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Biologia Celular 2001/2002Prof.Doutor José Cabeda Aula Teórica Nº 2 Organelos Celulares.

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Apresentação em tema: "Biologia Celular 2001/2002Prof.Doutor José Cabeda Aula Teórica Nº 2 Organelos Celulares."— Transcrição da apresentação:

1 Biologia Celular 2001/2002Prof.Doutor José Cabeda Aula Teórica Nº 2 Organelos Celulares

2 Biologia Celular 2001/2002Prof. Doutor José Cabeda Microscopia Ampliação, Contraste, Resolução Ampliação, Contraste, Resolução Microscopia óptica (200 nm) Microscopia óptica (200 nm) Campo claro Campo claro Fluorescente Fluorescente Avançada (M.Confocal, contraste fase, etc) Avançada (M.Confocal, contraste fase, etc) Microscopia electrónica (1 nm) Microscopia electrónica (1 nm) Transmissão (TEM) Transmissão (TEM) Scanning (SEM) Scanning (SEM)

3 Biologia Celular 2001/2002Prof. Doutor José Cabeda O Microscópio óptico

4 Biologia Celular 2001/2002Prof. Doutor José Cabeda O microscópio de campo claro Problem: Most cells are colorless & transparent Problem: Most cells are colorless & transparent To visualize structures stain with dyes To visualize structures stain with dyes Must preserve (fix), embed, section Must preserve (fix), embed, section New problem these actions New problem these actions Alter cell structure/molecules Alter cell structure/molecules Only give snapshot of dead cells Only give snapshot of dead cells

5 Biologia Celular 2001/2002Prof. Doutor José Cabeda Specimen preparation for brightfield microscopy

6 Biologia Celular 2001/2002Prof. Doutor José Cabeda Fluorescent microscopy Permits localization of specific cellular molecules Permits localization of specific cellular molecules Fluorescent dyes glow against dark background Fluorescent dyes glow against dark background Dye may be indirectly or directly associated with Dye may be indirectly or directly associated with the cellular molecule Multiple fluorescent dyes may be used simultaneously Multiple fluorescent dyes may be used simultaneously Cells may be fixed or living Cells may be fixed or living

7 Biologia Celular 2001/2002Prof. Doutor José Cabeda Figure 5-5 Figure 5-6 O Microscópio de Fluorescência

8 Biologia Celular 2001/2002Prof. Doutor José Cabeda 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)

9 Biologia Celular 2001/2002Prof. Doutor José Cabeda Advanced light microscopy Permits observation of transparent living cells Permits observation of transparent living cells Light phase shifts induced by specimen are used to Light phase shifts induced by specimen are used to generate contrast Phase contrast (refracted and unrefracted light) Phase contrast (refracted and unrefracted light) Differential interference contrast (two light beams) Differential interference contrast (two light beams)

10 Biologia Celular 2001/2002Prof. Doutor José Cabeda Transmission electron microscopy (TEM) Operates in vacuum Operates in vacuum Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material Specimen usually fixed, embedded, sectioned, and stained with an electron-dense material Special techniques: Metal shadowing: visualize surface structures, cell components Metal shadowing: visualize surface structures, cell components Cryoelectron: visualize unfixed, unstained samples Cryoelectron: visualize unfixed, unstained samples Freeze fracture, freeze etch: visualize membrane interior Freeze fracture, freeze etch: visualize membrane interior Freeze etch: visualize cell interior Freeze etch: visualize cell interior

11 Biologia Celular 2001/2002Prof. Doutor José Cabeda The transmission electron microscope

12 Biologia Celular 2001/2002Prof. Doutor José Cabeda Imunomarcação em TEM

13 Biologia Celular 2001/2002Prof. Doutor José Cabeda Scanning electron microscopy Can visualize surfaces of tissues, cells, isolated cell parts Can visualize surfaces of tissues, cells, isolated cell parts Specimen is fixed and coated with thin layer of heavy metal Specimen is fixed and coated with thin layer of heavy metal Images secondary electrons, resolution = 10 nm Images secondary electrons, resolution = 10 nm

