Histo Review

1
Histo Review 1 Mike Broman mbroma1_at_uic.edu
2
Microscopy

• Resolution
• Light 0.2 microns (mm)
• Scanning EM 2.5 nanometers
• Transmission EM 1 nm, theoretically 0.5 nm

3
Staining Terminology

• Acidophilia Reaction of cationic groups
  (protein amino grps.) with an acidic dye
• Basophilia Reaction of anionic groups
  (phosphate, sulfate) with a basic dye
• Only Heterochromatin, Nucleoli, Ergastoplasm
  (RNA), and Extracellular Sulfate Sugar Moieties
  (GAGs)
• 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

4
H and E Stain
H Hematoxylin, basic dye, stains acidic groups
(Heterochromatin, Glycosaminoglycans) blue. E
Eosin, acidic dye. Stains proteins red.
5
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.
6
Silver Stain
Stains Reticular Fibers and Basement Membrane
Black.
7
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
8
Cell Surface Structures/ Membrane Proteins

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

9
Caveolae

• Vesicular Formations that arise out of lipid
  rafts.
• Are not clathrin-coated
• Do not fuse with lysosomes
• Function in transcytosis, potocytosis, and cell
  signaling

10
Caveolae
11
Lipid Rafts

• Highly ordered, thicker regions of the plasma
  membrane
• Rich in cholesterol and sphingolipids
• Caveolin (Cav-1) binds to lipid rafts without
  modification, indicating a link between lipid
  rafts and caveolae.

12
Caveolae vs. Clathrin Coated Pits
13
Clathrin
14
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

15
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.)

16
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

17
Dystrophin and DMD in Muscle Cells
18
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 (vinculin, actin) and
  hemidesmosomes (interm. fil., plectin).

19
Endocytosis

• Can occur in many ways
• Receptor-mediated clathrin-coated pits
• Transcytosis, potocytosis caveolae

20
Caveolae
21
Junctional Complex
22
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

23
Desmosome
24
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.
25
Endomembrane System

• ER
• Golgi
• Lysosomes

26
Smooth ER

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

27
rER

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

28
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

29
(No Transcript)
30
Quality control ubiquitin-proteasome pathway
31
Secretory pathways- Golgi
32
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

33
Protein targeting

• Proteins may be targeted back to ER KDEL
  sequence
• Also mitochondrial, peroxisomal, lysosomal,
  nuclear proteins have signal sequences

34
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
• COP-II anterograde transport

35
SNAREs and SNAPs Involved in Vesicular Fusion
36
Lysosomes
37
Tay-Sachs Dx
38
Nucleus

• Chromatin
• Nucleolus
• Envelope/Matrix

39
Chromatin
marginal
karyosome
4 nm
40
Nucleolus
NO- Nucleolar Organizing Center P. Fibrosa-
Denser, Newly Formed rRNA subunits P.
Granulosa- Ribonucleoprotein Particles
41

• Nuclear envelope
• Separates RNA synthesis from RNA processing
  prevents damage from cytoskeleton
• Nuclear pore complex
• Composed of nucleoporins
• Allow small molecules entry by diffusion large
  proteins, however, require importin (and both ATP
  and GTP)
• RanGTP import/export GTP ? importin/protein
  separation RanGTP ? exportin-protein binding

42
Cytoskeleton
43
Cytoskeletal elements

• Microtubules
• - ?- and ?-tubulin form dynamic, polar filaments
• - about 20-25 nm in diameter
• Intermediate filaments
• -desmin, keratin, vimentin expressed in
  different tissues
• - about 10 nm in diameter
• Microfilaments
• - actin monofilaments
• - about 6-8 nm in diameter
• Nickel, dime, quarter microfilaments,
  intermediate filaments, microtubules

44
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

45
(No Transcript)
46
Types of Intermediate filaments

• Types I and II Acidic Keratin and Basic Keratin,
  respectively.
• Produced by different types of epithelial cells
  (bladder, skin, etc).
• 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 in astrocytes and other types of
  glia, and peripherin in peripheral nerve fibers.
• Type IV Neurofilament H (heavy), M (medium) and
  L (low).
• Modifiers refer to the molecular weight of the NF
  proteins. Another type IV is "internexin" and
  some nonstandard IV's are found in lens fibers of
  the eye (filensin and phakinin).
• Type V are the lamins which have a nuclear
  signal sequence so they can form a filamentous
  support inside the inner nuclear membrane.
• Lamins are vital to the re-formation of the
  nuclear envelope after cell division.

