Histology and Embryology Lecturers: Aleš Hampl, D.V.M., Ph.D., Assoc. Prof., Head of the Dept. Petr Vaňhara, RNDr., Ph.D., Assoc. Prof. Brno, 2023 Lecture 1 Introduction • The object and significance of histology. • Relevance of histology to other biomedical disciplines. • History, current state, and future of histology. • Methodologies to study a structure of cells and tissues. Cytology • The cell - definition, characteristics, compartmentalization. • Cell nucleus - ultrastructure and function, chromosomes, nucleolus. • Endoplasmic reticulum • Golgi apparatus • Centrosome • Mitochondria • Lyzosomes + Peroxisomes • Cytoplasmic inclusions • Cytoskeleton • Cell surface specialisations • Cell cycle, cell division, cell differentiaion Histology Microscopic and submicroscopic structure of the body (cells, extracellular matrix, fluid substances) Microscopic anatomy Composition and structure of organ systems & individual organs Which tissue types and how organized? Which special cell types? Which special structures? (e.g. tubules) How does it all work? Cytology General aspects of the structures composing the cells and their functioning General histology What are the main types of tissues? What are their functions? What cell types these tissues are made of? All this mirrors hierarchical organisation of living organisms Histology is no longer a static discipline dealing with just the structure !!! Histology Cell biology Physiology Pathology Biochemistry Molecular biology Gross anatomy Embryology Have the histology in action & in motion Learn thinking „histologically“ Studying histology was first made mandatory for medical students in 1893 by John’s Hopkins Medical School ! Most histologists are Germans primarily because they made great microscopes. Eponymously theirs….. Jan Evangelista Purkyně 1787 - 1869 Bohemian physiologist • Pioneer in histological techniques First to use something like a microtome • Introduced the term plasma • Found Purkinje fibers of the heart • Found Purkinje cells of the cerebellar cortex Schwann + Schleiden – 1839 – cell theory “Once the development was ended, the founts of growth and regeneration of the axons and dendrites dried up irrevocably. In the adult centers, the nerve paths are something fixed, ended, and immutable. Everything may die, nothing may be regenerated. It is for the science of the future to change, if possible, this harsh decree.” Santiago Ramón Y Cajal 1852 - 1934 Spanish physician and anatomist He established the neuron as the primary structural and functional unit of the nervous system. Nobel Prize in 1906 Making unexpected discoveries The existence of multipotent self-renewing progenitors residing in the postantal and adult nervous system • Subventricular zone of the lateral ventricle • Subgranular zone of the dentate gyrus of the hippocampus DEFINITELY IN: • Cortex ? • Amygdala ? POSSIBLY IN: (since early 1990s) Our view on the organization of the central nervous system has been dramatically changed !!! Methodologies to study cells and tissues 1 Making it observable Stabilization of the structure Fixation Making the objects smaller – transmissible for the light Embedding + Sectioning Making the structures well visible „Staining“ Enlargement Utility of Microscopes Light (optical) microscopes (interaction of photons with a matter) Resolution 0.1 mm • Equipped for visible light only • Equipped for fluorescence • Confocal laser scanning Electron microscopes (interaction of electrons with a matter) Resolution 0.1 nm (in practice 1 nm) • Transmission • Scanning Methodologies to study cells and tissues 2 Fixation (denaturation) • Organic solvents (EtOH, MetOH, Aceton,…) • Aldehydes (form-, paraform-, glutar-aldehyde, …) • Organic acids (acetic, picric, …) • Heavy metals (salts of mercury, chrome, osmium, …) Embedding + Sectioning • Paraffine wax • Celloidine (=cellulose nitrate) • Durcupan (synthetic polymer) • LR White (synthetic polymer) • others „Staining“ Chemical stains (H+E, Azan, van Gieson, …) Histochemical stains (for proteins/enzymes, sugars, lipids, …) Immunochemical visualization (labeled antibodies) Heavy metals (for TEM – salts of uranium, lead, wolfram, …) Understanding the complex systems can only be built on understanding its components Tissues Cells Cells Cells Fluids Extacellular matrix The cells make it all ! Fluids • Intersticial fluid • Plasma (in blood) • Lymph (in lymph vessels) • Cerebrospinal fluid • Intracellular fluid (cytosol) Living organisms are composed of cells Long way to this discovery: Robert Hooke 1665 He for the first time observed the structure of cork - cell Antonie van Leeuwenhoek 1678 He for the first time observed microscopical organisms (bacteria, protozoa) Rudolph Virchow 1855 Cell can develop only from preexisting cells „Omnis cellula e cellula“ Matthias Schleiden Theodor Schwann All organisms are composed of one or more cells 1839 Current cell theory – 6 principles on which it is built • Cell is the smallest structural and functional unit capable of life functions • Function of each cell is given by its specific structure • Cells are bulding units of all multicellular organisms – cells are responsible for all processes taking place in the organisms • Structure and function of all organisms is based on structural and functional properties of cells from which they are composed • All new cells originate from preexisting cells • Thanks to the continuity of life on the Earth, all cells are in principle the same (universal genetic code and its expression) Muscle fibre Neuron Neuromuscular junction Anaphase Telophase Cell is unifying theme/element of life (cells are very similar among each other: small + specialized functions) Despite of their common scheme, structural and functional diversity is a typical feature of all eukaryotic cell types The cells of human tissues and organs are also structurally and functionally very diverse Egg Sperm Neuron in brain Fat cell Cell of gut epithelium Blood cells Muscle cell Bone cells Such diversity is critical for an ability of cells to serve various functions in human body No cell is exactly like all others, but cells do have many common structural and functional features. Keep in mind that not all cells contain all the structures we will discuss ! All cells have 3 major parts: 1. PLASMA MEMBRANE 2. CYTOPLASM 3. NUCLEUS (eukaryotic) 1 2 3 Cellular organization is based on COMPARTMENTALIZATION Specialized functions can be carried out in different locations Membranes make up boundaries between the compartments Unique protein and lipid components and unique function Unique control of the movement of molecules Unique composition of the surrounded interior Compartments & Membranes Many small compartments are better More space for: • regulation • nutrients exchange • waste removal More membrane surface per volume surrounded Surface area is proportional to the square (r2) of its diameter. Volume is proportional to the cube (r3) of its diameter. Amplification X Reduction of selected compartments Cell differentiation Specialization of cells for different functions Rough ER in secretory cells Mitochondria in cardiac musle cells Biological membrane structure 1 ~2.5 nm ~2.5 nm 7-10 nm ~2.5 nm Electron dense Electron opaque Electron dense Unit membrane common to all membranes Cell membranes seen in electron microscope (pseudocolored) Biological membrane structure 2 Fluid mosaic - A bilayer of lipids with mobile globular proteins Polar phospholipid head Cholesterol Non-polar phospholipid tail Hydrophilic portion Hydrophobic portion Membrane structure 3 Membrane lipids Make up 90-99% of molecules in membrane (in numbers). • Phospholipids - 75% of lipids • Cholesterol - 20% • Glycolipids - 5% - only on cytoplasmic membrane - GLYCOCALYX Membrane proteins Constitute 1-10% of total molecules but 50% of the weight because of their larger size. Sensing signalsTransport Cell identity Cell adhesion Enzymatic activity PeripheralIntegral + Organelles Membranous • Endoplasmic reticulum • Golgi apparatus • Lysosomes • Endosomes • Peroxisomes • Mitochondria Specialized internal structures with specialized functions Non-membranous • Ribosomes • Centrosomes • Centrioles • Basal bodies Related to specific structure and function of the cell e.g., much