BMS550 - Histology (Quiz 1)

Methods of Study in Histology

1. Fixation: Small pieces of tissue are placed in solutions of chemicals that cross-link proteins and inactivate degradative enzymes → preserve cell and tissue structure

2. Dehydration: tissue is transferred through a series of increasingly concentrated alcohol solutions, ending in 100% → removes all water

3. Clearing: alcohol is removed in organic solvents in which both alcohol and paraffin are miscible

4. Infiltration: tissue is placed in melted paraffin until it becomes completely infiltrated with this substance

5. Embedding: paraffin-infiltrated tissue is placed in a small mold with melted paraffin and allowed to harden

6. Trimming: resulting paraffin block is trimmed to expose tissue for sectioning (slicing) on a microtome

Staining Techniques

Dyes make cells and tissues, and their components easier to distinguish

• React with chemical components of the structure being stained

• Reaction is more or less selective

Basic dyes (+ve charged)

• Stain cell and tissue components with net negative charges (anionic)

• Example: Nucleic acids & basophils → basophilic

• Hematoxylin (most common)

• Toluidine blue

• Alcian blue

• Methylene blue

Acidic dyes (–ve charged)

• Stain cell and tissue components with net positive charges (cationic)

• Example: Proteins with ionized amino groups → acidophilic

• Eosin

• Orange G

• Acid fuchsin

Most common acid-basic dye

Hematoxylin (basic) & Eosin (Acid)

Hematoxylin

• Basic dye

• Stains cell/tissue components with net negative charges (anionic)

• Stain purple

• Affinity with basic dyes → basophilic

Cell structures with acids in their composition stain well with hematoxylin

• Nucleic acids (DNA in nucleus)

• Glycosaminoglycans → ground substance of CT

Eosin

• Acidic dye

• Stains cell/tissue components with net positive charges (cationic)

• Stain pink

• Affinity with acidic dyes → acidophilic

Periodic acid–Schiff (PAS) reaction

• Stains carbohydrate-rich tissue and cellular components

• Color: dark purple or magenta

• Goblet cells (in small intestine) → secretory granules rich in carbs = PAS positive

Sudan Black

• Lipid–soluble dye

• Stains cells and tissue structures rich in lipids → black

• E.g., adipose cells

Electron vs Light Microscope

• More resolution (~3 nm): minimal distance between 2 objects to distinguish them

• Higher magnification → up to 400,000×

• Provides more detail

• Reveals ultrastructure of cell (e.g., organelles)

Cell

Basic structural and functional unit of any living thing

Cell Theory

1. All living things are made of one or more cells

2. All cells come from existing cells

3. Cells are the basic building blocks of life

Prokaryotic Cells (Bacteria, Archaea)

• Unicellular organisms

• Never form tissues

• No nucleus

• No organelles → only ribosomes

• Cell wall

• Capsule

Eukaryotic Cells (Nucleus enclosed in membranes)

• Unicellular and multicellular organisms

• Form tissues (in multicellular organisms)

• Have nucleus

• Have organelles

• Some have cell wall

Human cells

• Eukaryotic cells

• No cell wall

• Form human tissues

• Undergo differentiation

  • Specialization

  • Diversity

• Have nucleus

• Have organelles

Cell Differentiation

• Interaction between genes and cellular microenvironment → different genetic expression → leads to different structures

• One or more functions become more important: other basic functions still maintained (e.g., protein synthesis)

• Specific function(s) supported by different morphology → structure determines function

• Great morphological (structural) and functional cellular diversity

Cell Differentiation Pathway

Zygote → blastomeres (blastula) → differentiated (mature) cells → form cells of all human tissues

