Histology unit 1 objectives

what is the purpose of histological staining?

staining is done to visualize cellular components / micro anatomy of a cell

What are characteristics of hemotoxylin staining? (purple)

• positive, basic

• Binds (-) charged molecules

  • nuclei, histones

What are characteristics of Eosin staining? (Pink)

• negative, acidic

• binds (+) charged molecules

  • cytoplasm, cell membrane

What does cresyl violet stain do?

basic dye, stains neuron cell bodies (rER in cell body of nucleus)

what does Fuelgen reaction stain do?

stains DNA hydrolysis, but NOT RNA sugar (ribose)

useful for DNA quantification/localization

What does Giesma stain do?

Stains blood w/eosin, azure, + methylene blue

• similar to Wright’s stain

• classic lymphoma stainers

What does Gomori stain do?

Extremely broad, stains tons of stuff

• group of different histochemical techniques

• Example: the Gomori trichrome (mGT) uses multiple dyes to differentiate muscle fibers, connective tissue, and nuclei in tissue sections. An alternative method is called the Gomori methenamine silver (GMS), Gomori aldehyde fuchsin or Gomori reticulin stains.

What is H+E staining consist of?

Basophilic (blue/purple) + Acidophilic (pink) staining

• used in combination with metal ions (mordent - aluminum or iron NOT SILVER)

What is the application for Mallory staining?

Viewing connective tissue

it contains aniline blue, Orange G, and azocarmine (or acid fuchsin)

What is characteristic of MASON trichrome staining?

2+ dyes of different colors

acid fuchsin, orange G, and light green

nuclei appear BLACK

What is characteristic of orcein staining?

viewing elastic fibers

natural dye obtained from lichens

What is characteristic of period acid-schiff staining

viewing of proteins and glycogen/glycoproteins

What is characteristic of Toluidine blue?

basic stain that binds to nucleic acids, DNA & RNA

What is characteristic of wright blood stain?

Eosin + methylene blue to differentiate blood cell types

How does conventional histologic staining differ from immunohistochemistry as far as understanding and identifying molecules?

conventional staining looks at different types of molecules, while IMMUNOHISTOCHEMISTRY looks at specific molecules (usually a protein of RNA fragment)

What is the application for light microscopy?

cell-level visuals, is better for large visuals

What is the application for Transmission Electron Microscopy (TEM)?

Offers a higher resolution, finds smaller like proteins, organelle detail, etc.

electrons pass through ultra-thin specimens = image forms based on how electrons are scattered by atoms in a sample

What are characteristics of prokaryotic cells?

No true nucleus, organelles, or cytoskeleton

contain circular chromosomes (constructs nucleoid)

What are characteristics of Eukaryotic cells?

Think me and Eu-

Nucleus, organelles + cytoskeleton, nuclear membrane and nucleoli

linear chromosomes

How does compartmentation within eukaryotic cells contribute to cellular organization and function?

Compartmentation prevents mixing of biochemical reactions in the cell —> cells can have more specialized functions + grow to store molecules (hormones, neurotransmitters) for release

What are characteristics of passive diffusion?

moves easily through membrane directly, down concentration gradient

no energy required

What are characteristics of active transport?

requires energy

moves against concentration gradient

• symporters - carries in same directions, sodium-glucose co-transporter)

• antiporter - carries in opposite direction, sodium-potassium pump

What are characteristics of facilitated diffusion?

Moves easily via protein channels, as they are unable to dissolve in hydrophobic interior, down concentration gradient. Still passive

uniporters are passive

What are the functions of microfilaments?

• provide cytoskeletal support

• apical differentiations: projections (microvilli + stereo-cilia)

• movement (filapoda + lamellapoda)

What is the structure of microfilaments? What are they composed of?

Helical F-actin formation, made of G-actin monomers

What is the function of intermediate filaments?

stable support

What is the function of microtubules?

• mitotic spindle formation

• form cilia + flagella

what is the structure of Intermediate filaments? What are they made of?

end-to-end ULFs made of tetramers (8)

filamentous monomers

What is the structure of microtubules? What are they made of

Tube of protofilaments (13 in complete tubule)

alpha- and beta- tubulin

what is the role of B4-Thymosin in actin-binding proteins?

gathers g-actin (monomer) but does NOT add them

what is the role of Profilins in actin-binding proteins?

Facilitates addition (polymerization) via ATP-->ADP

what is the role of Cofilin (or ADF) in actin-binding proteins?

Depolarizes - removing individual monomer

what is the role of Gelosin in actin-binding proteins?

Caps F-actin preventing loss or addition of actin monomers. when Ca2+ present fragements F-actin and binds to - end, preventing further filament growth.

What is the role of microvilli and how is it differentiated?

• increases surface area for absorption

• Formins (w/ FH1 + FH2 domain) on the cap extend the filament

• Bundles of actin in the terminal web on cytoplasmic side to anchor filaments

• Unbranched, unlike stereocilia

What is the role of microvilli and how is it differentiated?

