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Lecture 8 Cell and Tissue Basics. Eukaryotes: group of organisms whose cells have a defined nucleus surrounded by a nuclear membrane. Cells are divided.

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Presentation on theme: "Lecture 8 Cell and Tissue Basics. Eukaryotes: group of organisms whose cells have a defined nucleus surrounded by a nuclear membrane. Cells are divided."— Presentation transcript:

1 Lecture 8 Cell and Tissue Basics

2 Eukaryotes: group of organisms whose cells have a defined nucleus surrounded by a nuclear membrane. Cells are divided into at least two components: the nucleus and the cytoplasm (cytosol+organelles) Cell size on the order of tens of microns. Cell: The functional unit of all living organisms

3 Plasma membrane Outer cell membrane, whose structure is not well known Fluid mosaic model is the prevalent model for its architecture Membrane consists of phospholipid bilayer (~10 nm); its major components are: -phospholipid molecules; amphipathic (polar, hydrophilic head; non- polar, hydrophobic tail) -cholesterol (1:1 cholesterol:phospholipids) * regulate fluidity * stability -protein molecules (half of the total mass of membrane) *intrinsic or integral proteins *transmembrane proteins *extrinsic or peripheral proteins -Glycocalyx *short chain of polysaccharides conjugated with membrane proteins and some of the membrane lipids

4 Plasma membrane Major functions: –Nutrient transport in/waste transport out –Barrier to unwanted materials –Maintenance of proper ionic composition, pH, osmotic pressure –Contact with other cells and extracellular matrix –Cell signaling receptors Spectroscopic features: –Refractive index (n=1.43-1.49) higher than water (n=1.33) leads to scattering Detected only by highly sensitive methods such as low- coherence interferometry

5 cytoskeleton Framework of minute filaments and tubules Provides structural support for PM, cellular organelles Responsible for locomotor mechanism for amoeboid movements and specialized structures such as cilia and flagella Responsible for contractility of cells in specialized tissues such as muscle Includes: –Microfilaments ~5nm in diameter; made out of actin Responsible for cell shape and motility; provide energy for contractile processes –Microtubules 25 nm in diameter; made up of globular protein subunits Can be readily assembled and disassembled Provide network onto which organelles such as mitochondria can move –Intermediate filaments 10-12 nm in diameter; stable fibrous structure Tough supporting network Distribute tensile forces across cell Spectroscopic features: Birefringence

6 Endoplasmic reticulum Rough ER –Interconnecting network of membranes, tubules and vesicles –Surfaces studded with ribosomes, where protein synthesis takes place –Proteins synthesized here are destined for lysosomes or export or incorporation in the PM –Continuous with outer lipid bilayer of nuclear envelope Smooth ER –Irregular network of membranes, tubules and vesicles devoid of ribosomes –Continuous with rough ER and Golgi apparatus –Function: lipid biosynthesis and membrane synthesis and repair

7 Golgi apparatus System of stacked, saucer shaped cisternae, with concave surface facing nucleus Function: glycosylation of proteins and packaging into secretory products or lysosomes Spectroscopic properties: It is expected that the Golgi apparatus along with the ER will contribute to light scattering from the cells because they consist of membranes (lipids) which have higher refractive index than water Golgi apparatus

8 Lysosomes Structure: Membrane bound organelles containing amorphous granular material (primary lysosomes) or electron dense particulates (secondary lysosomes) Function: degradation of worn-out cell constituents and foreign materials into monomeric units by acid hydrolases (e.g. proteases degrade proteins into peptides) Several hundred lysosomes may be present in eukoryotic cell Vary in size and appearance from hundreds of nm to a few  m. pH of lysosome ~ 4.8 –Protein denaturation –Protection in case of release of lysosomal contents Spectroscopic properties: scattering

9 mitochondria Structure: –Outer membrane Relatively permeable to small molecules –Inner membrane Folds into cristae and is much less permeable Contains lots of proteins including the cytochromes, enzymes involved in ATP production –Intermembrane space –Matrix (gel like; 50% protein content) Location of mitochondrial DNA and ribosomes Typical size: 200x600nm, but there is a lot of variation Their number varies from very few in metabolically inactive cells to a couple of thousand in liver and skeletal muscle cells

10 mitochondria Function: power-plant of the cell –Site of ATP production –ATP: energy currency for cells ATP is used for –Movement –Macromolecule synthesis –Molecule transport against concentration gradient Glucose metabolism: most efficient means of generating ATP