Outcomes of Cell Differentiation

1. Different appearance & structure

2. Functional specialization

Nucleus

• Command Center of the Cell

• Membrane-bound, highly specialized organelle

• Serves as information processing & administrative center of a eukaryotic cell

Functions of the Nucleus

1. Stores genetic (hereditary) material

2. Coordinates all cell’s activities

   • Growth

   • Metabolism

   • Protein synthesis

   • Cell division

3. Molecular machinery for

   • DNA synthesis & RNA synthesis

   • RNA processing: intron splicing, 5′ cap, 3′ poly-A tail

Components of the Nucleus

1. Nuclear envelope: pore riddled

2. Nucleoplasm: fluid

3. Chromatin: DNA + proteins (histones)

4. Nucleolus: dense cluster of RNA and proteins + inactive ribosomes

Nuclear envelope

• Formed by 2 membranes with pores

  • Outer membrane + perinuclear space are continuous with rER

• Selectively permeable barrier

• Separates nucleoplasm from cytoplasm

• Allows selective passage of molecules

  • Small lipophilic / hydrophobic molecules pass freely

  • Large hydrophilic molecules cross via nuclear pores

• Nuclear lamina stabilizes the envelope: meshwork of proteins closely associated with inner membrane

• Intermediate filament proteins (lamins) bound to membrane proteins

  • Lamins associated with chromatin in non-dividing cells

Components of the Nuclear Envelope

1. Inner membrane → associated with nuclear lamina

2. Outer membrane → binds ribosomes

3. Perinuclear space (cisterna) → lies between membranes

Nuclear Pores

• Nuclear pore complexes bridge inner & outer membranes

• Made of nucleoporins (core proteins)

• Show eightfold symmetry around a central lumen

Functions of Nuclear Pores

1. Allow passage of ions & small molecules by simple diffusion

2. Regulate movement of macromolecules between nucleoplasm & cytoplasm

Nucleoplasm

• AKA Karyoplasm

• Viscous fluid within the nucleus containing water, dissolved ions, and a complex mixture of other molecules

• Function: suspension medium for other nuclear components

Chromatin

• Components: DNA + associated proteins (histones)

• Divided among 46 chromosomes (23 pairs)

  • Gametes → 23 chromosomes

  • Homologous chromosomes = 1 pair (sperm + ovum)

Human DNA

• ~2 meters long per nucleus

• ~3.2 billion base pairs

Histones

• Basic proteins

• Histones + DNA form structural units called nucleosomes

Structure of Nucleosomes

• Core = 8 histones (2 copies each of H2A, H2B, H3, H4)

• 150 bp of DNA wrapped around core

• H1 histone associated with DNA at surface of core

• Linker DNA (50–80 bp) separates nucleosomes

Nucleosomes

• Dynamic structures

• Key to DNA replication and transcription

  • Histone modification

  • Histone rearrangement

Chromatin Folding

• DNA double helix → nucleosomes (DNA + histones) → fibers of packed nucleosomes → larger loops of coiled DNA

• Some unstable → transcriptional activity

• Many loops tethered to condensins (protein complexes)

• Occur during interphase

Euchromatin

• Decondensed

• Transcriptionally active

• Light microscope: slightly basophilic areas

• TEM: dispersed fine granules

• Predominates in metabolically active cells

Heterochromatin

• Condensed

• Transcriptionally inactive (or less active)

• Light microscope: intensely basophilic clumps

• TEM: coarse, electron-dense material

• Predominates in cells with little or no metabolic activity

Chromosomes

• Represent maximum coiling of DNA

• Visible during mitosis & meiosis only

Somatic Cells (Diploid – 2n)

• 22 pairs of autosomes + 1 pair of sex chromosomes

• Total = 46 chromosomes

Gametes (Haploid – n)

• 22 autosomes + 1 sex chromosome

• Sperm: either X or Y

• Oocytes: only X

Nucleolus

• Highly basophilic chromatin region (due to rRNA, –ve charge)

• Prominent in protein-synthesizing cells (more than one nucleolus possible)

• Dense concentration of rRNA

Ribosome Production

• Transcription of rRNA molecules

• rRNAs associate with ribosomal proteins

• Assembly of ribosomal subunits

Cell Cycle

• Produces new cells

• Four phases:

  1. Mitosis (M phase): cell division

  2. G1 phase: cell growth & normal functions

  3. S phase: DNA replication

  4. G2 phase: preparation for mitosis

Interphase

90% of cell cycle

• G1

  • Usually the longest phase

  • RNA & protein synthesis

  • Cells recover size (after mitosis)

• S (Synthesis)

  • DNA replication (46 chromosomes duplicated)

  • Histone synthesis

  • Beginning of centrosome duplication

• G2 (Gap 2)

  • Relatively short

  • Accumulation of proteins required for mitosis

  • General preparation for mitosis

Cell division (Mitosis)

10% of cell cycle

• G0 Phase

  • Quiescent state

  • Cellular differentiation

  • Cell cycle suspended → may or may not restart

  • Only occurs in nucleated cells

Mitosis

• All somatic cells

• Parent cell divides: two identical daughter cells (clones) → same DNA

• 4 main phases:

  1. Prophase

  2. Metaphase

  3. Anaphase

  4. Telophase

Prophase

1. Nucleolus disappears

2. Chromatin condenses → chromosomes

3. Mitotic spindle forms: centrosomes migrate to opposed poles

4. Nuclear envelope vanishes: phosphorylation of lamins and inner nuclear

membrane proteins → nuclear lamina and nuclear pores disassemble

Metaphase

1. Chromosomes further condense

2. Kinetochore proteins attach to chromosome center

3. Cell is more spherical

4. Chromosomes align in the equator of the cell (metaphase plate)

Anaphase

1. Sister chromatids separate → migrate to opposite poles

2. Each sister chromatid is now a single chromosome = daughter chromosome

3. This stage prepares the genetic material to be apportioned between two future daughter cells

Telophase

• 2 sets of chromosomes (1 at each spindle pole)

• Chromosomes decondense back to uncondensed state

• Spindle microtubules depolymerize

• Nuclear envelope reassembles around each set of daughter chromosomes (→ 2 nuclear envelopes form)

• Actin & myosin filaments form belt at equator

• Cytokinesis begins (end of telophase)

• Constriction of ring → cleavage furrow → results in two daughter cells

Stem cells

• Undifferentiated cells

• Renewal of differentiated cells as needed

• Divide infrequently

• Divisions are asymmetric

  • One remains as stem cell

  • One becomes a progenitor cell → committed to differentiation

Locations of stem cells

• In many tissues:

  • Bone marrow

  • Skin

  • Mucosa of digestive & respiratory tracts

• In specific niches: microenvironment helps maintain undifferentiated state

Progenitor cells

• AKA transit amplifying cells: intermediate stage (from undifferentiated → terminally differentiated cells)

• Frequent mitosis → increases number of new cells

• Terminally differentiated cells

  • Low or no potential for further division

  • Renewal depends on stem cells

  • Some cells in G0 may reenter the cycle (e.g., hepatocytes – liver can renew)

Meiosis

• Specialized process

• 2 unique cell divisions

• Only in germ cells (cells that will become sperm or oocytes – gametes)

Characteristics of Meiosis

1. Crossovers between homologous chromosomes

• Homologous chromosomes come together → synapsis

• DNA undergoes double-stranded breaks & repairs

• Reciprocal DNA exchanges → crossover / DNA recombination

2. Formation of four haploid cells: each cell has one chromosome from each pair

3. Fertilization: 2 haploid cells (egg + sperm) unite → forms a zygote (diploid undifferentiated cell)

Prophase I

• Longer than in mitosis

• Same basic events as mitosis, plus unique steps:

  • Homologous chromosomes form synapsis → tetrads (4 copies of each genetic sequence)

  • Crossover / recombination occurs

• Human Spermatogenesis: prophase I lasts ~3 weeks

• Human Oogenesis: oocytes arrest in Prophase I for 12+ years

Metaphase I

• Random arrangement of homologous chromosomes at metaphase plate

• Leads to:

  • Two possible chromosome arrangements

  • Four possible genetic arrangements

Anaphase I

• Homologous chromosomes separate

• Migrate to opposed poles

Meiosis I

• Telophase I and Cytokinesis are same as in mitosis

• Result:

  1. Haploid cells (n)

  2. Diploid chromosomes (each chromosome retain two sister chromatids)

     • Parental cells = diploid (2n = 46)

     • Daughter cells = haploid (n = 23)

Meiosis II

Same as mitosis in sequence of events

1. Prophase II → very fast, almost absent

2. Metaphase II → chromosomes align

3. Anaphase II → sister chromatids separate → become daughter chromosomes

4. Telophase II

5. Cytokinesis

• Result of Meiosis II: 4 haploid daughter cells → each with haploid chromosomes (DNA)

Cytoplasm

Cellular compartment between nuclear envelope and cell membrane

Contents of the Cytoplasm

• Cytosol

  • Hundreds of enzymes

  • Oxygen → respiration in mitochondria

  • Carbon dioxide

  • Ions

  • Substrates

  • Metabolites & waste products

• Organelles

• Inclusions: not enclosed in membranes

Membranous Organelles

• Plasma membrane

• RER

• SER

• Golgi apparatus

• Lysosomes

• Mitochondria

• Peroxisomes

Non-membranous Organelles

• Microtubules

• Filaments

• Centrioles

• Ribosomes

• Proteosomes → degrade non-functional proteins

What forms centrioles?

Microtubules

Site of protein synthesis

Ribosomes

Ribosomes

• Composition: 60% rRNA + 40% proteins

• Two subunits: made in nucleolus

  • Small subunit (40S): highly folded rRNA chain + 30+ proteins

  • Large subunit (60S): three rRNA molecules + ~50 proteins

• Active ribosome: two subunits assembled

Ribosomes in a Light Microscope

• Cytoplasmic basophilia = stain purple → due to rRNA of ribosomes (-ve charge)

• Observed in actively synthesizing cells

Polyribosomes

Many ribosomes bound to a single mRNA during protein synthesis

Endoplasmic Reticulum

• Convoluted membranous network in cytoplasm

• Extends from nucleus throughout cytoplasm

• Cisternae = interconnected channels of ER

• Membrane surface ~30× plasma membrane

• Site of major metabolic activities: lipid & protein synthesis

• Two types: Rough ER & Smooth ER

Functions of the ER

1. Synthesis

   • Smooth ER → lipid synthesis + carbohydrate metabolism

   • Rough ER → protein synthesis (for secretion, plasma membrane, lysosomes)

2. Transport: moves molecules through cisternal space from one part of the cell to another

3. Storage: stores newly synthesized molecules

4. Detoxification: smooth ER detoxifies drugs and alcohol

Where do lysosomes originate from?

Golgi apparatus

Rough ER

• Membranes form flat cisternae

• Continuous with outer nuclear membrane

• Functions:

  • Protein synthesis e.g., enzymes of lysosomes

  • Glycosylation + some post-translational modifications

  • Assembly of multichain proteins

Smooth ER

• Membranes form tubular or sac-like cisternae with no ribosomes

• Functions (depend on cellular specialization)

  • Synthesis of phospholipids & steroids (Leydig cells, adrenal cortex → testosterone, steroid hormones)

  • Detoxification of harmful exogenous molecules (alcohol, barbiturates → liver cells)

  • Calcium sequestration & release (sarcoplasmic reticulum in skeletal muscle cells)

What are the 3 locations that proteins from the RER are transported to?

• Plasma membrane

• Membranous organelles (golgi apparatus)

• Secretion via exocytosis

RER staining

Cells with well-developed rER → cytoplasmic basophilia (purple staining due to rRNA of ribosomes)

SER staining

• Cells with well-developed sER → negative image / pale zone

• sER does not stain with H&E

Golgi Apparatus

• Structure: membranous vesicular/flattened saccules

• 3 functional regions with different sets of enzymes

  1. Cis face: Receiving region

  2. Middle face

  3. Trans face: Shipping region

• Does not stain well with H&E

Where is the Golgi Apparatus located?

