8/13/2019 Histology-1_2006-1

1/73

Histo Review 1 2004

8/13/2019 Histology-1_2006-1

2/73

Your Test

Monday, 9/20 1:30 30questions from Microscopy to Cell

Bio

~10Image-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 inthe lectures, notes posted on Bb and

in CH 1,2 in the text.

Lecture notes and handouts!

8/13/2019 Histology-1_2006-1

3/73

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

8/13/2019 Histology-1_2006-1

4/73

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 adye based upon high concentration of thatdyes ligand in a cell e.g. toluidine Blue stains mast cell granules

purple- high [heparin sulfate]

8/13/2019 Histology-1_2006-1

5/73

H and E Stain

H: Hematoxylin, basic

dye, stains acidic groups

(Heterochromatin,

Glycosaminoglycans)

blue.

E: Eosin, acidic dye.

Stains proteins red.

8/13/2019 Histology-1_2006-1

6/73

PAS (periodic acid-Schiff) Stain

Stains reducing sugars red.

(Cleaves Aldehyde Grps)

Stains Glycogen, Mucus,

Basement Membrane and

Reticular Fibers

PAS Reaction:

- Periodic Acid cleavessugars into aldehyde

groups. Aldehydes react with

Schiff Reagent- RED

Feulgen Reaction:

- DNA (not RNA) is cleaved

by HCl, reacts w/Schiff.

8/13/2019 Histology-1_2006-1

7/73

Silver Stain

Stains Reticular Fibers and

Basement Membrane Black.

8/13/2019 Histology-1_2006-1

8/73

Immunohistochemistry

Enzyme-linked

antibodies

Targets specific

proteins associatedwith disease

Useful for diagnosis

Example: oral tumor

(condyloma) biopsy

tests positive forHuman Papilloma

VirusHPV+

8/13/2019 Histology-1_2006-1

9/73

Freeze Fracture

E

P

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.

8/13/2019 Histology-1_2006-1

10/73

Nucleus

Chromatin

Nucleolus

Envelope/Matrix

8/13/2019 Histology-1_2006-1

11/73

Chromatin

marginalkaryosome

4 nm

Orders of Chromatin Organization: Nucleosomefundamental packing unit = linker DNA +

nucleosome bead (2 whorls of DNA + histones [4] + one otherhistone H1) -beads-on-a-string form

30 nm chromatin fiber

loops

clusters of looped domains

chromosome

Amount of Euchromatin =

Transcriptional Activity of the Cell!

http://cellbio.utmb.edu/_vti_bin/shtml.dll/CELLBIO/NUCLEUS2.HTM/map

8/13/2019 Histology-1_2006-1

12/73

8/13/2019 Histology-1_2006-1

13/73

Nuclear envelope:

Separates RNA synthesis fromRNA processing; preventsdamage from cytoskeleton

Remember that nuclear outermembrane is contiguous withrough ER!

Nuclear pore complex:

Composed ofnucleoporins Allow small molecule entry by

diffusion; large proteins,however, require importin,exportin (and both ATP and

GTP)

8/13/2019 Histology-1_2006-1

14/73

Cell Surface Structures/

Membrane Proteins Plasma Membrane

Lipid Rafts/Caveolae

Membrane Proteins

Junctions, Ion Channels

8/13/2019 Histology-1_2006-1

15/73

Plasma Membrane Components

Outer leaflet:

SM, PC

Inner Leaflet: PS, PI, PEtn

8/13/2019 Histology-1_2006-1

16/73

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

8/13/2019 Histology-1_2006-1

17/73

Caveolae vs. Clathrin Coated Pits

8/13/2019 Histology-1_2006-1

18/73

Clathrin

8/13/2019 Histology-1_2006-1

19/73

Clathrin-Coated

Pits/Vesicles Important for

Receptor-Mediated

Endocytosis Lysosomal enzyme

targeting

M6P receptor

Secretory Vesicle

Formation

8/13/2019 Histology-1_2006-1

20/73

8/13/2019 Histology-1_2006-1

21/73

Ion Channel Mutations/Diseases

Myasthenia GravisMuscle weakness

due to autoantibodies against the

acetylcholine receptor Cystic FibrosisDefect in the Cl-

channel CFTRleads to excessive

phlegm and static infections

8/13/2019 Histology-1_2006-1

22/73

Glycocalyx Made up of Glycoproteins, Proteoglycans, and Glycolipids

Remember that most sugars are on the outside of the cell.

