Aula Teórica Nº 2

1. Aula Teórica Nº 2 Organelos Celulares Prof.Doutor José Cabeda

2. Microscopia • Ampliação, Contraste, Resolução • Microscopia óptica (200 nm) • Campo claro • Fluorescente • Avançada (M.Confocal, contraste fase, etc) • Microscopia electrónica (1 nm) • Transmissão (TEM) • Scanning (SEM) Prof. Doutor José Cabeda

3. O Microscópio óptico Prof. Doutor José Cabeda

4. O microscópio de campo claro • Problem: Most cells are colorless & transparent • To visualize structures stain with dyes • Must preserve (fix), embed, section • New problem  these actions • Alter cell structure/molecules • Only give snapshot of dead cells Prof. Doutor José Cabeda

5. Specimen preparation for brightfield microscopy Prof. Doutor José Cabeda

6. Fluorescent microscopy • Permits localization of specific cellular molecules • Fluorescent dyes “glow” against dark background • Dye may be indirectly or directly associated with the cellular molecule • Multiple fluorescent dyes may be used simultaneously • Cells may be fixed or living Prof. Doutor José Cabeda

7. O Microscópio de Fluorescência Figure 5-6 Figure 5-5 Prof. Doutor José Cabeda

8. Microscopia óptica de objectos 3D • Confocal Scanning or Deconvolution Microscopy • Generates 3D images of living cells • Removes out-of-focus images  optical sectioning • Can look inside thick specimens (eggs, embryos, tissues) Prof. Doutor José Cabeda

9. Advanced light microscopy • Permits observation of transparent living cells • Light phase shifts induced by specimen are used to generate contrast • Phase contrast (refracted and unrefracted light) • Differential interference contrast (two light beams) Prof. Doutor José Cabeda

10. Transmission electron microscopy (TEM) • Operates in vacuum • Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material Special techniques: • Metal shadowing: visualize surface structures, cell components • Cryoelectron: visualize unfixed, unstained samples • Freeze fracture, freeze etch: visualize membrane interior • Freeze etch: visualize cell interior Prof. Doutor José Cabeda

11. The transmission electron microscope Prof. Doutor José Cabeda

12. Imunomarcação em TEM Prof. Doutor José Cabeda

13. Scanning electron microscopy • Can visualize surfaces of tissues, cells, isolated cell parts • Specimen is fixed and coated with thin layer of heavy metal • Images secondary electrons, resolution = 10 nm Prof. Doutor José Cabeda

14. SEM Prof. Doutor José Cabeda

15. Criofractura Prof. Doutor José Cabeda

16. Purification of specific cells by flow cytometry Requires fluorescent tag for desired cell Prof. Doutor José Cabeda

17. Purification of cell parts • Understanding the roles of each each cell component depends on methods to break open (lyse) cells and separate cell components for analysis • Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution • Separation of cell components generally involves centrifugation Prof. Doutor José Cabeda

18. Cell fractionation by differential centrifugation Prof. Doutor José Cabeda

19. Organelle separation by equilibrium density-gradient centrifugation Prof. Doutor José Cabeda

20. Biomembranas • Fundamental structure and function of all cell membranes depends on lipids (phospholipids, steroid derivatives) • Specific function of each membrane depends on the membrane proteins that are present in that specific membrane • Membrane lipids and proteins may be glycosylated Prof. Doutor José Cabeda

21. Biomembranas • Bicamada de fosfolípidos • Fluidez • Colesterol • Aumenta a resistência • Diminui a fluidez • Flip-flop • Assimetria • Glicolípidos • Proteínas • Integrais • periféricas • Ancoradas covalentemente em lípidos Prof. Doutor José Cabeda

22. Phospholipid structure Prof. Doutor José Cabeda

23. Due to the amphipathic nature of phospholipids, these molecules spontaneously assemble to form closed bilayers Prof. Doutor José Cabeda

24. Each closed compartment has two faces The two faces of a membrane are asymmetric in terms of lipid and protein composition Figure 5-31 Prof. Doutor José Cabeda

