Cytology 2 Aleš Hampl 2019 • Mitochondria • Lyzosomes + Peroxisomes • Cytoplasmic inclusions • Cytoskeleton • Cell surface specialisations • Cell cycle, cell division, cell differentiaion Mitochondria 1 • all cells except erythrocytes • double membrane • diameter cca 0,5 mm • length up to 50 (100) mm • oxidative metabolism (glucose – ATP + CO2 + H20) • cytochrome c – activation of apoptotic pathway • origin in oocyte • mtDNA (circular) • brown fat thermogenesis • both membranes with low fluidity • both membranes equipped with many protein molecules • growth and divission of mitochondria Intermembrane space Mitochondria 2 Mitochondria 3 with crists with tubuli (in steroid producing cells) mitochondria microtubuli Mitochondria 4 mitochondrial eosinophilia Lysosomes 1 endosome-lysosome system • in all cells except for erythrocytes • vesicles about 0,05 – 0,5 mm • membrane-bound • highly acidic internal space (cca pH 5) • hydrolytic enzymes inside (min. 50 types) • tagging by mannose-6-fosphate Lysosomes 2 primary x secondary • primary lysosomes • secondary lysosomes (fagolysosomes) • residual bodies (lipofuscin) secondary lysosomes Lysosomes 3 Peroxisomes • structuraly similar to lysosoms • functionally similar to mitochondria • „nucleus “ = nucleoid • degradation of fatty acids (H202, H2O, 02) • detoxification (complement SER) • origin: growth from ER or division peroxisomes lysosomes nucleoid Cytoplasmic inclusions 1 (no or only little metabolic activity on themselves) • secretory granules • storage compounds: sugars (glycogen), lipids • crystals (proteins) • pigments: endogenous (autogenic and hematogenic) + exogenous Cytoplasmic inclusions 2 Secretory granules Secretory granules Golgi apparatus Rough ER Cytoplasmic inclusions 3 Lipid inclusions Cytoplasmic inclusions 4 Glycogen β-granules (stratified epithelium) α-granules (liver) Cytoplasmic inclusions 5 Glycogen Glycogen in liver cells (light microscope; PAS reaction) Cytoplasmic inclusions 6 Crystals Protein inclusions in Leydig cells Cytoplasmic inclusions 7 Pigments (colour inclusions): Exogenous x Endogenous • Autogenous Specific functions – melanin • Hematogenous Hemoglobin decomposition – hemosiderin, biliverdin, bilirubin Pigment in aged cells lipofuscin – accumulation of residual bodies in long-lived cells (neurones, kardiomyocytes) Cytoskeleton 1 Microtubules Intermediate filaments Microfilaments (actin) 25 nm 10 nm 7 nm microtubules microfilaments - actin Cytoskeleton 2 Microfilaments (actin) G-actin (globular) F-actin (fibrilar) • actin isoformes (a, b, g) • fast polymerisation and depolymerisation • polarisation (+ a – ends) • stabilisation by associated proteins (tropomyosin – myofibrils) • crosslinking by associated proteins (fimbrin, filamin, …) • anchoring to cell membrane (vinculin, tallin, …) • cortical actin – membrane skeleton • myosin motors (analogous to dynein + kinesin on microtubuli) Cytoskeleton 3 Microtubules • hollow tubes • a-tubulin + b-tubulin – dimers • fast polymerisation and depolymerisation • polarisation (+ a – ends) • MAP (proteins associated with microtubuli) • MTOC – microtubules organizing centre (centrosome; g-tubulin) • mechanical support • intracellular transport • mitotic spindle • cilia and flagella • mitotic poisons (colchicin, taxol, …) a-tubulin b-tubulin Cytoskeleton 4 Microtubules - motors Kinesins • move towards „plus“ end of microtubuli • transport from centrosome Dyneins • move towards „minus“ end microtubuli • transport towards centrosome • axonal transport – long distance Cytoskeleton 5 Intermediate filaments • „chemically“ highly heterogenou group • common composition (tetramers) “thread like“ • more stable than actin and tubulin structures • cell type specific: Cytokeratins (epithelia) Vimentin (cells of mesenchymal origin) Desmin (muscle cells) Neurofilaments (neurons) Glial fibrial acidic protein (neuroglia) Lamins (nuclear envelope) Cytokeratin intermediate filaments in stratum basale of epidermis Cell surfaces 1 free surface lateral surface basal surface Free • microvilli (irregular, regular – striated border, brush border) • cilia Lateral Cell-to-cell junction: • sealing: tight junction=zonula occludens • adhesion: zonula adherens, desmosom • communication: nexus (Gap junction) Basal • focal adhesions • hemidesmosomes • basal labyrinth Cell surfaces 2 Free surface of cultured human embryonic stem cells Microvilli Cell surfaces 3 Microvilli Regularly organised microvilli = striated border + brush border Actin filaments in microvilli • 20 in microvilli of epithelial cells • several hundreds in stereocilia of hair cells Thickness about 0,1 mm Length about 1-6 mm Cell surfaces 4 Microvilli striated border (tops of enterocytes) brush border (proximal tibuli of kidney) Gluten – Celiac disease Cell surfaces 5 Cilia + Flagella Thickness about 0,25 mm Length about 7-10 mm Axonema 20 microtubuli (9x2 + 2) Dynein arms (movement) Basal body Centriole 9 x 3 Cell surfaces 6 Cilia + Flagella in light microscope