Cell Bio 1 Exam 1

What is a benefit of growing cells in culture?

• Allows researchers to grow and study cells outside of the organism under controlled conditions.

• Advantage includes being able to control the cells' environment, such as:

  • Nutrients

  • Temperature

  • pH

  • Growth factors

  • Exposure to drugs or other treatments

• Researchers can change one condition at a time and observe how the cells respond.

• This makes it easier to determine cause-and-effect relationships.

• Cells in culture can also be used to:

  • Study normal cell growth and behavior.

  • Study diseases.

  • Test drugs or treatments.

  • Study the function of specific genes or proteins.

  • Observe cellular processes directly.

• A large population of similar cells can be grown, which makes experiments easier to repeat and compare.

What would need to be included in cell culture medium for optimal cell growth?

• Essential amino acids

  • Used as building blocks to make proteins.

• Vitamins and minerals

  • Needed for normal enzyme activity and metabolism.

• Salts/ions

  • Help maintain proper osmotic balance and cellular function.

• Glucose or another energy source

  • Provides fuel for ATP production and cellular metabolism.

• Growth factors

  • Proteins/signaling molecules that stimulate cell growth and division.

• Serum, depending on the culture conditions

  • Can provide growth factors, hormones, attachment factors, and other molecules needed for survival.

• The medium also needs an appropriate:

  • pH

  • Temperature

  • Osmolarity

Discuss the benefits and drawbacks to growing cells in a 2D monolayer and 3D organoids.

• 2D monolayer

  • Cells grow as a flat, single layer attached to a culture surface.

  • Benefits:

    • Relatively simple and inexpensive.

    • Easy to maintain.

    • Easy to observe using a microscope.

    • Easy to expose all cells to the same drug or treatment.

    • Experiments are easier to reproduce.

  • Drawbacks:

    • Does not accurately reproduce the three-dimensional environment found in tissues

    • Cell-cell interactions and cell–extracellular matrix interactions are more limited.

    • Cells may behave differently than they would inside an actual tissue.

• 3D organoids

  • Cells grow in a 3D arrangement and can organize into structures that resemble features of real organs.

  • Benefits:

    • More closely mimics the environment inside the body.

    • Allows more realistic:

      • Cell-cell interactions

      • Cell-matrix interactions

      • Cell differentiation

      • Tissue organization

      • Can provide a better model for studying development, disease, and drug responses.

    • Drawbacks:

      • More complicated to grow.

      • More expensive and time-consuming.

      • Harder to image and analyze.

      • Cells in different areas of the organoid may receive different amounts of nutrients, oxygen, or treatment.

Easy comparison:

• 2D = easier but less realistic.

• 3D = more realistic but more complicated.

What is the difference between a cell line and a cell culture?

Cell culture

• Refers broadly to cells that are removed from an organism and maintained/grown under controlled laboratory conditions.

• A primary cell culture is established directly from cells taken from a tissue.

• Primary cells usually have a limited lifespan and can only divide a certain number of times.

Cell line

• A population of cultured cells that can be maintained through repeated rounds of growth and subculturing.

• Some cell lines can continue dividing for a very long time or indefinitely.

• Immortalized cell lines are especially useful because researchers can perform repeated experiments using a consistent population of cells.

Remember:

• Cell culture = the general process/population of growing cells in the lab.

• Cell line = a population of cultured cells that can be repeatedly maintained and propagated.

Describe the technique: flow cytometry

• A technique used to count and analyze a cell’s size, shape, and properties within a large population.

• Cells are suspended in liquid and passed through the instrument one cell at a time.

• Each cell passes through a laser beam

  • Forward scatter - cell size

  • Side scatter - shape and internal complexity

• The instrument measures properties of each cell, such as:

  • Light scattering, which provides information about cell size and internal complexity.

  • Fluorescence, if the cells have been labeled with fluorescent molecules, antibodies, or specific proteins on or inside cells

• Researchers can identify different cell populations within a mixed sample.

Basic sequence:

Cells in suspension → pass one at a time → laser → light/fluorescence measured → cells analyzed

What is FACS? Why would a researcher utilize this special type of flow cytometry?

