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

Prof. Doutor José Cabeda 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) 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda O Microscópio óptico 2001/2002 Prof. Doutor José Cabeda

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 2001/2002 Prof. Doutor José Cabeda

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

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 2001/2002 Prof. Doutor José Cabeda

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

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) 2001/2002 Prof. Doutor José Cabeda

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 2001/2002 Prof. Doutor José Cabeda

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

Prof. Doutor José Cabeda Imunomarcação em TEM 2001/2002 Prof. Doutor José Cabeda

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 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda SEM 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda Criofractura 2001/2002 Prof. Doutor José Cabeda

Purification of specific cells by flow cytometry Requires fluorescent tag for desired cell 2001/2002 Prof. 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 Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution Separation of cell components generally involves centrifugation 2001/2002 Prof. Doutor José Cabeda

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

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

Prof. Doutor José Cabeda 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 2001/2002 Prof. Doutor José Cabeda

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

Phospholipid structure 2001/2002 Prof. Doutor José Cabeda

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

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

Prof. 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” 2001/2002 Prof. Doutor José Cabeda

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

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

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

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) 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda Complexidade celular 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda Animal cell structure 2001/2002 Prof. Doutor José Cabeda

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

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

Prof. Doutor José Cabeda 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 2001/2002 Prof. Doutor José Cabeda

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

Prof. Doutor José Cabeda Peroxisoma 2001/2002 Prof. Doutor José Cabeda

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

Prof. Doutor José Cabeda Mitocondria 2001/2002 Prof. Doutor José Cabeda

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

Prof. Doutor José Cabeda Cloroplasto 2001/2002 Prof. Doutor José Cabeda

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 2001/2002 Prof. Doutor José Cabeda

Retículo Endoplasmático 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda Ribossomas 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda 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 2001/2002 Prof. Doutor José Cabeda Figure 5-49

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

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

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

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

Prof. 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” 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda 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 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda citoesqueleto 2001/2002 Prof. Doutor José Cabeda

Prof. Doutor José Cabeda Microtubulos 2001/2002 Prof. Doutor José Cabeda

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

Prof. Doutor José Cabeda Vírus 2001/2002 Prof. Doutor José Cabeda