Membrane Proteins

Integralhave transmembrane domains Peripheralhave noncovalent attachment to the

membrane or an integral protein

Lipid-anchoredCovalently bonded to either a

phospholipid or a fatty acid (farnesyl, GPI, etc.)

8/13/2019 Histology-1_2006-1

23/73

Erythrocyte Membrane Skeleton

Spectrin Filamentsattach to b-actin

junctional complexes

b-Actin bindsGlycophorin C

Spectrin is held to themembrane by Ankyrin,Band 3 proteins

HereditarySpherocytosis: Defectin one or more of theseproteins

8/13/2019 Histology-1_2006-1

24/73

Dystrophin and DMD in Muscle

Cells

Lack of functional dystrophin leads toDuchennes Musc. Dystrophy (DMD)

Muscle weakening, pseudohypertrophy

dystroglycans

8/13/2019 Histology-1_2006-1

25/73

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 integrinsfound on leukocytes

avb3found on endothelial cells, smcs, plts Found in focal adhesions (with vinculin, actin) and hemidesmosomes (interm.

fil., plectin).

8/13/2019 Histology-1_2006-1

26/73

8/13/2019 Histology-1_2006-1

27/73

Junctional Complex

8/13/2019 Histology-1_2006-1

28/73

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

8/13/2019 Histology-1_2006-1

29/73

Desmosome

http://web.mit.edu/7.19/www/lecture7/JPEGS/Desmo.jpg

8/13/2019 Histology-1_2006-1

30/73

Gap JunctionsOne Connexon

connects to a

connexon in

another cell.

Each connexon

is made of 6

connexin

subunits.

Gap junctionsallow the

selective

passage of ions

and small

molecules.

8/13/2019 Histology-1_2006-1

31/73

Know the chart

on page 13!

Tight junction

Prevents

intercellular

transport!

Integrins

Gap Junctions

Connexin vs.

connexon

Structure of microvillivs. stereocilia vs. cilia

vs. basal body vs.

centriole!

8/13/2019 Histology-1_2006-1

32/73

The Cytoskeleton

8/13/2019 Histology-1_2006-1

33/73

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

8/13/2019 Histology-1_2006-1

34/73

Microtubules

Each fiber is a hollow cylinder

Microtubules have polarity: a positive,fast-growing end and a slow-growingnegative end

Soluble tubulin dimers bind end-to-end, alpha- to beta-

Polymerization is dependent on GTPhydrolysis

Colchecine, vincristine and otheralkaloids inhibit binding

Associated proteins:

Motor proteins: kinesin and dynein

8/13/2019 Histology-1_2006-1

35/73

8/13/2019 Histology-1_2006-1

36/73

Types of Intermediate filaments

Types I and II: Acidic Keratinand Basic Keratin, respectively. Produced by different types of epithelial cells (bladder, skin, etc).

Epidermolysis Bullosakeratin deficiency- blistering diseases

Type III Intermediate filaments are distributed in a number of cell

types, including:

Vimentinin fibroblasts, endothelial cells and leukocytes; desmininmuscle; 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 celldivision.

8/13/2019 Histology-1_2006-1

37/73

8/13/2019 Histology-1_2006-1

38/73

Cell Motors, Motility, and

Mitosis

8/13/2019 Histology-1_2006-1

39/73

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.

8/13/2019 Histology-1_2006-1

40/73

Dynein

Found in cilia/flagellacause sliding of MTs gives

beating motion

Dynactinlinker between Dynein and other structures

(centrosomes, actin, et al.)

Kinesin

Kinesin Iused in cells to transport membrane-bound organelles along microtubules. (+) directed

Some Kinesin Related Proteins move cilia, organizemicrotubules, or bind DNA directly (chromokinesin)

8/13/2019 Histology-1_2006-1

41/73

What Molecular Motors Do

Movement of organelles/vesicles from one part of thecell to another (e.g. from ER to Golgi)

Cell Polarity: Bring different proteins to differentsides of cells (axon vs. dendrite, apical vs.basolateral)

Flagellar/Ciliary function, maintenance

Mitosis/Meiosis

8/13/2019 Histology-1_2006-1

42/73

The Mitotic Spindle

Know your PMAT!

8/13/2019 Histology-1_2006-1

43/73

Clinical Correlations of MT

Motors Microtubule-directed drugs (paclitaxel

(Taxol), vincristine)stop mitosis, kill cancercells

Kartageners Syndrome Dynein(or Kinesin)mutations Situs Inversus

Sterility in males

Sinus Infections Lissencephaly- dynein deficiency leading tosevere brain developmental deficiencies

8/13/2019 Histology-1_2006-1

44/73

Centrioles/Basal Bodies vs. Cilia

Cilia/Flagella: 9*2 +

2 Arrangement

Centrioles/BasalBodies: 9*3

8/13/2019 Histology-1_2006-1

45/73

Molecular Motors

8/13/2019 Histology-1_2006-1

46/73

MyosinsActin Motors

Many types, heavy chain is conserved.