• long, branching projections

• cross-linked core of actin in bundles

• NO axoneme, unlike regular cilia

How does the structure and polarity of microtubules contribute to its organization and function?

• it is made of tubulin heterodimers

  • polymerized on alpha-tubulin + beta-tubulin bound together via GTP

• formed into rows called protofilaments

• 13 protofilaments make a hollow microtubule

Functions: intracellular transport, mechanical support, cell shape, flagella + cilia motility, and chromosome segregation

What contributes to stabilization in microtubules?

Microtubule associated proteins (MAPs) help stabilize by phosphorylating —> they preserve the GTP region + protect the tubule

1. classical MAPs

2. Non-classical MAPs

What are the functions of centrosomes?

1. nucleate (begin) the polymerization of tubulin into microtubules

2. organize microtubules into functional units (like mitotic spindle)

3. duplicate contriolar pair

4. become the bases for cilia (basal bodies)

Can microfilaments grow in different directions?

Yes

What are centrosomes?

organizers of microtubules (microtubule organizing center MTOC) - made of a pair of centrioles (mother+daughter)

MTOC- contains pericentriolar material (PCM) - where microtubules grow

how are centrioles organized?

organized in a 9 + 0 triplet arrangement

What are characteristics of “trichrome” staining?

2+ or more dyes (typically acidic)

to selectively color different basic tissue components within the same tissue section

many types of trichrome stains

What is direct IHC?

simpler, same host species can be used for multiple targets, there is no signal amplification. Not a sensitive, best for highly expressed antigens

What is INdirect IHC?

more complex, considered more sensitive because MULTIPLE secondary antibodies can bind to single primary antibody, thus amplifying the detection signal

• used to ensure you are fully able to visualize a color/fluorescent product when performing a staining procedure

What is immunohistochemistry (IHC) staining?

lab technique that enables the detection and visualization of specific antigens in tissue sections through the utilization of antibodies specific to those antigens/target proteins

What is this artifact (middle left)?

cracking artifact

What is this artifact (top left)?

stain precipitate

what is this artifact (top right)?

Separation (space) artifact

what is this artifact (middle right)?

Knife marks and folds

What is the artifact on the bottom?

folds

What is eukaryotic cell structure and function highly dependent on?

membranes and membrane-bound structures

What are peripheral proteins?

• not inserted into the hydrophobic interior of the membrane —> indirectly associated with membranes through protein-protein ionic bond interactions

  • these interactions can be disrupted by high salt or extreme pH

What are integral proteins?

• portions of integral proteins are inserted into the lipid bilayer and can only be released via solubilization using detergents

  • many are transmembrane proteins = span the entirety of the lipid bilayer, with segments exposed on both sides of the membrane

What prevents/hinders lateral movement of phospholipids and proteins?

can readily move in lateral direction, but is difficult to flip-flop from inner to outer membrane leaflets.

hindered by:

1. lipid rafts - proteins get trapped in them

2. attachment of membrane proteins to cytoskeletal and matrix proteins

3. cellular junctions - block lateral movement

Which cellular components exhibit polarity? (Microfilaments, intermediate filaments, and/or microtubules)

Microfilaments and Microtubules

What are the energy substrate(s) of Microfilaments, intermediate filaments, and microtubules?

ATPase (ATP/ADP)

NONE

GTPase

respecitvely

How are microfilaments assembled and disassembled?

tread milling

How are intermediate filaments assembled and disassembled?

Regulated by phosphorylation and dephosphorylation 

How are microtubules assembled and disassembled?

Dynamic Instability

What is treadmilling?

associated with microfilaments, G-actin monomers are assembled at one end (+) of the filament and concurrently disassembled at the other end (-)

what is dynamic instability?

associated with microtubules, switching of microtubules between states of persistent growth of GTP caps at the plus end and persistent unfolding at the GDP-rich minus end

3 steps:

1. polymerization phase

2. release of hydrolyzed phosphate (Pi)

3. Depolymerization phase

What is the transition from growth to shrinkage in dynamic instability called?

catastrophe

What is the transition from shrinkage to growth in dynamic instability called?

rescue (restore their length to about 20 um)

Which cellular component(s) exhibits stability? (Microfilaments, intermediate filaments, and/or microtubules)

Intermediate filaments are the only ones that exhibit stability

what is the cytochemical approach to localize DNA and RNA and evaluate nucleic acid synthesis within cells?

Fuelgen reaction stains DNA + RNA

what is the autoradiographic approach to localize DNA and RNA and evaluate nucleic acid synthesis within cells?

Radiographic precursors: radioactively labels DNA or RNA

[H3]thymadine vs [H3]uridine

how do Exportins regulate the directionality of macromolecule transport between the nucleus and cytoplasm?

Bring things out of the nucleus

how do Importins regulate the directionality of macromolecule transport between the nucleus and cytoplasm?