FACS = Fluorescence-Activated Cell Sorting

• FACS is a specialized form of flow cytometry.

• It can physically separate/sort specific populations of cells.

• Steps

  • Cells are first labeled, usually using fluorescent markers.

  • The cells then pass through a laser beam.

  • The machine measures:

    • Fluorescent light emitted by the cell.

    • Light scattered from the cell, which provides information about its size and shape.

  • The cell suspension passes through a nozzle and forms tiny droplets containing at most one cell.

  • The droplets are electrically charged based on their fluorescence.

  • An electric field deflects the droplets into different collection bins.

  • As many as 10 million cells per hour can pass through the machine

Researchers use FACS when they need to:

• Isolate one particular type of cell from a mixture.

• Separate fluorescent cells from nonfluorescent cells.

• Obtain a purified cell population for further experiments.

Key distinction:

• Flow cytometry → analyzes cells.

• FACS → analyzes AND physically sorts cells.

What is the goal of immunofluorescence microscopy? How is this achieved?

• Determine the location and distribution of a specific protein or other antigen within a cell or tissue.

• It combines:

  • Specificity of antibodies

  • Visibility of fluorescent molecules

How it works

• An antibody is chosen that specifically recognizes the protein of interest.

• The antibody is either:

  • Directly attached to a fluorescent dye, or

  • Detected using a second fluorescently labeled antibody.

• Antibody binds to its specific target in the cell.

• The sample is illuminated with the appropriate wavelength of light.

• The fluorescent molecule absorbs the light and then emits light at a different wavelength.

• The emitted fluorescence can be observed using a fluorescence microscope.

• The location of the fluorescence tells the researcher where the protein is located in the cell.

  • Different fluorescent dyes can also be used at the same time to visualize multiple structures or proteins.

Simple idea:

Antibody finds protein → fluorescent label makes protein visible → microscope shows where protein is located.

Compare and contrast SEM vs TEM.

• Both SEM and TEM are forms of electron microscopy.

  • Electron microscopy provides much higher resolution than light microscopy.

  • Resolution of cellular ultrastructure down to about 1 nm.

• TEM — Transmission Electron Microscopy

  • Electrons are transmitted through a very thin specimen.

  • Some electrons pass through while others are absorbed/scattered.

  • Produces detailed images of the internal structures of cells.

  • Useful for viewing things like:

    • Organelles

    • Membranes

    • Internal cellular ultrastructure

  • Usually produces a 2D image of a thin section.

• SEM — Scanning Electron Microscopy

  • The specimen is typically coated with a thin layer of metal.

  • Electrons interact with/scatter from the surface.

  • Produces detailed information about surface structure and shape.

  • Images give a more 3D-like appearance.

Biggest difference

• TEM → internal structures

• SEM → surface structures

What techniques could be used to isolate organelles?

Step 1: Break open the cells

• The plasma membrane must first be ruptured to release the cell contents.

• Methods include:

  • Homogenization

  • Sonication

  • Other mechanical disruption methods

• Cells can also be placed in a hypotonic solution, causing swelling and making the membrane easier to rupture.

Step 2: Differential centrifugation

• The cell homogenate is centrifuged at progressively higher speeds.

• Cellular components separate based largely on differences in size and density.

• Larger/heavier structures pellet at lower speeds.

• Smaller components require higher speeds.

Step 3: Density-gradient centrifugation

• Organelles can be separated even further based on their density.

• The sample is centrifuged through a density gradient.

• Organelles move until they reach a region matching their own density.

• This produces more purified fractions of particular organelles.

Overall process:

Cells → disruption/homogenization → cell homogenate → centrifugation → organelles separated

Describe the basic structure of a biological membrane. How do the chemical properties of phospholipids cause them to spontaneously form bilayers?

• The basic structure of a biological membrane is a phospholipid bilayer containing proteins and other lipids.

• Phospholipids are amphipathic, meaning they contain both:

  • A hydrophilic/polar head group

  • Hydrophobic/nonpolar fatty-acid tails

• In an aqueous environment:

  • Hydrophilic heads interact with water.

  • Hydrophobic tails avoid water.