in electron microscope Cell surfaces 7 Cilia + Flagella oviduct Microvilli of secretory cells Cilia of ciliated cells Cell surfaces 8 Cilia + Flagella Adhesions and Junctions 1 lateral surface Basal surface Adhesion Sealing Communication • Macula adherens (desmosome) • Zonula adherens • Hemidesmosome • Focal adhesion • Zonula occludens (tight junction) • Gap junction (nexus) junction jomplex Adhesions and Junctions 2 Adhesion • Macula adherens (desmosom) • Zonula adherens • Hemidesmosome • Focal adhesion Lung ECM - alveolar region Unified composition • Transmembrane proteins (cadherins+ integrins) • Adaptor (plak) proteins • Cytoskelelal fibers cell-cellcell-ECM Adhesions and Junctions 3 Adhesion • Macula adherens (desmosome) Diameter about 0,3 mm Distance between membranes about 20-40 nm Adhesions and Junctions 4 Adhesion • Hemidesmosome • Focal adhesion Epidermolysis bullosa Adhesions and Junctions 5 • Focal adhesion basal lamina Adhesions and Junctions 6 Basal labyrinth Adhesions and Junctions 7 Sealing • Zonula occludens (tight junction) Damage by: Clostridium perfringens Helicobacter pylori (ZO-1) Adhesions and Junctions 8 Sealing • Zonula occludens (tight junction) Adhesions and Junctions 9 Communication • Gap junction (nexus) Diameter about 0,3 mm Distance between cell membranes about 3 nm Internal diameter of the channel about 2 nm Adhesions and Junctions 10 Communication • Gap junction (nexus) Activities of cells • Movement – intracellular, amoeboid, cilia, flagella • Metabolism – income, processing, outcome • Responsivenes • Growth • Differentiation • Division (amplification) Cell division & Cell specialization Cell division & Cell specialization CELLDIFFERENTIATION CELLDIVISION Division x Differentiation of cells 1 Zygote Multicellular embryo Dynamic multicellular organism + other regulations: • translation • posttranslational modification STABLE GENOME Genomic equivalence (= equal amount of DNA and the same nucletide sequence in all cells of a organism – cloning) VARIABLE TRANSCRiPTOME Transcription Regulators X CELLDIFFERENTIATION Division x Differentiation of cells 2 Stem cells • slowly dividing (usually) • multipotent Progenitor cells • „transit amplifying cells“ • fast proliferation • multipotent Terminally diferentiated cells • nondividing Tissue renewal and regeneration Oligodendrocyte Astrocyte Neuron Division x Differentiation of cells 3 Stem cells generate and regenerate our body 1. Undifferentiated growth 2. Differentiation Capability to differentite into specialized cell types Pluripotency Capability to produce identical copies of itself Self-renewal Different properties Fetal Organ Tissue Embryonic stem cells Adult stem cells Induced pluripotent stem cells Cancer stem cells Mother nature and scientists supply us with many Stem cell can be isolated from tissues and studied in vitro 1 Collect lung Chop lung Enzymatic digestion Differential centrifugation Lung epithelial organoids Mesenchymal cells Single-celled lungepithelialcells Culture in non-adherent conditions Enzymatic digestion Primary spheres Secondary spheres Enzymatic digestion Culture in 3D Matrigel / collagen Culture in 3D Matrigel SCs after isolation Spheroid growing from SC „lungosphere“ Stem cell can be isolated from tissues and studied in vitro 2 Basic concept 1 MITOSIS and CYTOKINESIS produce genetically identical cells Cell division 1 STABLE (non-changing) GENOME Due to semiconservative duplication of DNA Kondenzovaný duplikovaný chromozom chromatida centromera telomera telomera Cell division 2 ho homologous chr. pairs duplication duplication nonsister chromatids sesterské chromatidy sesterské chromatidy chromosome with one chromatid mathers chromosome Metabolism of chromosomes – Homologous chormosomes Cell division 3 fathers chromosome chromosome with one chromatid Pairs of homologous chromosomes (2N) organized into so called „KARYOTYPE“ Cell division 4 Basic concept 2 MITOSIS and CYTOKINESIS are parts of cell cycle CELL CYCLE • semi-modular character • equipped with checkpoints • among cells it is coordinated by signalling molecules G1 START M S DNA replication checkpoint G2 metaphase Checkpoint (SAC) G0 Cell division 5 DNA damage checkpoint Interphase begins in daughter cell G1 S G2 Chromozómy kondenzují, začíná se tvořit dělící vřeténko Chromozomy se seřazují v metafázní (ekvatoriální) rovině Chromatidy se přesunují k protilehlým pólům vřeténka Formují se nová jádra a chomozomy začínají dekondenzovat Cytoplasm divides, two cells arise Cell division 6 Mitotic spindle Cell division 7 Centrosomal metabolism Semiconservative duplicationCell division 8 Centrosome structureCell division 9 Diameter – 0.2 mm Length - 0.5 mm 1 2 3 4 56 7 8 9 Modified from the catalogue ofSanta Cruz Biomedicals, USA Regulation – Cyklin-Dependent Kinases (CDK) + Cyklins Cell division 10 Periodicity of cyclin expression Cell division 11 prophase metaphase Cell division 12 anaphase - telophase telophase Cell division 13 Thank you for your attention !