14 Biologia Celular 2001/2002Prof. Doutor José Cabeda SEM

15 Biologia Celular 2001/2002Prof. Doutor José Cabeda Criofractura

16 Biologia Celular 2001/2002Prof. Doutor José Cabeda Purification of specific cells by flow cytometry Requires fluorescent tag for desired cell

17 Biologia Celular 2001/2002Prof. Doutor José Cabeda 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 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: Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution Separation of cell components generally involves centrifugation Separation of cell components generally involves centrifugation

18 Biologia Celular 2001/2002Prof. Doutor José Cabeda Cell fractionation by differential centrifugation

19 Biologia Celular 2001/2002Prof. Doutor José Cabeda Organelle separation by equilibrium density-gradient centrifugation

20 Biologia Celular 2001/2002Prof. Doutor José Cabeda Biomembranas Fundamental structure and function of all cell membranes depends on lipids (phospholipids, steroid derivatives) 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 Specific function of each membrane depends on the membrane proteins that are present in that specific membrane Membrane lipids and proteins may be glycosylated Membrane lipids and proteins may be glycosylated

21 Biologia Celular 2001/2002Prof. Doutor José Cabeda Biomembranas Bicamada de fosfolípidos Bicamada de fosfolípidos Fluidez Fluidez Colesterol Colesterol Aumenta a resistência Aumenta a resistência Diminui a fluidez Diminui a fluidez Flip-flop Flip-flop Assimetria Assimetria Glicolípidos Glicolípidos Proteínas Proteínas Integrais Integrais periféricas periféricas Ancoradas covalentemente em lípidos Ancoradas covalentemente em lípidos

22 Biologia Celular 2001/2002Prof. Doutor José Cabeda Phospholipid structure

23 Biologia Celular 2001/2002Prof. Doutor José Cabeda Due to the amphipathic nature of phospholipids, these molecules spontaneously assemble to form closed bilayers

24 Biologia Celular 2001/2002Prof. Doutor José Cabeda Each closed compartment has two faces Figure 5-31 The two faces of a membrane are asymmetric in terms of lipid and protein composition

25 Biologia Celular 2001/2002Prof. Doutor José Cabeda 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

26 Biologia Celular 2001/2002Prof. Doutor José Cabeda Fluorescence recovery after photobleaching (FRAP)

27 Biologia Celular 2001/2002Prof. Doutor José Cabeda Concentração de Proteínas em domínios de membrana

28 Biologia Celular 2001/2002Prof. Doutor José Cabeda The freeze fracture, freeze etch method

29 Biologia Celular 2001/2002Prof. Doutor José Cabeda Functions of the plasma membrane Regulate transport of nutrients into the cell Regulate transport of nutrients into the cell Regulate transport of waste out of the cell Regulate transport of waste out of the cell Maintain proper chemical conditions in the cell Maintain proper chemical conditions in the cell Provide a site for chemical reactions not likely to occur in an aqueous environment Provide a site for chemical reactions not likely to occur in an aqueous environment Detect signals in the extracellular environment Detect signals in the extracellular environment Interact with other cells or the extracellular matrix Interact with other cells or the extracellular matrix (in multicellular organisms)

30 Biologia Celular 2001/2002Prof. Doutor José Cabeda Complexidade celular

31 Biologia Celular 2001/2002Prof. Doutor José Cabeda Animal cell structure

32 Biologia Celular 2001/2002Prof. Doutor José Cabeda Plant cell structure Figure 5-43

33 Biologia Celular 2001/2002Prof. Doutor José Cabeda Organelles of the eukaryotic cell Lysosomes Lysosomes Peroxisomes Peroxisomes Mitochondria Mitochondria Chloroplasts Chloroplasts the Endoplasmic Reticulum (ER) the Endoplasmic Reticulum (ER) the Golgi complex the Golgi complex the Nucleus the Nucleus the Cytosol the Cytosol

34 Biologia Celular 2001/2002Prof. Doutor José Cabeda Lysosomes Responsible for degrading Responsible for degrading certain cell components certain cell components material internalized from the extracellular environment material internalized from the extracellular environment Key Features Key Features single membrane single membrane pH of lumen 5 pH of lumen 5 acid hydrolases carry out degradation reactions acid hydrolases carry out degradation reactions