• This causes the phospholipids to arrange themselves into a bilayer:

  • Heads face the aqueous environments on both sides.

  • Tails point inward toward one another.

  • This arrangement happens spontaneously because of the hydrophobic effect.

• Exposed hydrophobic edges are energetically unfavorable, so bilayers also tend to self-seal.

• The result is a:

  • Stable

  • Closed

  • Selectively permeable boundary

What does it mean to say that membranes are asymmetric? How can the two leaflets differ in lipid, protein, and carbohydrate composition?

• Membrane asymmetry means the two leaflets of a membrane are not identical.

• The cytosolic leaflet and exoplasmic/noncytosolic leaflet can differ in their:

  • Lipid composition

  • Protein orientation

  • Carbohydrate composition

• Lipids

  • Certain phospholipids are concentrated more strongly in one leaflet than the other.

  • This unequal distribution is actively maintained.

• Proteins

  • Integral membrane proteins have a specific orientation or topology.

  • The part of a protein facing the cytosol remains cytosolic.

  • The portion facing the noncytosolic/exoplasmic side remains on that side

• Carbohydrates

  • Carbohydrate groups attached to proteins and lipids are found mainly on the noncytosolic/extracellular surface of the plasma membrane.

  • This creates the carbohydrate-rich surface called the glycocalyx.

Main point: A membrane is not simply the same material on both sides — each side has its own composition and function.

What factors influence membrane fluidity? Be able to predict how changes in fatty-acid chain length, saturation, temperature, and cholesterol affect membrane properties.

Fatty-acid chain length

• Shorter chains → MORE fluid

  • Fewer interactions between tails.

• Longer chains → LESS fluid

  • More hydrophobic interactions hold the tails together.

Saturation

• More unsaturated fatty acids → MORE fluid

  • Cis double bonds create kinks in the tails.

  • The phospholipids cannot pack tightly together.

• More saturated fatty acids → LESS fluid

  • Straight tails pack together tightly.

Temperature

• Higher temperature → more fluid

• Lower temperature → less fluid

Cholesterol

• Cholesterol acts as a fluidity buffer.

  • At higher temperatures, cholesterol limits phospholipid movement → prevents the membrane from becoming too fluid.

  • At lower temperatures, cholesterol prevents phospholipids from packing too closely → prevents the membrane from becoming too rigid.

Quick memory:

• Short tails = ↑ fluidity

• Unsaturated = ↑ fluidity

• High temperature = ↑ fluidity

• Cholesterol = stabilizes/buffers fluidity

Compare integral membrane proteins, peripheral membrane proteins, and lipid-anchored proteins. How is each associated with the membrane?

Integral membrane proteins

• Are embedded directly within the lipid bilayer.

• Many span the membrane completely and are therefore transmembrane proteins.

• Their hydrophobic amino-acid regions interact with the hydrophobic interior of the bilayer.

• Usually require detergents to remove them from the membrane.

Peripheral membrane proteins

• Do not enter the hydrophobic interior of the bilayer.

• Loosely associate with:

  • Membrane surfaces

  • Integral membrane proteins

  • Lipid head groups

• Usually held by noncovalent interactions.

• Easier to remove than integral proteins.

Lipid-anchored proteins

• The protein itself does not have to pass through the bilayer.

• Instead, it is covalently attached to a lipid molecule that is inserted into the membrane.

• The lipid acts like an anchor holding the protein to the membrane.

Easy comparison:

• Integral = inside the membrane

• Peripheral = attached to the surface

• Lipid-anchored = attached to a lipid inserted into the membrane

Explain why some molecules can cross a lipid bilayer readily whereas others require membrane transport proteins. Consider size, polarity, charge, and the hydrophobic membrane interior

• The center of the membrane is hydrophobic because it contains the fatty-acid tails of phospholipids.

  • Because of this, permeability depends on:

    • Size

    • Polarity

    • Charge

  • Cross easily

  • Small nonpolar molecules, such as:

    • O₂

    • CO₂

• These dissolve in the hydrophobic membrane interior and move across by simple diffusion

  • Cross slowly

    • Small uncharged polar molecules can sometimes cross, but more slowly

  • Cannot readily cross

    • Large polar molecules

    • Charged molecules

    • Ions such as:

      • Na⁺

      • K⁺

      • Ca²⁺

      • Cl⁻

    • Need membrane transport proteins.