Bring things into the nucleus

how do NLS (nuclear localization signals) regulate the directionality of macromolecule transport between the nucleus and cytoplasm?

cargo proteins must be tagged w/this to go through pores

how do RAN-GTP regulate the directionality of macromolecule transport between the nucleus and cytoplasm?

Controls importins and exportins

How can small molecules diffuse through nuclear pore complexes?

small molecules can diffuse passively through the nuclear pore complex

How can proteins of ANY SIZE diffuse through nuclear pore complexes?

they must possess and NLS (nuclear localization amino acid sequence), they can be imported or exported into the cytoplasm through pore complex by an ENERGY-DEPENDENT mechanism requiring GTP

How is rRNA synthesized and processed within the nucleolus?

each gene has a Fibril: /cleaved/

each fibril= 45s rRNA // 18s rRNA // 28s rRNA // 5.8s rRNA

How does processed rRNAs contribute to the formation of small and large ribosomal subunits?

rRNA genes repeat, look like Xmas trees separated by spacers; each gene has a fibril

acts as the physical core and structural scaffolding that bind with ribosomal proteins to assemble the small and large ribosomal subunits

How does the Fibrillar Center of the nucleolus contribute to rRNA synthesis, processing, and ribosomal subunit assembly?

location of RNA pol I + SRP-RNA

How does the Fibrillar Component of the nucleolus contribute to rRNA synthesis, processing, and ribosomal subunit assembly?

Where nascent rRNA is present and undergoing processing (fibrillarin and nucleolin are found in the fibrillar dense component)

How does the Granular Component of the nucleolus contribute to rRNA synthesis, processing, and ribosomal subunit assembly?

where 18s rRNA + 28s RNA subunits are assembled/completed

DNA accessibility and transcriptional activity of less packed chromatin?

Transcription, S phase

DNA accessibility and transcriptional activity of more packed chromatin?

Inaccessible, cell division

what is the basic unit of chromatin condensation?

nucleosome: formed by 200 bp or 1.5 turns of double stranded DNA wrapped around a histone octamer core

What consists the histone octamer core?

composed of two molecules each of histone proteins H2A, H2B, H3, and H4

stabilized by linker histone (H1) protein cross-linking with DNA molecule which is wrapped around the octamer

What is the primary structure of chromatin characterized by?

(10 nm)consists of: nucleosomes + linker DNA

“beads on a string” appearance (nucleosome-nucleosome or fiber-fiber interactions) these allow for higher degrees of chromosomal condensation

What is the secondary structure of chromatin characterized by?

coils into 30 nm chromatin fiber: also stabilized by H1

fiber-fiber interactions result in coiling of chromatin

What is the Tertiary structure of chromatin characterized by?

further folding/coiling of secondary chromatin, also stabilized by H1, characteristic of condensed chromosomes seen during metaphase

gene transcription CAN OCCUR when chromatin is arranged in tertiary structure

Define euchromatin

diffuse chromatin; open for transcription - 10% of chromatin

electron lucent, stains light

Define heterochromatin

dense chromatin; transcriptionally inactive - 90% of chromatin

stains dark, electron-dense

what is the role of nuclear lamins?

Type V intermediate filaments: A1, B1, B2, + C

1. organize chromatin

2. space nuclear pores out

3. reassemble nucleus after cell division

what inner nuclear membrane proteins do nuclear lamins bind with?

1. LBR

2. Emerin

3. LAP1c

4. LAP 1 B

what is the role of the nuclear envelope?

enclosed + regulates nucleus

What is the role of chromatin?

DNA packaged w/ proteins

What is the role of nucleolus?

Produces rRNA + assembles ribosomes

How does the endomembrane system facilitate the compartmentalization, modification, packaging, and transport of cellular materials?

it separates the various processes + reactions into specific locations in the cell via membrane-bound organelles

What is the morphological characteristics, predominant functions, and tissue locations of simple squamous epithelium?

flat round nucleus; Barrier that allows for diffusion

alveoli, blood vessel lining, bowman’s capsule (kidney), serous

What is the morphological characteristics, predominant functions, and tissue locations of simple cuboidal?

round/square cell body: secretion

ducts and glands, PCT and DCT (kidney)

What is the morphological characteristics, predominant functions, and tissue locations of simple columnar?

long rectangular cell body; absorption

villi in the intestines

what are zonula adherens?

*belt desmosomes

forms circumferential belt around apical domain; associated w/ microfilaments via catenin complex to cytoplasmic desmosome cadherins

What are macula adherens?

*spot desmosomes

cytoplasmic plaques liek plakoglobin bind desmosome cadherins

What are hemidesmosomes?

link intermediate filaments to basal lamina

mode of:

inner cytoplasmic plate bound to tonofilaments

outer membrane plaque bound via laminins + integrins

What is the basal lamina?

Top layer of basal membrane; made of laminin, collagen, and proteoglycans

What is the reticular lamina?

the bottom layer of the basement membrane: made of Type III collagen