47
(No Transcript)
48
Cell Motors, Motility, and Mitosis
49
Microtubular Motors

• KinesinMoves from () end to () end.
• Dynein Moves from () end to () end.
• Carry organelles along MTs (mitochondria,
  vesicles)
• ATPases

50
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)

51
What Molecular Motors Do

• Interphase 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

52
Clinical Correlations of MT Motors

• Microtubule-directed drugs (paclitaxel,
  vincristine) stop mitosis, kill cancer cells
• Kartageners Syndrome Dynein (or Kinesin)
  mutations
• Situs Inversus
• Sterility
• Sinus Infections

53
The Mitotic Spindle
Know your PMAT!
54
Centrioles/Basal Bodies vs. Cilia

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

55
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

56
Cell Motility

• Microfilaments (actin fibers) form membranes
  structures such as microspikes and lamellopodia
• Microtubules are also necessary for cell movement
  and guidance.

57
Cytokinesis

• Microfilaments form a contractile ring, myosins
  cause the division between two daughter cells
  after telophase.

Structural Support

• Microvilli and Stereocilia Actin!
• Cilia -Microtubules

58
Clinical Correlations of Actin/Myosin

• Phallotoxin (phalloidin) binds and freezes
  F-actin
• Latrunculin disrupts actin organization
• Listeria and Shigella use actin to travel through
  the cell
• Usher Syndrome mutation in Myosin VII, hearing
  loss, retinitis pigmentosa
• Griscelli Syndrome Myosin V deficiency
  albinism

59
Blood
60
Lymphocytes

• T-cell- Cell-mediated immunity,
• Helper-T and Cytotoxic-t
• Mature in the Thymus
• B-cell Humoral immunity
• Mature in Bone Marrow
• Release IgG in extravascular tissue

61
Granulocytes

• PMN
• Neutrophil, Multi-Lobed Nucleus, 10-12um
• Granules
• Specific- Lysozyme, collagenase, etc.
  Azurophilic- MPO, Defensins
• Monocyte
• Macrophage precursor
• Kidney Bean Nucleus, 10-20 um
• Lysosomal Granules
• Eosinophil
• Bilobed Nucleus, Red Granules, 10-12 um
• Basophil inhibitor
• Histaminase, Aryl Sulfatase
• Basophil
• Bilobed Nucleus, Blue Granules, 10-12 um
• Vasoactive Traits
• 1.Bind IgE
• 2. Secrete histamine and SRS (slow releasing
  substance of anaphylaxis)
• 3. Heparin sulfate is an anti-coagulant

62
Platelets

• 2-5 um, anucleate
• 2-400,000/ mL blood
• Granules
• Alpha- fibrinogen, coag. Factors
• Delta- serotonin, ADP, histamine
• Know Clotting Cascade
• Thromboplastin activates Thrombin
• Thrombin activates Fibrin
• tPA or pPA activates Plasmin
• Plasmin breaks the clot

63
Erythrocytes

• Anucleate, 7.5 um
• 4-5 million/ mL blood
• Know Hb, Spectrin, Glycophorin C, Band 3
• Basis of Blood Groups
• Life Span 120d.

64
Population

• Never
• Let
• Monkeys
• Eat
• Bananas
• Above is in the order of population

WBCs
65
(No Transcript)
66
(No Transcript)
67
(No Transcript)
68
(No Transcript)
69
(No Transcript)
70
(No Transcript)
71
(No Transcript)
72
(No Transcript)
73
(No Transcript)
74
(No Transcript)
75
(No Transcript)