Compare simple diffusion, facilitated diffusion, primary active transport, and secondary active transport. What provides the driving force for each?

Simple diffusion

• Molecule passes directly through the lipid bilayer.

• Moves down its concentration gradient:

  • High → low concentration

• Does NOT require:

  • Transport protein

  • ATP

• Driving force = concentration gradient.

Facilitated diffusion

• Molecule moves through a channel or transporter.

• Still moves down its concentration/electrochemical gradient.

• Does NOT directly require ATP.

• Driving force = existing gradient.

Primary active transport

• Uses an ATP-powered pump.

• ATP hydrolysis directly provides energy.

• Molecules can move against their electrochemical gradient.

• Driving force = ATP hydrolysis.

Secondary active transport

• Does NOT directly hydrolyze ATP at the transporter.

• Instead, movement of one substance down its electrochemical gradient provides the energy to move another substance against its gradient.

• Includes:

  • Symport — substances move in the same direction.

  • Antiport — substances move in opposite directions.

Driving force = energy stored in an ion gradient, which was usually created by primary active transport.

What is an electrochemical gradient? For an ion, explain how both its concentration gradient and the membrane potential determine the direction in which it tends to move.

For an ion, two forces determine which way it wants to move.

• Chemical gradient

  • Depends on the concentration of the ion.

  • Favors movement

    • High concentration → low concentration

• Electrical gradient

  • Depends on:

    • Charge of the ion

    • Membrane potential

  • Opposite charges attract.

  • Like charges repel.

Together:

• Chemical gradient + electrical gradient = electrochemical gradient

• electrochemical gradient determines the energetically favorable direction for ion movement.

  • For example, if the inside of a cell is negative:

    • A positively charged ion may be electrically attracted inward.

    • But its concentration gradient might favor movement outward.

Compare channels and carrier proteins. How do they differ in mechanism, selectivity, rate of transport, and regulation?

Channels

• Form a continuous hydrophilic pore through the membrane.

• Allow ions or water to move rapidly.

• Movement is passive and down an electrochemical gradient.

• Very fast.

• Selectivity depends on properties of the pore/selectivity filter.

• Can be:

  • Gated

  • Nongated

Carrier proteins/transporters

• Have specific binding sites for their solutes.

• Solute binds → transporter undergoes a conformational change → solute is released on the opposite side.

• Slower than channels.

• Highly specific.

• Can function as:

  • Uniporters

  • Symporters

  • Antiporters

• Remember:

  Channel = tunnel

  Carrier = bind → change shape → release

Explain how the Na⁺/K⁺ ATPase works and why it is important. How can an ATP-driven pump create an ion gradient that is subsequently used to drive secondary active transport?

• The Na⁺/K⁺ ATPase is a P-class ATP-powered pump.

  • For each transport cycle:

    • 3 Na⁺ OUT + 2 K⁺ IN + ATP consumed

Steps

• Three Na⁺ ions bind to the pump on the cytosolic side.

• ATP binds and phosphorylates the pump.

• Phosphorylation causes a conformational change.

• Three Na⁺ are released outside the cell.

• Two K⁺ bind from the extracellular side.

• The pump is dephosphorylated.

• Another conformational change returns it to its original orientation.

• Two K⁺ are released into the cytosol.

Why is it important?

• It establishes:

  • High Na⁺ outside the cell.

  • High K⁺ inside the cell.

These gradients help with:

• Membrane potential

• Cell volume

• Ion balance

• Secondary active transport

  • For ex, intestinal cells use the Na⁺ gradient created by the Na⁺/K⁺ ATPase to power Na⁺-coupled nutrient uptake.

So:

ATP → Na⁺ gradient → stored energy → secondary active transport

Predict the direction of ion movement across a membrane. Given information about an ion’s concentration on each side of the membrane and the membrane potential, explain how you would determine the direction the ion will tend to move when a selective channel opens.

