Growth and differentiation: tissue homeostasis

1. Growth and differentiation:tissue homeostasis James Going Pathology, GRI

2. One organism • Many different tissues • Many different cell types • Many special • functions • Things wear out

3. Self renewal • Some tissues renewed more rapidly than others • Red cells last longer than platelets which last longer than neutrophils • Cartilage, neural tissue ‘turn over’ slowly • Intestinal mucosa rapidly

4. Tissue homeostasisA lifelong need for new cells • Cell proliferation: new cells • Cell differentiation: specialisation • Cell destruction: old, unwanted • and dangerous cells • Programmed cell death / apoptosis

5. Stem cells provide for cell renewal

6. Renewal of tissues Stem cells divide slowly Rapidly proliferating ‘transit amplifying’ (TA) cell populations differentiate into various cell types Extracelluar matrix and microenvironment strongly influential - stem cell ‘niches’

7. Hair follicle

9. ‘Immortal strand’ hypothesis • Cairns 1975 • “a change in sequence is not fixed irreversiblyuntil it is present in both strands, i.e., has been copiedat the next round of replication. It seemed possible, therefore,that stem cells might avoid accumulating mutations if, at mitosis,they always kept, for each chromosome, the chromatid with theolder template strand”

10. Asymmetrical stem cell division • Normal colon crypts - • orientated division of stem cells - • Retain DNA label

11. Asymmetrical stem cell division • APC colon crypts -random orientation of stem cell divisions even in heterozygotes • Lose ‘label-retaining’ ability

12. More commitment, less flexibility Totipotent stem cells can form any possible tissue Pluripotent stem cells can form many but not all tissues Multipotent stem cells further restricted eg haemopoietic, cutaneous

13. Stem cells have unexpected possibilities Livers of female patients with bone marrow transplants from male donors can contain hepatocytes with Y chromosomes - presumably of graft origin Female donor kidneys in male patients may contain renal (not blood) cells of male, ie recipient origin Therapeutic possibilities - tissue engineering etc

14. Differentiation and gene expression • Specific patterns of gene expression stable in differentiated cell lineages (RNA,Protein) • Environment, Epigenetic modifications • eg DNA methylation, Histone configuration and modifications - the ‘Histone code’

15. Stem cell and differentiation control Important genes beginning to be identified Basement membrane-integrin interactions Inductive signalling - diffusible signals, cell/cell contacts, gap junctions etc Pattern formation - ‘morphogenetic gradients’

16. Cell Proliferation

17. Eukaryotic cell division • The ‘cell cycle’ - • Successive rounds of • Growth (G1) • Genome doubling (DNA synthesis, S) • More growth (G2) • Genome halving (Mitosis, M)

18. The Cell Cycle M G1 G0 G2 S Howard and Pelc 1951

19. G0 • A cell not actively proceeding round the cell cycle exits in G1 to a non-replicating state: G0 • G0 cells often ‘terminally differentiated’, will never re-enter cell cycle • Others can re-enter G1 - eg lymphocytes recruited by antigenic stimulation, hepatocytes following partialhepatectomy

20. Control of the cell cycle • Regulatory ‘checkpoints’ • ‘Start’: G1/S checkpoint • DNA replication, centrosome duplication • The G2/M checkpoint • Spindle assembly to Metaphase • Metaphase/Anaphase transition • Separation of sister chromatids, cytokinesis

21. Control of the cell cycle • Key regulatory elements • Cyclin-dependent kinases (CDKs) activate downstream events by phosphorylation. CDK levels stable during cell cycle • Cyclins activate CDKs by binding to them. Cyclin+Cdk = Holoenzymes • Cyclin levels vary during the cell cycle

22. CDK Inhibitors Two families of CDK inhibitors INK4 p15p16p18p19 Cip/Kip p21p27p57 p21 under transcriptional control ofp53 p15andp57upregulated by inhibitory growth factorTGF-beta

23. pRb Predicted by Knudson 1971 pRb mutated or inactivated in many cancers Activated by dephosphorylation: silences genes required for G1/S transition

24. The G1 / S checkpoint Kinases e.g.AT, rad3recognise DNA damage & phosphorylate p53 - stabilised p53 causes p21 p21 inhibits CDKs - cell cycle arrest Severedamage leads to p53-mediated apoptosis

25. HPV and carcinogenesis Viral proteins E6 and E7 E7 interferes with Rb E6 with p53

26. The G2/M checkpoint DNA damage also causes G2/M arrest via inhibitory phosphorylation and extranuclear sequestration of CDK1/Cyclin B.