25. Lipids and integral proteins demonstrate lateral mobility in biomembranes “The Fluid Mosaic Model” • Mobility (diffusion) of a given membrane • components depends on: • the size of the molecule • its interactions with other molecules • temperature • lipid composition (tails, cholesterol) • Mobility can be measured by “FRAP” Prof. Doutor José Cabeda

26. Fluorescence recovery after photobleaching (FRAP) Prof. Doutor José Cabeda

27. Concentração de Proteínas em domínios de membrana Prof. Doutor José Cabeda

28. The freeze fracture, freeze etch method Prof. Doutor José Cabeda

29. Functions of the plasma membrane • Regulate transport of nutrients into the cell • Regulate transport of waste out of the cell • Maintain “proper” chemical conditions in the cell • Provide a site for chemical reactions not likely to occur in an aqueous environment • Detect signals in the extracellular environment • Interact with other cells or the extracellular matrix (in multicellular organisms) Prof. Doutor José Cabeda

30. Complexidade celular Prof. Doutor José Cabeda

31. Animal cell structure Prof. Doutor José Cabeda

32. Plant cell structure Figure 5-43 Prof. Doutor José Cabeda

33. Organelles of the eukaryotic cell • Lysosomes • Peroxisomes • Mitochondria • Chloroplasts • the Endoplasmic Reticulum (ER) • the Golgi complex • the Nucleus • the Cytosol Prof. Doutor José Cabeda

34. Lysosomes • Responsible for degrading • certain cell components • material internalized from the extracellular environment • Key Features • single membrane • pH of lumen  5 • acid hydrolases carry out degradation reactions Prof. Doutor José Cabeda

35. Peroxisomes • Responsible for degrading • fatty acids • toxic compounds • Key Features • single membrane • contain oxidases and catalase Prof. Doutor José Cabeda

36. Peroxisoma Prof. Doutor José Cabeda

37. Mitochondria • Site of ATP production via aerobic metabolism • Key Features • outer membrane • intermembrane space • inner membrane • matrix Prof. Doutor José Cabeda

38. Mitocondria Prof. Doutor José Cabeda

39. Cloroplasto • Site of photosynthesis in plants and green algae • Key Features • outer membrane • intermembrane space • inner membrane • stroma • thylakoid membrane • thylakoid lumen Prof. Doutor José Cabeda

40. Cloroplasto Prof. Doutor José Cabeda

41. O Retículo endoplasmático (ER) • Responsible for • most lipid synthesis • most membrane protein synthesis • Ca++ ion storage • detoxification • Key Features • network of interconnected closed membrane tubules and vesicles • composed of smooth and rough regions Prof. Doutor José Cabeda

42. Retículo Endoplasmático Prof. Doutor José Cabeda

43. Ribossomas Prof. Doutor José Cabeda

44. O complexo de Golgi • Modifies and sorts most ER products • Key Features • series of flattened compartments & vesicles • composed of 3 regions: cis (entry), medial, trans (exit) • each region contains different set of modifying enzymes Prof. Doutor José Cabeda Figure 5-49

45. O complexo de Golgi Prof. Doutor José Cabeda

46. Secretory proteins are synthesized in the ER and pass through the Golgi on the way to the extracellular environment Prof. Doutor José Cabeda

47. O núcleo • Separa • DNA do citosol • Transcrição da tradução • Características essenciais • Dupla membrana • Lâmina nuclear • Poros nucleares • Nucléolo • cromatina Prof. Doutor José Cabeda

48. Núcleo • Territórios cromossómicos bem definidos • Cromatina altamente organizada • Nucléolo com domínios definidos Prof. Doutor José Cabeda

49. Poro Nuclear • Estrutura supramolecular • 2 aneis coaxiais • Ligados em estrutura octogonal • Grânulo central • Filamentos ligam ao citoplasma • 1 anel intranuclear • Menor • Ligado aos 2 maiores • Forma um “cesto” Prof. Doutor José Cabeda

50. The cytosol • The portion of the cell enclosed by the plasma membrane but not part of any organelle • Key Features • the cytoskeleton • polyribosomes • metabolic enzymes Prof. Doutor José Cabeda