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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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)

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Animal cell structure

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Mitocondria

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Retículo Endoplasmático

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Ribossomas

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Núcleo

Territórios cromossómicos bem Territórios cromossómicos bem definidosdefinidos

Cromatina altamente Cromatina altamente organizadaorganizada

Nucléolo com domínios Nucléolo com domínios definidosdefinidos

2001/2002 Prof. Doutor José Cabeda Biologia Celular

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

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Microtubulos

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Parede Celular

2001/2002 Prof. Doutor José Cabeda Biologia Celular

Vírus