Técnicas de Imunologia Prof.Doutor José Cabeda Estudo do complemento.
Biologia Celular 2001/2002Prof.Doutor José Cabeda Aula Teórica Nº 2 Organelos Celulares.
Transcript of Biologia Celular 2001/2002Prof.Doutor José Cabeda Aula Teórica Nº 2 Organelos Celulares.
2001/2002 Prof.Doutor José Cabeda Biologia Celular
Aula Teórica Nº 2
Organelos CelularesOrganelos Celulares
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Microscopia Ampliação, Contraste, ResoluçãoAmpliação, Contraste, Resolução Microscopia óptica (200 nm)Microscopia óptica (200 nm)
Campo claroCampo claro FluorescenteFluorescente Avançada Avançada (M.Confocal, contraste fase, etc)(M.Confocal, contraste fase, etc)
Microscopia electrónica (1 nm)Microscopia electrónica (1 nm) Transmissão (TEM)Transmissão (TEM) Scanning (SEM)Scanning (SEM)
2001/2002 Prof. Doutor José Cabeda Biologia Celular
O Microscópio óptico
2001/2002 Prof. Doutor José Cabeda Biologia Celular
O microscópio de campo claro
Problem: Most cells are colorless & transparentProblem: Most cells are colorless & transparent To visualize structures To visualize structures stain with dyesstain with dyes Must preserve (fix), embed, sectionMust preserve (fix), embed, section
New problem New problem these actions these actions Alter cell structure/molecules Alter cell structure/molecules Only give snapshot of dead cellsOnly give snapshot of dead cells
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Specimen preparation for brightfield microscopy
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Fluorescent microscopy
Permits localization of specific cellular moleculesPermits localization of specific cellular molecules Fluorescent dyes “glow” against dark backgroundFluorescent dyes “glow” against dark background Dye may be indirectly or directly associated with Dye may be indirectly or directly associated with
the cellular moleculethe cellular molecule Multiple fluorescent dyes may be used simultaneouslyMultiple fluorescent dyes may be used simultaneously Cells may be fixed or livingCells may be fixed or living
2001/2002 Prof. Doutor José Cabeda Biologia CelularFigure 5-5
Figure 5-6
O Microscópio de Fluorescência
2001/2002 Prof. Doutor José Cabeda Biologia Celular
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 Biologia Celular
Advanced light microscopy Permits observation of transparent living cellsPermits observation of transparent living cells Light phase shifts induced by specimen are used to Light phase shifts induced by specimen are used to
generate contrastgenerate 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)
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Transmission electron microscopy (TEM)
Operates in vacuumOperates in vacuum Specimen usually fixed, embedded, sectioned, and Specimen usually fixed, embedded, sectioned, and
stained stained with an electron-dense materialwith an electron-dense material
Special techniques:Special techniques: Metal shadowing: visualize surface structures, cell Metal shadowing: visualize surface structures, cell
componentscomponents Cryoelectron: visualize unfixed, unstained samplesCryoelectron: visualize unfixed, unstained samples Freeze fracture, freeze etch: visualize membrane Freeze fracture, freeze etch: visualize membrane
interiorinterior Freeze etch: visualize cell interiorFreeze etch: visualize cell interior
2001/2002 Prof. Doutor José Cabeda Biologia Celular
The transmission electron microscope
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Imunomarcação em TEM
2001/2002 Prof. Doutor José Cabeda Biologia Celular
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 Specimen is fixed and coated with thin layer of heavy
metal metal Images secondary electrons, resolution = 10 nmImages secondary electrons, resolution = 10 nm
2001/2002 Prof. Doutor José Cabeda Biologia Celular
SEM
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Criofractura
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Purification of specific cells by flow cytometry
Requires fluorescent tag for desired cell
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Purification of cell parts
Understanding the roles of each each cell component Understanding the roles of each each cell component depends on methods to break open (lyse) cells and depends on methods to break open (lyse) cells and separate cell components for analysisseparate cell components for analysis
Cell lysis is accomplished by various techniques: Cell lysis is accomplished by various techniques:
blender, sonication, tissue homogenizer, hypotonic blender, sonication, tissue homogenizer, hypotonic solutionsolution
Separation of cell components generally involves Separation of cell components generally involves centrifugationcentrifugation
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Cell fractionation by differential centrifugation
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Organelle separation by equilibrium density-gradient centrifugation
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Biomembranas
Fundamental structure and function of all cell Fundamental structure and function of all cell membranes depends on lipids (phospholipids, steroid membranes depends on lipids (phospholipids, steroid derivatives) derivatives)
Specific function of each membrane depends on the Specific function of each membrane depends on the membrane proteins that are present in that specific membrane proteins that are present in that specific membrane membrane
Membrane lipids and proteins may be glycosylatedMembrane lipids and proteins may be glycosylated
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Biomembranas
Bicamada de fosfolípidosBicamada de fosfolípidos FluidezFluidez
ColesterolColesterol Aumenta a resistênciaAumenta a resistência Diminui a fluidezDiminui a fluidez Flip-flopFlip-flop
AssimetriaAssimetria GlicolípidosGlicolípidos ProteínasProteínas
IntegraisIntegrais periféricasperiféricas Ancoradas Ancoradas
covalentemente em covalentemente em lípidoslípidos
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Phospholipid structure
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Due to the amphipathic nature of phospholipids, these molecules spontaneously
