Cytology

What are the main topics in the cell biology course outline (part 1)?

Biomembranes, ER & protein sorting, Golgi apparatus, vesicular network, mitochondria (and diseases), peroxisomes, nucleus, cytoskeleton, extracellular matrix, cell signaling/proliferation/differentiation/death, cancer biology

What are the main topics in the course outline (part 2)?

Nucleic acids & electrophoresis, (de/re)naturation, dot blotting & Southern blotting, restriction enzymes, recombinant DNA, cloning, RFLP, central dogma, PCR & sequencing, genome, transcription & regulation, coding/non-coding RNAs, RNA quantification, translation, yeast two-hybrid, mutations

Which organisms are commonly used to study cells?

E. coli, yeast (Saccharomyces cerevisiae), C. elegans, Drosophila melanogaster, mice, cultured cells/tissues

Why is E. coli used in research?

Prokaryote; simple; single circular chromosome; ideal for studying replication, transcription, translation

Why is yeast (S. cerevisiae) used?

Eukaryotic; multiple linear chromosomes like humans; model for studying cell death/apoptosis

Why is C. elegans useful?

Multicellular worm; short lifespan (~2 weeks); transparent body; shares many mechanisms (e.g., apoptosis) with humans

Why is Drosophila melanogaster important?

Short lifespan; similar systems to humans; first model for genetics

Why are mice used?

Complex systems; share genes/systems with humans; used for disease & immune response studies

Why do we use cultured human cells/tissues?

Closest to human biology; show programmed cell death; used for transplantation, drug testing

What are the four major molecular components of cells?

Nucleic acids, carbohydrates, proteins, lipids

What % of plasma membranes are lipids?

~50% of mass

What % of mitochondrial membranes are lipids?

~30%

What are noncovalent interactions?

Hydrogen bonds, ionic bonds, van der Waals forces, hydrophobic interactions

Are noncovalent interactions reversible? Why?

Yes, weaker than covalent bonds → reversible under physiological conditions

What are main microscopy types for cell studies?

Light microscopy, fluorescence microscopy, electron microscopy, scanning EM

What is immunofluorescence used for?

Detects proteins using antibodies tagged with fluorescent dyes

What is cell fractionation?

Breaking cells → centrifugation → separating organelles by size/density

What is the order of pellet formation in centrifugation?

1. Low speed → nuclei, cytoskeleton

2. Medium speed → mitochondria

3. High speed → ER fragments (microsomes), ribosomal pieces

4. Very high speed → ribosomes, viruses, macromolecules

What is the protein:lipid ratio in the retina?

Roughly 50:50 → ensures dynamic membrane for fast signal transmission

What are sterols vs. cholesterol?

Sterols = family of steroid alcohols; cholesterol = major sterol in animals; absent in bacteria/plants (plants use other sterols)

Why does myelin contain high glycolipids + cholesterol?

Makes stable, insulating, low-permeability membranes → supports rapid signal transmission

Which lipids are found in the outer leaflet of the bilayer?

Phosphatidylcholine, sphingomyelin

Which lipids are found in the inner leaflet?

Phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol (minor)

Which lipid plays a role in signaling?

Phosphatidylinositol

Where are glycolipids located?

Exclusively in outer leaflet

What are lipid rafts?

Membrane regions rich in cholesterol & sphingolipids; cluster proteins (e.g., GPI-anchored) for signaling & trafficking

What are caveolae?

Subset of lipid rafts; small invaginations requiring cholesterol + caveolin + cavin

What is caveolin?

Integral membrane protein bending membrane inward

What is cavin?

Cytoplasmic protein stabilizing caveolae

What are caveolae functions?

Endocytosis, signaling, lipid transport regulation, protection against mechanical stress

What are lipid rafts?

Specialized membrane microdomains rich in cholesterol and sphingolipids (sphingomyelin, glycolipids). They cluster GPI-anchored proteins and signal transduction molecules, organizing membrane signaling and trafficking.

