Histo Review #1 2004

Your Test
Monday, 9/20 1:30 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!

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

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]

H and E Stain
H: Hematoxylin, basic dye, stains acidic groups (Heterochromatin, Glycosaminoglycans) blue.

E: Eosin, acidic dye. Stains proteins red.

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.

Silver Stain

Stains Reticular Fibers and Basement Membrane Black.

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+

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

Nucleus
Chromatin Nucleolus Envelope/Matrix

Chromatin

karyosome

marginal

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

Nucleolus
NO- Nucleolar Organizing Center P. Fibrosa- Denser, Newly Formed rRNA subunits

P. GranulosaRibonucleoprotein Particles (proteins are imported from the cytosol) Remember that ribosome subunits are assembled in the nucleolus!!!
Final assembly of ribosomes occurs in the cytosol.

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)

Cell Surface Structures/ Membrane Proteins

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

Plasma Membrane Components

Outer leaflet:
SM, PC

Inner Leaflet:
PS, PI, PEtn

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

Caveolae vs. Clathrin Coated Pits

Clathrin

Clathrin-Coated Pits/Vesicles
Important for Receptor-Mediated Endocytosis Lysosomal enzyme targeting
M6P receptor

Secretory Vesicle Formation

Ion Channel Mutations/Diseases

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

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.)

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

Dystrophin and DMD in Muscle Cells

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

dystroglycans

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).

Junctional Complex

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

Desmosome

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.

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!

The Cytoskeleton

Cytoskeletal elements
Microtubules:
- a- and b-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

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-toend, alpha- to betaPolymerization is dependent on GTP hydrolysis Colchecine, vincristine and other alkaloids inhibit binding Associated proteins:
Motor proteins: kinesin and dynein

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.

Cell Motors, Motility, and Mitosis

Microtubular Motors Kinesin:Moves 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.

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 membranebound organelles along microtubules. (+) directed Some Kinesin Related Proteins move cilia, organize microtubules, or bind DNA directly (chromokinesin)

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

The Mitotic Spindle

Know your PMAT!

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

Centrioles/Basal Bodies vs. Cilia

Cilia/Flagella: 9*2 + 2 Arrangement

Centrioles/Basal Bodies: 9*3

Molecular Motors

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

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.

Clinical Correlations of Actin/Myosin

Cytochalasin D prevents F-actin elongation Phallotoxin (phalloidin) binds and freezes F-actin, prevents depolymerization 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

Endomembrane System
ER Golgi Lysosomes

Smooth ER
-Steroid Production -Detoxification/ Drug Metabolism -Connected to rER

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

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

Quality control: ubiquitin-proteasome pathway

Protein Synthesis/ Signal Sequences

Protein modification
Co- vs. Posttranslational
Golgi is post-, ER is cotranslational

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

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 retroanterograde as needed

COP-I: retrograde transport- binds KDEL receptor COP-II: anterograde transport

Exocytosis
Vesicles fuse with outer plasma membrane

Lysosomal Targeting
*- KFERQ sequence is a destruction signal for senescent organelles

Clathrin

Lysosomes

Tay-Sachs Disease

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 nonfunctional peroxisomes.

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

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

Electron Micrographs