1. What does the concentration gradient favor?

• Determine where the ion concentration is higher.

  • Ion naturally tends to move: High → low

2. What does the electrical gradient favor?

• Look at:

  • Charge of the ion.

  • Membrane potential.

• For example, if the inside is negative:

  • Positive ions are attracted inward.

  • Negative ions are pushed outward.

Then combine the two forces.

• If both forces point in same direction → movement is strongly favored in that direction.

• If the forces oppose each other → determine which force is stronger.

• The combined force is the electrochemical gradient.

  • A more exact way is to compare the membrane potential (Vm) with that ion's equilibrium/Nernst potential (Eion).

    • When Vm = Eion, no net movement of that ion.

    • When different, ion moves in the direction that brings Vm toward Eion.

Compare protein import into the nucleus, ER, mitochondria. Which organelle can import a protein in its folded state, and which generally require the protein to be unfolded or extended?

• Nucleus

  • Proteins enter through the nuclear pore complex (NPC).

  • Can import proteins in their fully folded state.

  • Proteins contain an NLS (nuclear localization signal) that is recognized by import proteins.

• ER

  • Proteins are generally imported in an unfolded/extended state.

  • The protein is usually transported into the ER while it is being synthesized (co-translational translocation).

  • Uses an ER signal sequence, SRP, SRP receptor, and translocon.

• Mitochondria

  • Proteins are generally imported in an unfolded state.

  • Proteins are first synthesized on cytosolic ribosomes and then imported (post-translational import).

  • Mitochondrial targeting sequences help direct the protein to the mitochondria.

Main thing to remember:

Nucleus → folded proteins CAN enter
ER → unfolded/extended
Mitochondria → unfolded

What is protein sorting/targeting? When does it typically occur?

• Protein targeting/sorting is the delivery of newly synthesized proteins to their proper location in the cell.

• Proteins have many possible destinations, including:

  • ER

  • Golgi

  • Lysosome

  • Plasma membrane

  • Mitochondria

  • Nucleus

  • Secretion outside the cell

• All nuclear DNA-encoded mRNAs are initially translated on cytosolic ribosomes.

• Targeting occurs:

  • During translation, or

  • Soon after protein synthesis.

• The targeting information is generally encoded in the protein's own amino-acid sequence as a targeting sequence.

Describe the process of protein targeting to the ER membrane. Your answer should include the terms: signal recognition sequence, SRP, SRP receptor, and translocon.

Step 1 — Signal recognition sequence

• Translation begins on a free cytosolic ribosome.

• The growing protein contains an ER signal recognition sequence.

  • It is usually:

    • Near the N-terminus & sometimes in the middle

    • About 16–30 amino acids.

    • Rich in hydrophobic amino acids.

    • Primary sequence

Step 2 — SRP binds

• SRP = Signal Recognition Particle

• SRP binds:

  • The ER signal sequence.

  • The ribosome.

• This temporarily pauses/slows translation.

Step 3 — SRP receptor

• SRP directs the ribosome/nascent protein complex to the ER.

• SRP binds the SRP receptor in the ER membrane.

Step 4 — Translocon

• The ribosome becomes associated with the translocon, a protein-conducting channel in the ER membrane.

• SRP is released.

• Translation resumes.

• As the protein is synthesized, the growing polypeptide is fed through the translocon into the ER lumen or inserted into the ER membrane, depending on the protein.

Memorize the order:

Signal sequence → SRP → SRP receptor → translocon → ER

Proteins are modified and checked for proper folding in the ER. Name 2 ways this is done.

Molecular chaperones

• ER chaperone proteins bind newly synthesized proteins.

• They help proteins achieve the correct 3D conformation.

• Improperly folded proteins are prevented from continuing through the secretory pathway.

Disulfide bond formation

• Disulfide bonds form between cysteine residues.

• These covalent bonds help stabilize the protein's final structure.

Another important ER modification is:

• N-linked glycosylation

  • An oligosaccharide is attached to an asparagine (Asn) residue.

    • These carbohydrates can assist with:

      • Protein folding

      • Quality control

      • Later protein sorting

• Improperly folded proteins can be retained in the ER and eventually targeted for degradation.