11 Glucose metabolism Overall reaction: C 6 H 12 O 6 +6O 2 +36P i 2- +36ADP 3- +36H + →6CO 2 +36ATP 4- +42H 2 O 1 st stage: anaerobic glycolysis (in cytosol) C 6 H 12 O 6 +2NAD + + 2ADP 3- +2P i 2- →C 3 H 4 O 3 +2NADH+2ATP 4- 2 nd stage: aerobic glycolysis –Pyruvate transferred to mitochondria and oxidized to CO2 by O2. In the process 34 ATP molecules produced C 6 H 12 O 6 : glucose P i 2- : inorganic phosphate ADP: adenosine diphosphate ATP: adenosine triphosphate C 3 H 4 O 3 : pyruvate NAD + : nicotinamide adenine dinucleotide (oxidized form) NADH: nicotinamide adenine dinucleotide (reduced form)

12 Aerobic glycolysis 3 reaction groups: –Oxidation of pyruvate and fatty acids to CO 2, coupled to reduction of NAD + and FAD into NADH and FADH 2 (Kreb’s cycle or citric acid cycle) Matrix of inner membrane –Electron transfer from NADH/FADH 2 to O 2 coupled to generation of proton-motive force Inner membrane –Production of ATP using electrochemical H + gradient across inner membrane

13 Aerobic glycolysis

14 Electron transport chain As electrons are transported via four enzyme complexes from NADH and FADH 2 to O 2, protons are pumped across the inner mitochondrial membrane, generating a proton- motive force (~220 mV), which is due to –proton concentration gradient (~60 mV) –Electric potential (matrix becomes negative with respect to intermembrane space) -160 mV

15 Mitochondrial spectroscopic signatures Scattering Fluorescence –NADH and FAD are fluorescent molecules –NAD+ and FADH2 are essentially non-fluorescent –Handle on metabolic activity of the cell through redox ratio: FAD/(NADH+FAD) NADH image: exc:360 nm/em: 450 nmFAD image: exc: 455 nm/em:520 nm Epithelial Keratinocytes From human foreskins

16 Nucleus: command center of the cell Contents: –DNA (20% mass)-deoxyribonucleic acid Usually arranged as tangled strands –Nucleoproteins DNA binding proteins –Histones (DNA folding, regulation of DNA activity) –Non-histones (regulation of gene activity) DNA/RNA synthesis proteins –DNA usually arranged as tangled strands Heterochromatin: DNA and associated proteins not involved in RNA synthesis (electron-dense) Euchromatin: DNA and associated proteins involved in RNA synthesis (electron-lucent)

17 Structure of the nucleus The nucleus consists of the nucleoplasm and the nucleolus (may be more than one per nucleus) It is surrounded by two membranes, comprising the nuclear envelope (NE) –Each membrane has a phospholipid bilayer structure –the outer membrane is continuous with the ER and is studded with ribosomes –At the inner membrane there is a fibrillar layer, the nuclear lamina, which links membrane proteins and heterochromatin –The envelope contains numerous nuclear pores, at the margins of which the inner and outer membranes become continuous –Nuclear pores (NP) permit and regulate the exchange of metabolites, macromolecules and ribosomal subunits between the nucleus and the cytoplasm The nuclear cytoskeleton is attached to the nuclear lamina and provides support to nuclear contents The nucleolus is the site of ribosomal RNA synthesis and ribosomal assembly heterochromatin euchromatin

18 Nuclear spectroscopic signatures The nucleus doesn’t have any significant autofluorescence However, it has a refractive index of ~1.42, which is significantly higher than the surrounding cytosol (n~1.36). Its high refractive index in combination with its large size (typically 5- 10  m) yield highly directional scattering properties This scattering is used in light scattering spectroscopic measurements to assess nuclear morphology Changes in nuclear morphology constitute hallmarks that are used routinely by histopathologists to determine the presence of pre- cancerous and early cancerous changes. These changes include: –Nuclear enlargement (increase in the overall nuclear size) –Nuclear pleomorphism (variation in the size of the nuclei among cells) –Hyperchromasia (increased staining density with standard DNA dyes) –Increase in the number of nuclei per unit area –Increased roughness in nuclear texture