assemble to form closed bilayers
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Each closed compartment has two faces
Figure 5-31
The two faces of a membrane are asymmetric in terms of lipid and protein composition
2001/2002 Prof. Doutor José Cabeda Biologia Celular
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 Biologia Celular
Fluorescence recovery after photobleaching (FRAP)
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Concentração de Proteínas em domínios de membrana
2001/2002 Prof. Doutor José Cabeda Biologia Celular
The freeze fracture, freeze etch method
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Functions of the plasma membrane
Regulate transport of nutrients into the cellRegulate transport of nutrients into the cell Regulate transport of waste out of the cellRegulate transport of waste out of the cell Maintain “proper” chemical conditions in the cellMaintain “proper” chemical conditions in the cell Provide a site for chemical reactions not likely to occur Provide a site for chemical reactions not likely to occur
in an aqueous environmentin 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)(in multicellular organisms)
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Complexidade celular
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Animal cell structure
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Plant cell structure
Figure 5-43
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Organelles of the eukaryotic cell
LysosomesLysosomes PeroxisomesPeroxisomes MitochondriaMitochondria ChloroplastsChloroplasts the Endoplasmic Reticulum (ER) the Endoplasmic Reticulum (ER) the Golgi complexthe Golgi complex the Nucleusthe Nucleus the Cytosolthe Cytosol
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Lysosomes Responsible for degrading Responsible for degrading
certain cell certain cell components components
material internalized material internalized from the extracellular from the extracellular environmentenvironment
Key FeaturesKey Features single membranesingle membrane pH of lumen pH of lumen 5 5 acid hydrolases carry acid hydrolases carry
out degradation out degradation reactionsreactions
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Peroxisomes
Responsible for degrading Responsible for degrading fatty acids fatty acids toxic compoundstoxic compounds
Key FeaturesKey Features single membrane single membrane contain oxidases and catalasecontain oxidases and catalase
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Peroxisoma
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Mitochondria
Site of ATP production via Site of ATP production via aerobic metabolismaerobic metabolism
Key FeaturesKey Features outer membraneouter membrane intermembrane spaceintermembrane space inner membraneinner membrane matrix matrix
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Mitocondria
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Cloroplasto
Site of photosynthesis in plants Site of photosynthesis in plants and green algaeand green algae
Key FeaturesKey Features outer membraneouter membrane intermembrane spaceintermembrane space inner membraneinner membrane stromastroma thylakoid membranethylakoid membrane thylakoid lumenthylakoid lumen
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Cloroplasto
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O Retículo endoplasmático (ER)
Responsible forResponsible for most lipid synthesis most lipid synthesis most membrane most membrane
protein synthesisprotein synthesis CaCa++++ ion storage ion storage detoxificationdetoxification
Key FeaturesKey Features network of network of
interconnected closed interconnected closed membrane tubules and membrane tubules and vesicles vesicles
composed of smooth composed of smooth and rough regionsand rough regions
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Retículo Endoplasmático
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Ribossomas
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O complexo de Golgi
Modifies and sorts most ER Modifies and sorts most ER products products
Key FeaturesKey Features series of flattened series of flattened
compartments & vesiclescompartments & vesicles composed of 3 regions: composed of 3 regions:
cis (entry), medial, trans cis (entry), medial, trans (exit)(exit)
each region contains each region contains different set of modifying different set of modifying enzymesenzymes
Figure 5-49
2001/2002 Prof. Doutor José Cabeda Biologia Celular
O complexo de Golgi
2001/2002 Prof. Doutor José Cabeda Biologia Celular
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 Biologia Celular
O núcleo
Separa Separa DNA do citosol DNA do citosol Transcrição da traduçãoTranscrição da tradução
Características essenciaisCaracterísticas essenciais Dupla membranaDupla membrana Lâmina nuclearLâmina nuclear Poros nuclearesPoros nucleares NucléoloNucléolo cromatinacromatina
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Núcleo
Territórios cromossómicos bem Territórios cromossómicos bem definidosdefinidos
Cromatina altamente Cromatina altamente organizadaorganizada
Nucléolo com domínios Nucléolo com domínios definidosdefinidos
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Poro Nuclear
Estrutura supramolecularEstrutura supramolecular 2 aneis coaxiais2 aneis coaxiais
Ligados em estrutura Ligados em estrutura octogonaloctogonal
Grânulo centralGrânulo central Filamentos ligam ao citoplasmaFilamentos ligam ao citoplasma 1 anel intranuclear1 anel intranuclear
MenorMenor Ligado aos 2 maioresLigado aos 2 maiores Forma um “cesto”Forma um “cesto”
2001/2002 Prof. Doutor José Cabeda Biologia Celular
The cytosol
The portion of the cell enclosed by the The portion of the cell enclosed by the plasma membrane but not part of any plasma membrane but not part of any organelle organelle
Key FeaturesKey Features the cytoskeleton the cytoskeleton polyribosomespolyribosomes metabolic enzymesmetabolic enzymes
2001/2002 Prof. Doutor José Cabeda Biologia Celular
citoesqueleto
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Microtubulos
2001/2002 Prof. Doutor José Cabeda Biologia Celular
Parede Celular
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Vírus