What is unique about the targeting sequences in proteins destined for the mitochondria?

• Most mitochondrial proteins are encoded by nuclear DNA.

• They are synthesized on free cytosolic ribosomes.

• Mitochondrial proteins usually contain an N-terminal targeting sequence.

• Unlike the strongly hydrophobic ER signal sequence, mitochondrial targeting sequences commonly form an amphipathic α-helix.

• One side contains positively charged/basic amino acids.

• The other side contains hydrophobic residues.

• They generally lack negatively charged amino acids.

• Receptors on the mitochondrial surface recognize the targeting sequence.

• Proteins are usually imported in an unfolded state through translocation complexes of the outer and inner mitochondrial membranes.

• The targeting sequence is often removed after import.

Important distinction:

• ER signal sequence → hydrophobic

• Mitochondrial targeting sequence → amphipathic α-helix

Describe the structure of the nuclear pore complex (NPC). What types of molecules can move through the NPC?

• The Nuclear Pore Complex (NPC) is a very large protein complex embedded where the inner and outer nuclear membranes join.

• It is built from proteins called nucleoporins.

• NPCs form selective channels between:

  • Cytoplasm

  • Nucleus

• The NPC has a central transport channel and structures extending toward both the cytoplasm and nucleus.

• Small molecules can pass through by passive diffusion.

• Large macromolecules require selective, receptor-mediated transport.

• Selectively controls nuclear traffic

Examples transported through NPCs:

• Nuclear proteins

• RNAs

• Ribosomal subunits

• Protein-RNA complexes

What are NLS and NES and why are they important in nuclear transport?

• NLS — Nuclear Localization Signal

  • An amino-acid sequence that directs a protein INTO the nucleus.

  • Recognized by nuclear import receptors called importins.

  • NLS = IN

• NES — Nuclear Export Signal

  • Directs proteins OUT of the nucleus.

  • Recognized by nuclear export receptors called exportins.

  • NES = EXIT

• These signals are important because large proteins cannot simply diffuse freely through the NPC.

Instead:

Targeting signal + transport receptor → selective transport through NPC

Ran G-proteins, GAPs, and GEFs are essential in nuclear transport. Explain in detail how they contribute to the process of moving proteins in and out of the nucleus.

• Ran gradient tells the transport system gives nuclear transport its directionality.

• Ran is a small GTP-binding protein that exists as:

  • Ran-GTP

  • Ran-GDP

• The cell maintains:

  • High Ran-GTP in the nucleus

  • High Ran-GDP in the cytoplasm

• This difference is created by:

  • Ran-GEF

    • GEF = Guanine nucleotide Exchange Factor

    • Located primarily in the nucleus.

    • Converts: Ran-GDP → Ran-GTP

  • Ran-GAP

    • GAP = GTPase-Activating Protein

    • Located primarily on the cytoplasmic side.

    • Stimulates: Ran-GTP → Ran-GDP

• Nuclear import

  • In the cytoplasm:

    • Protein containing an NLS binds an importin.

    • Importin carries the cargo through the NPC.

  • Inside the nucleus:

    • Ran-GTP binds importin.

    • This causes importin to release its cargo.

    • Importin + Ran-GTP returns to the cytoplasm.

    • Ran-GAP causes GTP hydrolysis: Ran-GTP → Ran-GDP

    • Ran separates from importin, allowing importin to be reused.

• Nuclear export

  • In the nucleus:

    • Protein with an NES binds an exportin.

    • Ran-GTP also binds.

    • The: Cargo + exportin + Ran-GTP

  • Complex travels through the NPC into the cytoplasm.

  • In the cytoplasm:

    • Ran-GAP causes: Ran-GTP → Ran-GDP

      • The complex falls apart.

      • The exported cargo is released.

Big picture:

Nucleus = high Ran-GTP

Cytoplasm = high Ran-GDP

What are the steps of the secretory pathway?

