focal adhesions

22. Gap junctions: intercellular communication • 1 subunit = connexin • Pore with 6 connexins • = connexon • permit passage of ions • and small metabolites • between cells • highly selective (20 diff • connexin genes, each for • different flow-through)

23. Cell adhesion molecules • All intercellular connections consist of cell adhesion • molecules (CAMs) • 4 broad families: integrins, cadherins, selectins and • IgG adhesion molecules • Not just for adhesion, but also for signalling: • cells that lose contact with other cells undergo • dissociation-induced apoptosis (anoikis) • collagen-integrin interaction essential for osteoblast • differentiation

24. Part 3: transport across cell membranes

25. Exo- and endocytosis Note that the cytoplasmic side of the membrane always remains the cytoplasmic side

26. Endocytosis continued • Phagocytosis: eating of bacteria, dead tissue by leukocytes • Pinocytosis: drinking of solutes • Both processes involve invagination of the plasma membrane before pinching off vesicle inside the cell • Clathrin-mediated endocytosis: three-legged clathrin molecules cover endocytotic vesicle (NB for receptor internalization and synaptic function)

27. How do molecules move across the cell membrane? • Small non-polar and neutral polar molecules diffuse directly across (O2, N2 CO2) • Everything else needs help! • Transport proteins form channels for transport of various molecules • Even water! (through aquaporins) • Some are non-selective ion channels, some are very selective

28. How do molecules move across the cell membrane? • Some channels are gated (opened upon a particular stimulus):

29. How do molecules move across the cell membrane? • Carrier proteins transport molecules WITH a concentration or electrical gradient: facilitated diffusion, does not require energy (example: glucose) • Other carriers transport molecules AGAINST a gradient: active transport, requires energy • Many carrier proteins are therefore ATPases: hydrolyses ATP for energy for transport • Secondary active transport: transport of one molecule coupled to the transport of another (often Na+) • Symport: two molecules moving in the same direction • Antiport: exchange of molecules in opposite directions

30. Ion channels Possible configurations:

31. Part 4: Energy (ATP) production

32. ATP hydrolysis = energy ATP → ADP + Pi + 30-50 kJ energy Energetically unfavourable (unstable) Energetically more stable Interesting factoid: 60% of energy goes towards maintenance of body temp

33. Main site of ATP production: the citric acid cycle cytoplasm mitochondria But before we get to this point…..

34. Glycolysis (Embden-Meyerhof pathway) 1x 6-carbon 2x 3-carbon Net gain (1 mol glucose): 4 ATP – 2 ATP = 2 ATP; 2 pyruvate; 2 NADH

35. Or….Glycogen breakdown Net gain from 1 mol glucose-6-phosphate: 4 ATP – 1 ATP = 3 ATP; 2 pyruvate; 2 NADH

36. Or…Beta-oxidation of fatty acids • Takes place in mitochondria: long-chain fatty acids • transported in by carnitine • - 18-C fatty acid generates 8 acetyl-CoA

37. Main site of ATP production: the citric acid cycle cytoplasm mitochondria

38. From NADH/FADH2 to ATP

39. ATP production: adding it up • 1 pyruvate generates 4 NADH, 1 FADH2 and 1 GTP (ATP) • 1 NADH = 3 ATP, 1 FADH2 = 2 ATP • 1 pyruvate = (4x3) + (1x2) + 1 = 15 ATP • 1 glucose (2 ATP; 2 pyruvate; 2 NADH) = 2 + (2x15) + (2x3) = 38 ATP • 1 glucose-6-P (from glycogen) = 39 ATP • 1 18-C fatty acid = 8 x 15 = 120 ATP • 1 triglyceride ≥ 360 ATP