35 Biologia Celular 2001/2002Prof. Doutor José Cabeda Peroxisomes Responsible for degrading Responsible for degrading fatty acids fatty acids toxic compounds toxic compounds Key Features Key Features single membrane single membrane contain oxidases and catalase contain oxidases and catalase

36 Biologia Celular 2001/2002Prof. Doutor José Cabeda Peroxisoma

37 Biologia Celular 2001/2002Prof. Doutor José Cabeda Mitochondria Site of ATP production via aerobic metabolism Site of ATP production via aerobic metabolism Key Features Key Features outer membrane outer membrane intermembrane space intermembrane space inner membrane inner membrane matrix matrix

38 Biologia Celular 2001/2002Prof. Doutor José Cabeda Mitocondria

39 Biologia Celular 2001/2002Prof. Doutor José Cabeda Cloroplasto Site of photosynthesis in plants and green algae Site of photosynthesis in plants and green algae Key Features Key Features outer membrane outer membrane intermembrane space intermembrane space inner membrane inner membrane stroma stroma thylakoid membrane thylakoid membrane thylakoid lumen thylakoid lumen

40 Biologia Celular 2001/2002Prof. Doutor José Cabeda Cloroplasto

41 Biologia Celular 2001/2002Prof. Doutor José Cabeda O Retículo endoplasmático (ER) Responsible for Responsible for most lipid synthesis most lipid synthesis most membrane protein synthesis most membrane protein synthesis Ca ++ ion storage Ca ++ ion storage detoxification detoxification Key Features Key Features network of interconnected closed membrane tubules and vesicles network of interconnected closed membrane tubules and vesicles composed of smooth and rough regions composed of smooth and rough regions

42 Biologia Celular 2001/2002Prof. Doutor José Cabeda Retículo Endoplasmático

43 Biologia Celular 2001/2002Prof. Doutor José Cabeda Ribossomas

44 Biologia Celular 2001/2002Prof. Doutor José Cabeda O complexo de Golgi Modifies and sorts most ER products Modifies and sorts most ER products Key Features Key Features series of flattened compartments & vesicles series of flattened compartments & vesicles composed of 3 regions: composed of 3 regions: cis (entry), medial, trans (exit) each region contains different set of modifying enzymes each region contains different set of modifying enzymes Figure 5-49

45 Biologia Celular 2001/2002Prof. Doutor José Cabeda O complexo de Golgi

46 Biologia Celular 2001/2002Prof. Doutor José Cabeda Secretory proteins are synthesized in the ER and pass through the Golgi on the way to the extracellular environment

47 Biologia Celular 2001/2002Prof. Doutor José Cabeda O núcleo Separa Separa DNA do citosol DNA do citosol Transcrição da tradução Transcrição da tradução Características essenciais Características essenciais Dupla membrana Dupla membrana Lâmina nuclear Lâmina nuclear Poros nucleares Poros nucleares Nucléolo Nucléolo cromatina cromatina

48 Biologia Celular 2001/2002Prof. Doutor José Cabeda Núcleo Territórios cromossómicos bem definidos Cromatina altamente organizada Nucléolo com domínios definidos

49 Biologia Celular 2001/2002Prof. Doutor José Cabeda 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

50 Biologia Celular 2001/2002Prof. Doutor José Cabeda The cytosol The portion of the cell enclosed by the plasma membrane but not part of any organelle The portion of the cell enclosed by the plasma membrane but not part of any organelle Key Features Key Features the cytoskeleton the cytoskeleton polyribosomes polyribosomes metabolic enzymes metabolic enzymes

51 Biologia Celular 2001/2002Prof. Doutor José Cabeda citoesqueleto

52 Biologia Celular 2001/2002Prof. Doutor José Cabeda Microtubulos

53 Biologia Celular 2001/2002Prof. Doutor José Cabeda Parede Celular

54 Biologia Celular 2001/2002Prof. Doutor José Cabeda Vírus


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