Histo Review

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Histo Review 1 2004
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Your Test

• Monday, 9/20 130
• 30 questions from Microscopy to Cell Bio
• 10 Image-Based (LM and EM)
• Pass Level is 55-65 (Hell probably throw out
  some questions)
• 10 of your total grade
• Dr. B says
• there will be 8 or 9 questions on EMs
• pay some attention to clinical refs in the
  lectures, notes posted on Bb and in CH 1,2 in the
  text.
• Lecture notes and handouts!

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Microscopy

• Resolution
• Eye 200?m 0.2mm 200,000nm
• Light microscope 0.2 microns (mm)
• Electrons
• Scanning EM 2.5 nanometers
• Transmission EM 1 nm, theoretically 0.5 nm

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Staining Terminology

• Acidophilia Reaction of cationic groups
  (protein amino grps.) with an acidic dye
• Proteins are acidophilic
• Basophilia Reaction of anionic groups
  (phosphate, sulfate) with a basic dye
• Only Heterochromatin, Nucleoli, Ergastoplasm
  (RNA), and Extracellular Sulfate Sugar Moieties
  (GAGs) are highly basophilic
• Metachromasia A change in the color of a dye
  based upon high concentration of that dyes
  ligand in a cell
• e.g. toluidine Blue stains mast cell granules
  purple- high heparin sulfate

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H and E Stain
H Hematoxylin, basic dye, stains acidic groups
(Heterochromatin, Glycosaminoglycans) blue. E
Eosin, acidic dye. Stains proteins red.
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PAS (periodic acid-Schiff) Stain
Stains reducing sugars red. (Cleaves Aldehyde
Grps) Stains Glycogen, Mucus, Basement Membrane
and Reticular Fibers
PAS Reaction - Periodic Acid cleaves sugars
into aldehyde groups. Aldehydes react with
Schiff Reagent- RED Feulgen Reaction - DNA (not
RNA) is cleaved by HCl, reacts w/Schiff.
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Silver Stain
Stains Reticular Fibers and Basement Membrane
Black.
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Immunohistochemistry

• Enzyme-linked antibodies
• Targets specific proteins associated with disease
• Useful for diagnosis
• Example oral tumor (condyloma) biopsy tests
  positive for Human Papilloma Virus

HPV
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Freeze Fracture

• The Plasma Membrane is Split in Half, making two
  faces, the E and P face. On Scanning EM, the
  P-face generally has more proteins associated.

E P
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Nucleus

• Chromatin
• Nucleolus
• Envelope/Matrix

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Chromatin
marginal
karyosome
Orders of Chromatin Organization Nucleosome
fundamental packing unit linker DNA
nucleosome bead (2 whorls of DNA histones 4
one other histone H1) -beads-on-a-string form
30 nm chromatin fiber loops clusters of
looped domains chromosome Amount of Euchromatin
Transcriptional Activity of the Cell!
4 nm
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Nucleolus
NO- Nucleolar Organizing Center P. Fibrosa-
Denser, Newly Formed rRNA subunits P.
Granulosa- Ribonucleoprotein Particles
(proteins are imported from the
cytosol) Remember that ribosome subunits are
assembled in the nucleolus!!! Final assembly of
ribosomes occurs in the cytosol.
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• Nuclear envelope
• Separates RNA synthesis from RNA processing
  prevents damage from cytoskeleton
• Remember that nuclear outer membrane is
  contiguous with rough ER!
• Nuclear pore complex
• Composed of nucleoporins
• Allow small molecule entry by diffusion large
  proteins, however, require importin, exportin
  (and both ATP and GTP)

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Cell Surface Structures/ Membrane Proteins

• Plasma Membrane
• Lipid Rafts/Caveolae
• Membrane Proteins
• Junctions, Ion Channels

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Plasma Membrane Components

• Outer leaflet
• SM, PC
• Inner Leaflet
• PS, PI, PEtn

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Caveolae

• Not clathrin coated
• Arise from Lipid Rafts (thickenings of PM)
• Contain Cav-1, Cholesterol, Sphingolipids,
• certain GPI-anchored proteins
• -Activated by src-kinase
• -Important for potocytosis, transcytosis

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Caveolae vs. Clathrin Coated Pits
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Clathrin
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Clathrin-Coated Pits/Vesicles

• Important for Receptor-Mediated Endocytosis
• Lysosomal enzyme targeting
• M6P receptor
• Secretory Vesicle Formation

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Ion Channel Mutations/Diseases

• Myasthenia Gravis Muscle weakness due to
  autoantibodies against the acetylcholine receptor
• Cystic Fibrosis Defect in the Cl- channel CFTR
  leads to excessive phlegm and static infections

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Glycocalyx

• Made up of Glycoproteins, Proteoglycans, and
  Glycolipids
• Remember that most sugars are on the outside of
  the cell.

