Notes

Outline

Microscopic Anatomy Cell Structural Level

Outline Visualizing tissues and cells Light, fluorescence and electron microscopy Tissue types Epithelia, connective tissue Cellular structures and organelles Subcellular fractionation

Structures and scales Structure Scale (mm) Unit Organ 10 to 100 cm Tissue 1 to 10 mm Tissue layer .1 to 1 mm Cell .01 to .1 mm Organelle .001 to .01 nm

Microscopy Light (100 mm) Bright-field Phase contrast, differential interference Fluorescence Wide-field Laser confocal (optical sectioning) Electron Scanning (10 mm) Transmission (1 mm) High voltage transmission tomography (10 mm)

Conventional vs confocal microscopy

Histological preparation Tissue fixation (cross link proteins) Formalin, methanol Gluteraldehyde Embedding Freezing Paraffin Sectioning Cryostat (thick) Microtome (thin) Ultramicrotome (very thin)

Staining To enhance contrast Various satins bind to different cellular components Hematoxylin and eosin (H&E) for LM Hematoxylin is blue, basic, and binds to negatively-charged substances such as nucleic acids Eosin is red, acidic, and binds to positively charged substances, usually proteins Heavy metal density labels for EM

Structures visible with LM Tissue layers Extracellular matrix Basement membrane Cells Cell nuclei Mitotic chromosomes Nucleoli Cytoskeleton Cilia and microvilli

Hematoxylin & Eosin stained tracheal epithelium

Antibody labeling Labels are covalently attached to antibodies Directly conjugated Attached to a secondary antibody Different types of labels for various visualization techniques Radioactive (35S, 3H, 33P) Enzymatic (HRP or AP) Fluorescent stains Colloidal gold

Fluorescence images

Structures visible with EM Mitochondria Plasma membrane Internal membranes Endoplasmic reticulum Golgi stacks Lysosomes, peroxisomes, synaptosomes, etc Ribosomes and polysomes Granules

Mitochondrion Reconstructed by Electron Tomography (T.G. Frey)

Scanning EM of cells with cilia and microvilli

Tissue types Epithelia Organized  in tightly packed sheets Connective tissue Extracellular matrix of protein fibers (collagen, elastin, laminin) Ground material (proteoglycans and glycosaminoglycans) Muscle Skeletal (striated), smooth, cardiac Nervous tissue Neurons, glia, adipose cells

Characteristics of epithelia Come in a variety of types Layers  (simple, stratified) Cell shape (columnar, cuboidal, squamous) Usually line a free surface External body surfaces and internal cavities Mostly comprised of cells with little extracellular matrix material Distinct boundary (the basal lamina) between underlying connective tissue Polarized with apical surface toward the lumen, and basal surface toward the basal lamina

Cell-cell junctions in epithelial sheets Epithelial cells are tightly joined together Tight junctions – seal neighboring cells together Create a barrier to movement of cells, viruses, and molecules Other cell junctions Adherens – joins actin bundles in adjacent cells Desmosome – anchors intermediate filaments in neighboring cells Gap – allows passage of small water-soluble ions and molecules

Three views of epithelial cells

Transport across epithelia: the intestinal epithelium is highly polarized

Characteristics of connective tissue Primarily comprised of  extracellular matrix Cells are scattered and embedded in the matrix Some of these secrete the matrix (fibroblasts, osteoblasts) The matrix rather than the cells carry the mechanical load Tensile strength is provided by the fibrous protein collagen Resiliency is provided by elastin

Types of connective tissue Connective tissues have different varieties of collagen and modes of collagen organization Loose connective tissue Sub-epithelial layer in mucous membranes Adipose tissue Dense regular connective tissue Ligaments and tendons Reticular connective tissue Lymph nodes

Cell developmental lineages Ectodermal Epithelial cells Mesodermal Fibroblasts Muscle cells Blood cells Endodermal Vascular cells

Eukaryotic cellular structures Plasma membrane Nucleus Cytoplasm Intracellular membrane system Ribosomes Mitochondria Cytoskeleton

Plasma Membrane External boundary of cell 5 nm thick Phospholipid bilayer Hydrophilic exterior Hydrophobic interior Fluid mosaic model Proteins are attached to the surface and embedded in the membrane bilayer