Myosin I- interacts with membranes,

important for endocytosis, inner ear function Myosin IIfound in many types of cells,

regulates cell contraction, locomotion,

cytokinesis.

Myosin Vfunctions in delivery of vesicles to

membrane

8/13/2019 Histology-1_2006-1

47/73

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 aend

similar to MTs.

8/13/2019 Histology-1_2006-1

48/73

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 Syndromemutation in Myosin VII, hearing loss, retinitispigmentosa (deaf/blind)

Griscelli SyndromeMyosin V deficiencyalbinism

http://www.nifg.org.uk/species/photos/ri.htm

8/13/2019 Histology-1_2006-1

49/73

Endomembrane System

ER

Golgi

Lysosomes

8/13/2019 Histology-1_2006-1

50/73

Smooth ER

-Steroid Production

-Detoxification/ DrugMetabolism

-Connected to rER

8/13/2019 Histology-1_2006-1

51/73

rER

Interconnected

tubules, vesicles

and sacs

Associates withribosomes,

Protein synthesis

http://cellbio.utmb.edu/cellbio/cit2.htm

8/13/2019 Histology-1_2006-1

52/73

ER, signal sequence, protein translation Hydrophobic sequence targets ribosome to ER

SRP: signal recognition peptidebinds 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 ERmembrane

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

8/13/2019 Histology-1_2006-1

53/73

8/13/2019 Histology-1_2006-1

54/73

Quality control: ubiquitin-proteasome pathway

8/13/2019 Histology-1_2006-1

55/73

Protein Synthesis/ Signal Sequences

P t i difi ti

8/13/2019 Histology-1_2006-1

56/73

Protein modification

Co- vs. Post-translational Golgi is post-, ER is co-

translational

Golgi is functionallycompartmentalized;each cisternae containscertain enzymes thatcan modify proteins inspecific ways Glycosylation,

phosphorylation,sulfation

Proteolytic modification

Glycolipid synthesis

Sorting of vesicles;clathrin-coated

pits/adaptors

G

8/13/2019 Histology-1_2006-1

57/73

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

trans ort

8/13/2019 Histology-1_2006-1

58/73

Exocytosis

Vesicles fuse with outer plasma

membrane

8/13/2019 Histology-1_2006-1

59/73

Lysosomal Targeting

*- KFERQ

sequence is a

destruction

signal for

senescentorganelles

Clathrin

8/13/2019 Histology-1_2006-1

60/73

Lysosomes

8/13/2019 Histology-1_2006-1

61/73

Tay-Sachs Disease

8/13/2019 Histology-1_2006-1

62/73

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 inclusionsin non-humans Zellweger Syndrome: early death due to non-

functional peroxisomes.

8/13/2019 Histology-1_2006-1

63/73

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,F1F0ATP synthase are in theinner membrane

Matrix is within the innermembrane, houses the Krebs

cycle Mitochondria have their own DNA,

ribosomes, division process

8/13/2019 Histology-1_2006-1

64/73

Mitochondria and

Apoptosis Opening of PTP

(permeability transitionpore) leads to

Cytochrome C escapefrom mito

Cyt C activates Apaf-1,which activates the

Caspase Cascade Intracellular proteases

degrade cellularcomponents

8/13/2019 Histology-1_2006-1

65/73

Electron Micrographs

8/13/2019 Histology-1_2006-1

66/73

8/13/2019 Histology-1_2006-1

67/73

8/13/2019 Histology-1_2006-1

68/73

http://imc.gsm.com/integrated/maonline/maonline/ma/picts/20000/1000/200/21251.jpghttp://imc.gsm.com/integrated/maonline/maonline/ma/picts/20000/1000/200/21251.jpg

8/13/2019 Histology-1_2006-1

69/73

8/13/2019 Histology-1_2006-1

70/73

8/13/2019 Histology-1_2006-1

71/73

8/13/2019 Histology-1_2006-1

72/73

http://137.222.110.150/restricted/gallery/album94/junctional_complex_cuboidal_cell

8/13/2019 Histology-1_2006-1

73/73

http://137.222.110.150/restricted/gallery/album94/junctional_complex_cuboidal_cell