Membrane Proteins

• Integral have transmembrane domains
• Peripheral have noncovalent attachment to the
  membrane or an integral protein
• Lipid-anchored Covalently bonded to either a
  phospholipid or a fatty acid (farnesyl, GPI,
  etc.)

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Erythrocyte Membrane Skeleton

• Spectrin Filaments attach to b-actin junctional
  complexes
• b-Actin binds Glycophorin C
• Spectrin is held to the membrane by Ankyrin, Band
  3 proteins
• Hereditary Spherocytosis Defect in one or more
  of these proteins

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Dystrophin and DMD in Muscle Cells

• Lack of functional dystrophin leads to Duchennes
  Musc. Dystrophy (DMD)
• Muscle weakening, pseudohypertrophy

dystroglycans
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Integrins

• Integral Membrane proteins that link the cell to
  the ECM.
• Have a and b subunits, many types found in
  different cells with different functions
• b2 integrins found on leukocytes
• avb3 found on endothelial cells, smcs, plts
• Found in focal adhesions (with vinculin, actin)
  and hemidesmosomes (interm. fil., plectin).

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Junctional Complex
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Junctional Complex

• Zona Occludens
• ZO-1,2, Occludin, Claudin
• Most Apical, Functions in preventing stuff from
  getting between two cells
• Zona Adherens
• Cadherins, Catenins, Actin, Plakoglobin
• Ca-dependent Cell-Cell adhesion. Very strong.
  
• Macula Adherens (Desmosome)
• Cadherins, Desmoglein, collin, Intermediate
  Filaments
• Virtually permanent cell-cell adhesion

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Desmosome
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Gap Junctions
One Connexon connects to a connexon in another
cell. Each connexon is made of 6 connexin
subunits. Gap junctions allow the selective
passage of ions and small molecules.
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Know the chart on page 13!

• Tight junction
• Prevents intercellular transport!
• Integrins
• Gap Junctions
• Connexin vs. connexon
• Structure of microvilli vs. stereocilia vs. cilia
  vs. basal body vs. centriole!

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The Cytoskeleton
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Cytoskeletal elements

• Microtubules
• - ?- and ?-tubulin form dynamic, polar filaments
• - about 20-25 nm in diameter
• - require GTP for assembly
• Intermediate filaments
• -desmin, keratin, vimentin expressed in
  different tissues
• - about 10 nm in diameter
• Microfilaments
• - actin monofilaments
• - about 6-8 nm in diameter
• - require ATP for assembly

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Microtubules

• Each fiber is a hollow cylinder
• Microtubules have polarity a positive,
  fast-growing end and a slow-growing negative end
• Soluble tubulin dimers bind end-to-end, alpha- to
  beta-
• Polymerization is dependent on GTP hydrolysis
• Colchecine, vincristine and other alkaloids
  inhibit binding
• Associated proteins
• Motor proteins kinesin and dynein

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Types of Intermediate filaments

• Types I and II Acidic Keratin and Basic Keratin,
  respectively.
• Produced by different types of epithelial cells
  (bladder, skin, etc).
• Epidermolysis Bullosa keratin deficiency-
  blistering diseases
• Type III Intermediate filaments are distributed
  in a number of cell types, including
• Vimentin in fibroblasts, endothelial cells and
  leukocytes desmin in muscle glial fibrillary
  acidic factor (GFAP) in astrocytes and other
  types of glia
• Type IV Neurofilament H (heavy), M (medium) and
  L (low).
• Type V Lamins
• Lamins are vital to the re-formation of the
  nuclear envelope after cell division.

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Cell Motors, Motility, and Mitosis
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Microtubular Motors

• KinesinMoves from () end to () end.
• Dynein Moves from () end to () end.
• ATPases
• Carry organelles along MTs (mitochondria,
  vesicles)
• () end of MTs is usually at the periphery of
  the cell, (-) end is usually near the MTOC
  centrally.

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Dynein

• Found in cilia/flagella cause sliding of MTs
  gives beating motion
• Dynactin linker between Dynein and other
  structures (centrosomes, actin, et al.)