What are caveolae and how do they differ from lipid rafts?

Caveolae (“little caves”) are invaginated lipid rafts requiring cholesterol. Formed by caveolin (membrane protein) and cavin (cytoplasmic stabilizer). Functions: endocytosis, signal regulation, lipid transport, and mechanical protection. Mnemonic: Rafts are flat islands; caveolae are caves.

What are the main types of membrane proteins?

1. Integral membrane proteins – embedded within the bilayer (single-pass or multipass).

2. Peripheral proteins – loosely attached via ionic bonds to other proteins/lipids.

3. Lipid-anchored proteins – covalently attached to membrane lipids (e.g., myristoylation, palmitoylation, GPI).

How does the glycocalyx protect the cell?

It’s a carbohydrate coat made of glycolipids and glycoproteins’ oligosaccharides. Functions:

• Protection from ionic/mechanical stress

• Barrier to microorganisms

• Cell-cell recognition (e.g., immune response)

• Protects epithelial surfaces (lungs, GI tract)

What is the role of the Endoplasmic Reticulum (ER)?

A network of cisternae connected to the nuclear envelope.

• Rough ER (RER): Protein synthesis and processing.

• Smooth ER (SER): Lipid metabolism and detoxification.

• Transitional ER: Vesicle exit sites to Golgi.

What determines whether a protein is synthesized on free or bound ribosomes?

• Free ribosomes: make proteins for the cytosol, nucleus, mitochondria, peroxisomes.

• Bound ribosomes (RER): make proteins for secretion, membranes, lysosomes, Golgi. A signal sequence directs the ribosome to the ER membrane.

What is the signal sequence?

A short N-terminal amino acid sequence directing ribosomes to the ER. Recognized by a receptor; the nascent (developing) protein is inserted into the ER lumen through a translocon, and the sequence is cleaved by signal peptidase.

What are the fates of ER-synthesized proteins?

1. Secreted outside the cell

2. Inserted into membranes (ER, Golgi, plasma membrane)

3. Sent to organelles (endosomes, lysosomes)

How do transmembrane proteins insert into the ER membrane?

The transmembrane sequence halts translocation and embeds into the bilayer. Orientation (N- or C-terminal facing cytosol) depends on sequence charge and position.

What happens to multi-pass membrane proteins during synthesis?

Each new transmembrane region is inserted into the ER membrane as it appears; synthesis continues until all passes are embedded.

What modifications occur inside the ER?

• Folding (with chaperones)

• Disulfide bond formation (by protein disulfide isomerase)

• Glycosylation

• Lipid anchoring

• Subunit assembly (quaternary structure)

What happens to misfolded proteins in the ER?

They undergo ER-associated degradation (ERAD): Misfolded proteins → ubiquitylated → exported to cytosol → degraded by proteasomes.

What lipids are synthesized in the Smooth ER?

• Glycerophospholipids → membrane lipids

• Ceramide → precursor for sphingolipids

• Steroids → especially in testis/ovary

• Detoxification enzymes abundant in liver SER

What is the ER–Golgi Intermediate Compartment (ERGIC)?

A transit station where ER vesicles fuse before reaching Golgi. ER proteins accidentally sent to ERGIC are retrieved back to the ER.

What is the KDEL sequence and its function?

A C-terminal sequence (Lys–Asp–Glu–Leu) that retains proteins in the ER. If missing → protein secreted; if added → retained in ER.

What are the functions of the Golgi apparatus?

• Protein modification and sorting

• Synthesis of glycolipids and sphingomyelin

• Processing N-linked and O-linked glycosylation

What are the structural regions of the Golgi?

1. Cis-Golgi – receives vesicles from ER

2. Medial-Golgi – modification site

3. Trans-Golgi – packaging and sorting

4. Trans-Golgi Network (TGN) – distribution hub

How does the Golgi modify glycoproteins?