energy needed → many mitochondria Nucleus 1 Envelop-bounded structure Liver cell nucleus Mostly: • Spheherical (5-10 mm) (lobular, twisted, disk-shaped,…) • Located centrally • One per cell (osteoclast more, erythrocyte none) Nucleus Perinuclear cisterna Unit membrane Lamina Unit membrane 20-50 nm 80-100 nm Outer nuc. membr. Inner nuc. membrane Nucleus 2 Continuation on nuclear envelop Perinuclear cisterna Unit membrane Lamina Unit membrane 20-50 nm 80-100 nm Outer nuc. membr. Inner nuc. membrane Lamins: • Intermadiate filament proteins (A, B, C) • Form meshwork inside of INM, some extend into nucleoplasm • Nuclear strength and architecture • Anchorage sites for chromatin • DNA replication and mRNA transcription • Involved in apoptosis Laminopathies • Human diseases (at least 13 known) • Mutations in lamin genes (almost 200 mutations known) • Deregulated gene expression • Premature aging Hutchinson-Gilford progeria Rare - 1-4 per 8 milion of newborns Missense mutation in A-type lamin Nucleus 3 Nuclear pore complex Diameter ~ 100 – 125 nm Three rings (8 subunits each) Inner filamentous basket Distal ring Nuclear ring Cytoplasmic ring Transport via nuclear pores (Nucleocytoplasmic shuttling) • Proteins, RNAs, ribosome subunits • Bidirectional • Needs nuclear localization/export signals • Helped by importins/exportins • Regulated by Ran GTPases Nucleus 4 Chromatin Heterochromatin Feulgen positive – dark in light microscope Dark/dense granular in TEM Transcriptionally inactive Euchromatin Invisible in light microscope Relaxed uncoiled chromosomes Transcriptionally active Interphase nucleus 2 nm DNA double helix 30 nm Chromatin fiber of packed nucleosomes Nucleus 5 Nucleolus nucleolusnuclear membrane nucleoplasm cytoplasm non-membrane-bounded structure Main functions Synthesis of rRNA Assembly of ribosomes Pars fibrosa Primary transcripts of rRNA Pars granulosa Assembly of ribosomes Nucleolar-organizing regions of DNA on five chromosomes in human cells (chrs. 13, 14, 15, 21, 22) Endoplasmic reticulum 1 „within cell“ „net“ Majority of the membrane within cells. Interconnected tubuli and vesicles Cisterns Endoplasmic reticulum 2 Rough ERSmooth ER 200 nm NO attached ribosomes → No protein-synthesis functions! Manufactures phospholipids and cholesterol • Liver – lipid and cholesterol metabolism, breakdown of glycogen and, along with the kidneys, detoxification of drugs • Testes – synthesis of steroid-based hormones (testosterone) • Intestinal cells – absorption, synthesis, and transport of lipids • Skeletal and cardiac muscle – storage and release of calcium (sarcoplasmic reticulum) External surface has ribosomes attached • Manufactures all secreted proteins • Synthesizes integral membrane proteins • Modifies proteins Ribosomes ribosomes mRNA 100 nm POLYRIBOSOME (cluster of ribosomes translating certain segment of mRNA) mRNA 5` AUG 3` START kodon 3`UAC 5` Met-tRNA Beginning of translation mRNA 5` UAG 3` End of translation mRNA 5` UAA 3` mRNA 5` UGA 3` STOP kodony bind „release factor“E P A 5` 3` reading of mRNA = movement of ribosomes on mRNA codons Aminoacyl tRNA free tRNA Growing polypeptide chain Ribosomes - Translation Golgi apparatus – Transgolgi pathway Cis Trans Centrosome Diameter – 0.2 mm Length - 0.5 mm 1 2 3 4 56 7 8 9 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 Basic concept 1 MITOSIS and CYTOKINESIS produce genetically identical cells Cell division 1 STABLE (non-changing) GENOME Due to semiconservative duplication of DNA Condensed duplicated chromosome chromatid centromere telomere telomere Cell division 2 ho homologous chr. pairs duplication duplication nonsister chromatids sister chromatides sister chromatides chromosome with one chromatid mathers chromosome Metabolism of chromosomes – Homologous chromosomes 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 ! ahampl@med.muni.cz Building A1 – 1st floor Histology lectures Key elements of the microscopic structure of tissues and organs and their relevance to the function Very latest discoveries in the field of tissue structure and maintenance and their relevance to the disease development and therapy