19 Epithelial tissues

20 Basic properties of epithelia Epithelia cover or line all body surfaces, cavities and tubes. They form continuous sheets of cells, comprising one or more cell layers. Epithelial cells are closely bound to one another All epithelia are supported by a basement membrane of variable thickness, which separates them from underlying supporting tissues and blood vessels. They function as interfaces between different biological compartments. Thus, they mediate –Selective diffusion, absorption, and/or secretion –Physical protection –Containment

21 Classification of epithelia Epithelia are classified according to three morphological characteristics –The number of cell layers Simple (single layer) Stratified (many layers) –The shape of the component cells Squamous Columnar Cuboidal transitional –The presence of surface specializations Ciliated keratinizing

22 Simple epithelia Simple epithelia consist of a single layer of cells Almost always found at interfaces involved in selective diffusion, absorption or secretion. They provide little protection against mechanical abrasion

23 Simple squamous epithelia Composed of flattened, irregularly shaped cells They are supported by basement membrane Found lining surfaces involved in passive transport of either gases or fluids Cells frequently have specialized receptors that control secretion of locally acting chemical messengers Found in the lining of: –Lungs –Blood vessels (endothelium) –Peritoneal, pleural and pericardial cavity (mesothelium) endothelium Mesothelium-peritoneum

24 Simple columnar epithelium Cells are tall and appear like columns in sections at right angles to the basement membrane Height of the cells may vary depending on site and degree of functional activity Nuclei are elongated and polar, i.e. they are located towards the base, center or apex of the cell Most often found on highly absorptive or secretory surfaces The luminal plasma membranes of highly absorptive epithelial cells have a brush or striated border made up of microvilli, minute finger like projections, mainly for the purpose of increasing surface area, by as much as 30-fold. –Small intestine –Stomach –Gall bladder –Renal tubules –colon –Larger breast ducts

25 Simple columnar ciliated epithelium Simple columnar epithelium with surface specializations called cilia on the majority of the cells Among ciliated cells, non-ciliated cells can be found that usually have secretory function Cilia are much larger than microvilli and are visible with light microscopy. Each cell may have 300 cilia beating in synchrony with cilia from neighboring cells to propel fluid of minute particles over the epithelial surface Found in the human reproductive tract –Fallopian tube (propel ovum and secretions from ovary to uterus)

26 Pseudostratified columnar ciliated epithelium Just like simple columnar epithelium, with nuclei disposed at different levels, but typically still confined to the basal two-thirds of the epithelium Found in larger airways of respiratory system Often referred to as respiratory epithelium Cilia propel mucous towards the pharynx

27 Simple cuboidal epithelium Intermediate form between simple squamous and simple columnar epithelium Cells appear square in section perpendicular to the basement membrane Usually round nucleus in the center of the cell Cuboidal epithelium lines small ducts and tubules –Kidney –Salivary glands –Pancreas –Breast ducts

28 Stratified epithelia Consist of two of more layers of cells Mainly protective function Degree and nature of stratification related to the types of stresses that epithelium is exposed to Poorly suited for absorption and secretion

29 Stratified squamous epithelium Consists of a variable number of cell layers which exhibit transition from a cuboidal basal layer to a flattened surface layer. Basal cells divide continuously, with the offspring being pushed progressively towards the free surface, where they are ultimately shed. Nuclei become progressively condensed and flattened, before ultimately disintegrating Well adapted to withstand abrasion, since loss of surface cells doesn’t compromise the underlying tissue Poorly adapted to withstand desiccation Found in sites subject to mechanical abrasion but kept moist by glandular secretions, such as: –Oral cavity –Pharynx –Esophagus –Anal canal –Uterine cervix –vagina

30 Stratified squamous keratinizing epithelium During maturation, cells accumulate cross-linked cytoskeletal proteins in a process called keratinization, resulting in the formation of a tough, non-living surface layer consisting of the protein keratin Keratin is highly fluorescent and scattering Squamous keratinizing epithelia are found in the –Skin –Oral cavity

31 Stratified cuboidal Thin stratified epithelium, consisting of two or three layers of cuboidal or low columnar cells. Found in the lining of excretory ducts of glands, such as the salivary glands

32 Transitional epithelium Stratified epithelium almost exclusively confined to the urinary tract Highly specialized to accommodate a great degree of stretch and to withstand the toxicity of urine. In relaxed (contracted) state, it appears to be about four to five cell layers thick Basal cells are roughly cuboidal, intermediate cells are polygonal and surface cells are large and rounded and may contain two nuclei In the stretched state, the epithelium appears only two or three layers thick and top layers are extremely flattened Found in – bladder