27. Mitosis • ‘S phase’ produces dyads of identical sister chromatids linked by cohesins • Chromosomes condense • Sister chromatids are separated • Distributed equally between daughters

28. Mitosis Each daughter cell must receive One pair of centrioles Enough cytoplasm / organelles One complete set of genes (2 genomes) Paulson / Laemmli. About 3% of one human chromosome

29. Prophase Centrosomes polarise Mitotic spindle forms from tubulin monomers Chromosomes shorten and become compact

30. Prometaphase Nuclear membrane vanishes Kinetochore forms at the centromere of each chromatid Kinetochores attach to spindle fibres so chromatids of each dyad are attached to opposite poles of the mitotic spindle

31. Anaphase -Telophase Cohesindegraded - sister kinetochores separate and move to their respective poles - Chromatids dragged with them Nuclear envelope reforms Chromosomes return to elongated conformation

32. Programmed Cell Death&Apoptosis

33. The fate of cells All cells die in the end - either they die when the organism dies or they are killed by lethal injury- Necrosis or they kill themselves - PCD or Apoptosis

34. Necrosis v Apoptosis • Lethally injured cells swell, leak or burst, causing inflammation. Energy, protein synthesis, gene transcription not required • Apoptotic cells shrink and fragment. The fragments are ingested by adjacent cells without inflammation. Energy, protein synthesis, gene transcription required

35. Necrosis - Anterior MI

36. Necrosis - MI histology Normal 4 days post MI

37. ‘Apoptosis’

38. Apoptosis morphology 1: Normal. 2: Condensation of chromatin on nuclear envelope, convoluted nuclear and cell outline, condensation the cytoplasm. 3: Nucleus fragments. Surface blebbing, separation into apoptotic bodies 4: phagocytosed by nearby cells 5: degraded by lysosomal enzymes

39. Apoptosis biochemistry Internucleosomal cleavage of DNA Caspase*-mediated proteolysis Phosphatidylserine exposure on outer plasma membrane - M *Cysteine-dependent aspartate-specific proteases

40. Why is apoptosis necessary? • Development and morphogenesis • eg tadpole tail resorption, digit formation • Brain development - surplus cells eliminated • Mullerian / Wolffian ducts • Tissue homeostasis: menstrual shedding, normal breast • Eliminating undesirable cells • Virus infected cells (cytotoxic T cells) • Unwanted clones of immune cells • Cells with damaged DNA

41. Apoptosis significanceNormal development

42. Apoptosis significanceNormal development

43. Apoptosis really is ‘programmed’ During C. elegans development, 1,090 cells are formed… of which 131 are deleted by apoptosis. Mutants affecting apoptosis in at least 14 C. elegans genes 18,000 genes

44. Apoptosis signalling - external • Fas and TNF-R are membrane proteins with extracellular receptor domains • Signalling via ligands (FasL, TNF) activates a proteolytic enzyme cascade via Caspase-8 • Cytotoxic T cells use FasL to deliver ‘coup de grace’ to cells expressing appropriate target antigen

45. Apoptosis signalling - internal • Internal ‘cell death’ signals release Apaf-1 and cytochrome C from mitochondria • Apaf-1 and cytochrome C complex with Caspase-9 • Caspase activation degrades proteins and DNA • The remnants of cell are phagocytosed

46. Apoptosis - a normal event Bcl-2

47. Apoptosis and neoplasia • In many tumours, apoptotic machanisms are subverted • Bcl2 upregulation in follicular lymphoma • Apaf-1 inhibition in melanoma • Soluble fas ligand - lung and colon cancer - blocking cytotoxic T cells • Many tumours have p53 mutations

48. Apoptosis and neoplasia • Follicular lymphoma

49. the end

50. A morphogenetic gradientin DrosophilaBicoid - blueHunchback - greenDNA - redShallow bicoid gradient determines sharp cutoff in Hunchback expression Thomas Gregor, David W. Tank, Eric F. Wieschaus, and William Bialek, Probing the Limits to Positional Information, Cell 130, 153-164 (2007).