Close to cell nucleus

Functions of the Golgi Apparatus

• Terminal site of post-translational modification of RER-synthesized proteins

• Sorting, packaging & distribution of proteins from RER

Cis Face of the Golgi Apparatus

• Adds mannose-6-phosphate → lysosomal enzymes

• Trims N-linked oligosaccharides & adds other sugars

Medial Face of the Golgi Apparatus

• Glycoslyation on -OH groups of lipids and O-linked Ser/Thr residues

• Further modification of N-linked oligosaccharides on proteins

• Sorting of glycoproteins & glycolipids into specific vesicles

Trans Face of the Golgi Apparatus

• Adds sialic acid as terminal sugar to some oligosacch.

• Sulfation of tyrosine & some sugars

• Sorting & separation of vesicles for different destinations

Lysosomes

• Membrane-bound, spherical, uniform granular content

• Main function: cellular digestion (contain >40 hydrolytic enzymes)

• Golgi terminates & packages them into vacuoles → form lysosomes

• Abundant in leukocytes

Heterolysosomes

Active lysosomes

Where are enzymes of the lysosome made?

RER

Functions of the Lysosomes

• Cellular digestion of extracellular material

• Release of nutrients

• Autophagy: cellular waste processing and recycling

• Secretion of hydrolytic enzymes

Mitochondria

• Can be seen with light microscope

• TEM: two membranes: outer + inner

• Outer membrane: sieve-like with porins (transmembrane proteins)

• Inner membrane: folds (cristae) with enzymes of oxidative phosphorylation (ETC → ATP production)

2 Regions of the Mitochondria

• Innermost matrix: enzymes for pyruvate oxidation, fatty acid oxidation, Krebs cycle → NADH, FADH₂, CO₂, ATP

• Intermembrane space (between outer and inner membrane) → contains H⁺ for ATP synthesis (proton gradient)

Functions of the Mitochondria

• ATP synthesis (oxidative phosphorylation)

• Apoptosis → releases cytochrome C into cytoplasm

  • Cytochrome C activates proteases → degradation of cellular components

• Lipid metabolism → β-oxidation of fatty acids in mitochondrial matrix

What does the mitochondrial matrix contain?

• Circular DNA

• Ribosomes, tRNA, mRNA

Mitochondrial Matrix

• Can synthesize some of their proteins

• Mitochondria divide by binary fission (similar to prokaryotes)

• During cell division → daughter cells receive half of mitochondria from parent cell

Mitochondria staining

Contain lots of proteins → stain with acidic dyes

Peroxisomes

• Spherical, membrane-bound organelles

• Detoxification

• Lipids metabolism (complementing SER and mitochondria)

• Peroxisomal enzymes produced by free

ribosomes.

Origin of Peroxisomes

• ER

• Budding from pre-existing peroxisomes

Where are peroxisomal enzymes produced?

In free ribosomes

Cytoskeleton

Complex array of:

• Microtubules

• Microfilaments (actin filaments)

• Intermediate filaments

Functions of the cytoskeleton

• Cellular shape

• Movement of organelles

• Cellular movement via rearrangement of the cytoskeleton

Microtubules

• Form axonemes of cilia and flagella (more stable arrangement)

• Found in the cytoplasm

• Hollow and rigid tubules

• Variable length

• Transport vesicles within cells

Which filament of the cytoskeleton is the thickest?

Microtubules

Which filament of the cytoskeleton is the thinnest?

Microfilaments (actin filaments)

Polymerization of Microtubules

• Protein subunit: heterodimer of α and β tubulin

• Polymerization occurs rapidly at (+) end (polarized)

• Directed by microtubule organizing centers (MTOC)

• Show dynamic instability: continuous cycles of polymerization and depolymerization

• Energy source: GTP