• N-linked sugars (from ER) are enzymatically processed.

• O-linked sugars added to Ser/Thr residues. → Determines protein destination and function.

What are lysosomes and their function?

Membrane-bound organelles with ~60 acid hydrolases active at pH \approx 5. They degrade macromolecules from inside and outside the cell.

How is the acidic pH of lysosomes maintained?

By an ATP-driven proton pump transporting H^+ ions into the lumen.

How are lysosomal enzymes targeted to lysosomes?

Enzymes receive mannose-6-phosphate (M6P) tags in the Golgi. Bind to M6P receptors, sorted into late endosomes → lysosomes.

What is I-cell disease (mucolipidosis II)?

A defect in the enzyme adding M6P to lysosomal enzymes. Results in mis-targeted enzymes → accumulation of undigested material → severe psychomotor retardation and death by 5–8 yrs.

What is glucocerebroside and why is it important?

A glycosphingolipid from RBC recycling in macrophages. Accumulation due to glucocerebrosidase deficiency causes Gaucher disease.

What are lysosomal storage diseases?

Inherited disorders with defective lysosomal hydrolases.
Examples:

• Gaucher disease – glucocerebrosidase deficiency
• Sphingolipidoses – glycolipid accumulation
• Mucopolysaccharidoses – defective GAG breakdown

What is vesicular transport?

Movement of cargo within membrane-bound vesicles between organelles. Selective for destination and contents.

What are the general steps of vesicular transport?

1. Cargo clustering in donor membrane

2. Bud formation

3. Vesicle pinching off

4. Transport along cytoskeleton

5. Fusion with target membrane

What are the types of exocytosis (secretory pathways)?

1. Constitutive secretion: continuous, unregulated.

2. Regulated secretion: stimulus-dependent (e.g., hormones, neurotransmitters).

How do polarized epithelial cells direct transport?

Proteins sorted to:

• Apical surface: GPI-sugar modified

• Basolateral surface: sequence-based targeting

Describe clathrin-dependent endocytosis.

Ligand-receptor binding triggers vesicle formation coated with clathrin. Acidic pH in early endosomes releases ligands; receptors recycled. Example: LDL receptor removes plasma cholesterol.

What is the function of clathrin?

• Shapes vesicle curvature

• Selectively sorts cargo

• Facilitates budding and uncoating

• Controls vesicle movement

What are the stages of endocytosis?

1. Early endosomes (pH \approx 6.5)

2. Late endosomes (pH \approx 5.5)

3. Lysosomes (pH \approx 4.5–5.0)

Describe phagocytosis.

Clathrin-independent engulfment of large particles (e.g., bacteria). Forms a phagosome, which fuses with lysosomes → phagolysosome.

What is macropinocytosis?

“Cell drinking” — uptake of extracellular fluid in small vesicles (also clathrin-independent).

What is autophagy?

“Self-eating” process forming autophagosomes (ER-derived vesicles via Atg9) that encapsulate damaged organelles or proteins. Fuse with lysosomes → phagolysosomes → degradation.

What are the physiological roles of autophagy?

Beneficial:

• Removes dysfunctional organelles

• Maintains energy during starvation

• Tissue remodeling during development
  Harmful:

• Cancer cell survival under stress

• Implicated in neurodegenerative diseases (e.g., Parkinson’s)

What are the three main functions of the Golgi apparatus?

Further protein processing and modification, synthesis of glycolipids and sphingomyelin, and protein sorting.

Describe the structure of the Golgi apparatus.

It consists of stacked flattened sacs called cisternae, divided into cis, medial, and trans compartments, plus the trans-Golgi network.

In which direction are proteins carried through the Golgi?

From the cis to the trans direction.

What is the function of transport vesicles in the Golgi?

They carry Golgi proteins back to earlier compartments for reuse.

How are N-linked oligosaccharides processed in the Golgi?