Kinesin

• Kinesin I used in cells to transport
  membrane-bound organelles along microtubules. ()
  directed
• Some Kinesin Related Proteins move cilia,
  organize microtubules, or bind DNA directly
  (chromokinesin)

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What Molecular Motors Do

• Movement of organelles/vesicles from one part of
  the cell to another (e.g. from ER to Golgi)
• Cell Polarity Bring different proteins to
  different sides of cells (axon vs. dendrite,
  apical vs. basolateral)
• Flagellar/Ciliary function, maintenance
• Mitosis/Meiosis

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The Mitotic Spindle
Know your PMAT!
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Clinical Correlations of MT Motors

• Microtubule-directed drugs (paclitaxel (Taxol),
  vincristine) stop mitosis, kill cancer cells
• Kartageners Syndrome Dynein (or Kinesin)
  mutations
• Situs Inversus
• Sterility in males
• Sinus Infections
• Lissencephaly- dynein deficiency leading to
  severe brain developmental deficiencies

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Centrioles/Basal Bodies vs. Cilia

• Cilia/Flagella 92 2 Arrangement
• Centrioles/Basal Bodies 93

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Molecular Motors
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Myosins Actin Motors

• Many types, heavy chain is conserved.
• Myosin I- interacts with membranes, important for
  endocytosis, inner ear function
• Myosin II found in many types of cells,
  regulates cell contraction, locomotion,
  cytokinesis.
• Myosin V functions in delivery of vesicles to
  membrane

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Actin Microfilaments

• G-actin (globular subunit) is converted to
  F-actin (fibers) under certain conditions
• (WASP activation (wiskott-aldrich syndrome
  protein) (dont memorize)
• Actin binding proteins regulate actin
  assembly/disassembly (gelsolin, thymosin),
  regulation (troponin). and organization (fimbrin,
  alpha-actinin, filamin).
• Actin Microfilaments have a end and a end
  similar to MTs.

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Clinical Correlations of Actin/Myosin

• Cytochalasin D prevents F-actin elongation
• Phallotoxin (phalloidin) binds and freezes
  F-actin, prevents de-polymerization
• Latrunculin binds and inhibits G-actin
• Listeria and Shigella use actin to travel through
  the cell
• Usher Syndrome mutation in Myosin VII, hearing
  loss, retinitis pigmentosa (deaf/blind)
• Griscelli Syndrome Myosin V deficiency
  albinism

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Endomembrane System

• ER
• Golgi
• Lysosomes

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Smooth ER

• Steroid Production
• Detoxification/ Drug
• Metabolism
• -Connected to rER

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rER

• Interconnected tubules, vesicles and sacs
• Associates with ribosomes, Protein synthesis

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ER, signal sequence, protein translation

• Hydrophobic sequence targets ribosome to ER
• SRP signal recognition peptide binds signal
  sequence and stops translation ribosome
  translocates to ER
• SRP Receptor SRP/ribosome/nascent protein binds
  to ER
• Sec61 protein translocation complex signal
  sequence is inserted into ER membrane
• Translation resumes, with growing peptide chain
  translocating across membrane
• BiP protein chaperone aids in proper folding and
  assembly within ER
• Peptide is cleaved after signal sequence and
  released into lumen of ER

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Quality control ubiquitin-proteasome pathway
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Protein Synthesis/ Signal Sequences
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Protein modification

• Co- vs. Post-translational
• Golgi is post-, ER is co-translational
• Golgi is functionally compartmentalized each
  cisternae contains certain enzymes that can
  modify proteins in specific ways
• Glycosylation, phosphorylation, sulfation
• Proteolytic modification
• Glycolipid synthesis
• Sorting of vesicles clathrin-coated
  pits/adaptors

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Golgi Maturation

• Vesicular transport
• Vesicles carry proteins toward trans-face
• Cisternal maturation
• Entire cisternae move toward PM and break up
• Combined
• Cisternae mature, but enzymes transported
  retro-anterograde as needed
• COP-I retrograde transport- binds KDEL receptor
• COP-II anterograde transport

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Exocytosis

• Vesicles fuse with outer plasma membrane

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Lysosomal Targeting

• - KFERQ sequence is a destruction signal for
  senescent organelles

Clathrin
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Lysosomes
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Tay-Sachs Disease
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Peroxisomes

• Small, Spherical Organelles
• Are more homogenous-appearing than lysosomes
• Contain Catalase, other enzymes
• Important for
• Ethanol oxidation (liver)
• b-oxidation of fatty acids
• Have crystalloid inclusions in non-humans
• Zellweger Syndrome early death due to
  non-functional peroxisomes.

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Mitochondria

• Originate from prokaryotes?
• Two membrane bilayers
• Cristae form from inner membrane
• Intermembrane space is contiguous with cristal
  lumen, contains H gradient
• Electron Transport Chain proteins, F1F0 ATP
  synthase are in the inner membrane
• Matrix is within the inner membrane, houses the
  Krebs cycle
• Mitochondria have their own DNA, ribosomes,
  division process

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Mitochondria and Apoptosis

• Opening of PTP (permeability transition pore)
  leads to Cytochrome C escape from mito
• Cyt C activates Apaf-1, which activates the
  Caspase Cascade
• Intracellular proteases degrade cellular
  components

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Electron Micrographs
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