33 Basement membrane The basement membrane provides structural support for the epithelium as well as binding to the underlying supporting tissue. Involved in control of epithelial growth and differentiation, forming an impenetrable barrier to downward epithelial growth; this is breached during malignant transformation Controls flow of nutrients, metabolites and other molecules to and from epithelium 50-300 nm thick Main constituents: –Glycosaminoglycan: heparan sulfate –Fibrous protein: collagen IV –Structural glycoproteins: fibronectin, laminin and entactin Glycosaminoglycans (GAG): unbranched polysaccharide chains, each composed of repeating disaccharide units

34 Supporting/connective tissues Basic type of tissue which provides structural and metabolic support for other tissues and organs Connective tissues usually contain blood bessels and mediate the exchange of nutrients, metabolites and waste products between tissues and the circulatory system. In most organs, loose supportive tissues act as a biological packing material between cells and other tissues with more specific functions Dense forms of supporting tissue provide tough physical support in the dermis of the skin, comprise the capsule of organs such as the liver and spleen, and are the source of great tensile strength in ligaments and tendons. Cartilage and bone are highly specialized forms of supporting tissue Important metabolic roles in the context of fat storage and temperature regulation Cells of the immune system enter supporting tissues to defend against pathogens Tissue repair is largely a function of supporting tissues

35 Supporting/connective tissues Three major components: –Cells Fibroblasts (synthesis and maintenance of extracellular material) Adipocytes (fat storage and metabolism) Immune system cells (macrophages, lymphocytes, all types of white blood cells) –Extracellular matrix Matrix of organic material called ground substance within which are embedded a variety of fibers Ground substance composed of GAGs, which are entangled and electrostatically linked to one another and their water of hydration to form a flexible gel through which metabolites can diffuse Fibers include –Collagen –elastin –Structural glycoproteins Fibrillin, fibronectin, laminin, entactin and tenascin Mediate interaction of cells with other constituents Fibronectin also plays a role in collagen deposition and orientation

36 Supportive tissue cells Fibroblasts –Synthesis and maintenance of extracellular material Mature fibroblasts –Condensed, elongated nuclei –Limited cytoplasmic volume active fibroblasts –Large, round nuclei with prominent nucleoli reflecting active protein synthesis –Extensive cytoplasm

37 Active fibroblasts are strongly autofluorescent fibroblasts Epithelial cells NADH fluorescence

38 Adipocytes Fat stored in adipocytes accumulates as lipid droplets which fuse to form a single large droplet, which occupies most of the cytoplasm The nucleus is compressed and diplaced to one side of the lipid droplet Cytoplasm is reduced to a small rim around the periphery In routine histological processing lipid content is extracted, so that adipocytes have large unstained space

39 Lipids are rich in beta carotene Beta carotene exhibits strong absorption features in the visible spectrum. Wavelength (nm) Molar extinction coef (M -1 cm -1 )

40 Collagen fibers Collagen is the main fiber type found in most supporting tissues It is the most abundant protein in the human body Its major function is to provide tensile strength At least 19 different types of collagen have been characterized Type I collagen is the major collagen type found in fibrous supporting tissue, the dermis of the skin, tendon, ligaments and bone Collagen is secreted in the extracellular matrix as tropocollagen (three polypeptide chains bound together to form a helical structure 300 nm long and 1.5 nm in diameter. Tropocollagen molecules are aggregated to form fibrils Parallel collagen fibrils are further arranged into strong bundles 2-10  m in diameter

41 Collagen has strong scattering and autofluorescent features Collagen contributes to highly scattering nature of most epithelial tissues, i.e. it diffuses the light It is an asymmetric molecule and exhibits a strong second harmonic generation signal It has very strong fluorescence in the near UV and visible range of the spectra Multi-photon excitation images acquired with 840 nm excitation TPEF: Two-photon excited fluorescence; SHG: second harmonic generation; FAD: flavin adenine dinucleotide TPEF+SHGSHG FAD TPEF SHG+FAD TPEF

42 Elastin fibers Elastin is found in varying proportions in most supporting tissues Its major function is to confer elasticity to enable recovery of tissue shape following normal physiological deformation Typically occurs in the form of short branching fibers with no recognizable periodicity Present prominently in lung, skin, urinary bladder and blood vessel walls Skin dermis elastin

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