They are enzymatically modified after being added to asparagine residues in the ER.

What are O-linked sugars and where are they added?

Carbohydrates added to the hydroxyl groups of serine and threonine residues in proteins.

What lipids are synthesized in the ER before modification in the Golgi?

Glycerol phospholipids, cholesterol, and ceramide.

Into which molecules can ceramide be converted in the Golgi?

Sphingomyelin (a phospholipid) or glycolipids.

What are lysosomes and what is their function?

Membrane-enclosed organelles containing enzymes that degrade all types of biological macromolecules from inside and outside the cell.

How many types of enzymes are found in lysosomes?

Around 60 different acid hydrolases.

What maintains the acidic pH of lysosomes (~pH 5)?

An ATP-dependent proton pump.

How does the cell protect itself from lysosomal enzymes?

By containing them within lysosomes and because they are inactive at cytosolic pH (~7.4).

What modification directs lysosomal enzymes to lysosomes?

Addition of mannose-6-phosphate (M6P) to specific proteins.

What receptor binds M6P-tagged enzymes in the Golgi?

The Mannose-6-phosphate receptor, which directs them to late endosomes that mature into lysosomes.

What causes I-cell disease (Mucolipidosis II)?

Deficiency of the enzyme that phosphorylates mannose, leading to defective targeting of lysosomal enzymes.

What are the main symptoms of I-cell disease?

Severe psychomotor retardation progressing to death between 5–8 years.

What is glucocerebroside and where does it come from?

A glycosphingolipid formed during normal RBC recycling by macrophages.

Which enzyme deficiency causes Gaucher disease?

Glucocerebrosidase deficiency, leading to accumulation of glucocerebroside in tissues.

Name four categories of lysosomal storage diseases.

Gaucher disease, glycolipidoses (sphingolipidoses), oligosaccharidoses, and mucopolysaccharidoses.

What is the general definition of vesicular transport?

The movement of molecules in membrane sacs (vesicles) either into, out of, or within the cell.

What are two main examples of vesicular transport?

(1) Endocytosis from the cell surface, and (2) transport of lysosomal enzymes from the Golgi to lysosomes.

List the five general steps of vesicular transport.

(1) Cargo alignment, (2) Vesicle budding, (3) Vesicle pinching, (4) Cytoskeletal transport, (5) Membrane fusion.

What are the two types of exocytosis?

Continuous (constitutive) secretion and regulated, signal-stimulated secretion.

Give examples of regulated secretion.

Release of neurotransmitters and digestive enzymes.

What determines vesicle targeting in epithelial cells?

Special amino acid sequences (for basolateral targeting) or GPI sugar modifications (for apical targeting).

What is the sequence of compartments in endocytosis?

Endocytic vesicle → Early endosome → Late endosome → Lysosome.

What are the pH values of early endosomes, late endosomes, and lysosomes?

Early endosome ~ 6.5, Late endosome ~ 5.5, Lysosome ~ 4.5–5.0.

What is clathrin and what does it do?

A coating protein that shapes vesicles, helps cargo sorting, facilitates budding, and regulates vesicle movement.

What happens to clathrin after vesicle docking?

The vesicle becomes uncoated before fusing with the target membrane.

Describe receptor-mediated endocytosis using LDL as an example.

LDL binds to its receptor, forms clathrin-coated vesicles, dissociates in acidic endosomes, and the receptor is recycled.

What is phagocytosis?

Clathrin-independent process where cells engulf large particles like bacteria via pseudopodia to form phagosomes.

What is a phagolysosome?

A fusion product of a phagosome and lysosome, where the engulfed material is digested.

What is macropinocytosis?

A clathrin-independent process where cells ingest small droplets of extracellular fluid (“cell drinking”).

What is autophagy?

A “self-eating” process forming an autophagosome around damaged organelles or proteins, which then fuses with a lysosome.

Which protein assists autophagosome formation?

Atg9, located on the ER surface.