Bilaminar membrane model

A pure phospholipid bilayer acts as a selectively permeable barrier

Transmission EM image of plasma membrane

Nucleus Large ovoid structure (10 mm) Stains darkly with hematoxylin because of large amount of nucleic acid (DNA) Chromosomes are visible during mitosis Surrounded by nuclear envelope Double membrane with pores Breaks down during mitosis Contains dense bodies, nucleoli (2 mm), that are the sites of rRNA synthesis and ribosome assembly

Transmission EM of liver cell nucleus with prominent nucleolus

Cytoplasm Cytosol (concentrated aqueous gel) Less dense than nucleus Packed with molecules and organelles Granular material (ribosomes, glycosomes) Laminar structures (endoplasmic reticulum, Golgi stacks) Membrane bound vesicles (lysosomes, endosomes, peroxisomes) Double-membrane structures (mitochondria)

Internal membrane system Endoplasmic reticulum Transport vesicles Golgi apparatus Endosomes Lysosomes Peroxisomes Synaptosomes

Endoplasmic reticulum (ER) Complex network of sheets and tubes Proximal to nucleus and continuous with outer nuclear membrane Rough ER (site of synthesis of proteins delivered into ER lumen or membranes) Smooth ER (site of protein maturation, modification, and sorting) Disulfide bond formation Chaperone mediated folding Initiation of glycosylation

Rough endoplasmic reticulum

Transport vesicles Bud off from ER and fuse with cis face of Golgi network Bud off of trans face of Golgi to fuse with the plasma membrane As secretory vesicles To carry newly synthesized lipid and proteins to the plasma membrane Bud off of trans face of Golgi to fuse with endosomes to form lysosomes and peroxisomes

Golgi apparatus Layered parallel stacked sacs cis Golgi network cis cisterna medial cisterna trans cisterna trans Golgi network Further protein modification and sorting Addition of oligosaccharides

3D model of Golgi

Golgi apparatus

Ribosomes Complex of RNA and proteins 20 nm diameter Some are loosely attached to the endoplasmic reticulum (rough ER) Others are free Form polysomal complexes with mRNA and newly synthesized protein Proteins synthesized in the cytosol can be taken up by the smooth ER.

E. Coli ribosome reconstruction from EM images at at 25Å

Mitochondria Prominent structures in cytoplasm Sausage or worm shaped 1 or more mm long Smooth outer membrane Convoluted inner membrane Inner membrane contains proteins responsible for cellular respiration

Mitochondrion & rough ER

Cytoskeleton Critical for cell organization, shape, movements, attachments Dynamic assembly and disassembly Thin filaments (7 nm diameter) Twisted chains of two-stranded actin molecules Intermediate filaments (10 nm) Strong knots of coiled-coil dimers and tetramers Keratin, vimentin, nuclear lamins Microtubles (25 nm) Bundles of tubulin

This cell has large numbers of intermediate or 10 nm filaments

Microtubular bundles in cilia

Cell fractionation To determine intracellular localization of proteins and other cellular components Tissue disruption Mechanical (freeze/thaw, pressure cell, sonication, homogenization) Chemical (detergents, ionic shock) Enzymatic (proteases)

Centrifugation To separate components in an induced gravitational field Cellular homogenates are fractionated into supernatants  and pellets in a series of steps Low, medium, high, super speed, ultra centrifuges (100,000 rpm = 600K g) Different sizes and shapes of swinging bucket and fixed angle rotors Different sizes and types of tubes

Centrifugation procedures Differential centrifugation Separates by size and density Supernatants and pellets at different speeds and run times Velocity sedimentation Separates by density Shallow sucrose gradient Components of similar size form bands Equilibrium sedimentation Separates on basis of density Steep sucrose or cesium chloride gradient

Cell fractionation by differential centrifugation

Velocity sedimentation

Analytical centrifugation Centrifugation is used to analyze cellular structures and functions Marker proteins, enzymes, and antibodies can be used to define and resolve structural and functional compartments

Putting it together Cellular structural and functional analysis is based on a marriage of diverse technologies Visualization Fractionation Biochemistry Molecular biology

TEM of rat liver organelles