• Step 1

  • Synthesis of proteins with an ER signal/target sequence

• Step 2

  • Protein packaged into vesicles bud from the ER and fuse together to form a new cis-Golgi cisternae

• Step 3

  • ER enzymes or structural proteins returned to ER

• Step 4

  • cis-Golgi cisterna and contents moves from the cis to the trans face of the Golgi complex

• Step 5

  • Retrograde transport vesicles move Golgi-resident proteins to the previous Golgi compartment

• Step 6

  • Constitutive secretion (all cells) - transport vesicles move continuously and fuse with the PM

• Step 7

  • Regulated secretion (certain cell types) - Proteins accumulated and stored in regulated secretory vesicles

  • Vesicles fuse with PM and secrete proteins only when cell receives a neuronal or hormonal signal secretion signal

• Step 8

  • Lysosome-destined membrane and soluble proteins

What are the steps of the endocytic pathway?

• Step 1 - plasma membrane

  • Material from outside the cell binds to receptors or is taken up from the cell surface

• Step 2 - clathrin-coated vesicle

  • In receptor-mediated endocytosis, ligand-receptor complexes are collected into clathrin/AP2-coated pits and internalized

• Step 3 - Early endosome

  • The vesicle delivers its contents to an early endosome, which acts as a sorting station

• Step 4 - material can either

  • Be recycled back to the plasma membrane

  • Continue toward late endosomes

• Step 5 - lysosome

  • Material that is not recycled is delivered to the lysosome for degradation and recycling

Compare and contrast the general features of the secretory and endocytic pathways.

• Secretory pathway

  • Moves material generally from inside the cell toward the cell surface or other organelles.

  • Used to:

    • Secrete soluble proteins outside the cell.

    • Deliver membrane proteins to:

      • Plasma membrane

      • ER

      • Golgi

      • Lysosomes

  • A general route is: ER → Golgi → final destination

• Endocytic pathway

  • Moves material from the cell surface into the cell.

  • General route: Plasma membrane → endosome → lysosome/recycling pathway

• Both use:

  • Transport vesicles

  • Cargo selection

  • Vesicle budding

  • Rab proteins

  • SNARE proteins

  • Specific donor and target membranes

Describe in detail the process of vesicle budding and fusion. Your answer should include the terms: Rab, SNARE, cargo, donor membrane, and target membrane.

Step 1 — Cargo selection

• Specific cargo molecules must be transported.

• Cargo can include:

  • Membrane proteins

  • Soluble proteins

• Soluble cargo may bind to cargo receptors in the donor membrane.

Step 2 — Coat assembly

• Small GTP-binding proteins help recruit coat proteins to the donor membrane.

• Coat proteins:

  • Select cargo.

  • Bend the membrane.

  • Help form the budding vesicle.

• Produces membrane curvature and acts as a filter determining which proteins enter the vesicle.

Step 3 — Vesicle buds

• The membrane curves outward.

• A transport vesicle eventually separates from the donor membrane.

Step 4 — Coat is removed

• The vesicle sheds its coat.

• This exposes proteins needed for targeting and fusion, including Rab and SNARE proteins.

Step 5 — Rab directs targeting

• Rab GTPases help the vesicle recognize and dock with the correct target membrane.

• This helps make vesicular transport specific.

Step 6 — SNARE pairing

• Vesicle contains a v-SNARE.

• Target membrane contains the matching t-SNARE.

• Correct SNAREs pair together.

Step 7 — Fusion

• SNARE interactions pull the two membranes very close together.

  • v-SNAREs interact with matching t-SNAREs

• The vesicle membrane fuses with the target membrane.

• Cargo is delivered.

Memorize:

Cargo selection → coat → budding → uncoating → Rab targeting → SNARE docking → fusion

Why are there 3 different transport coat proteins and not just 1 type?

The three major coats are:

• COPII

• COPI

• Clathrin

Different coat proteins are necessary because transport vesicles:

• Bud from different membranes.

• Travel to different destinations.

• Carry different types of cargo.

The coat is therefore not just packaging — it helps determine:

• Where the vesicle forms

• Which cargo enters

• Which pathway the vesicle participates in

Having different coats allows the cell to keep different trafficking routes organized and specific.

What does KDEL represent and where do proteins with this signal go?

KDEL = Lys-Asp-Glu-Leu

• It is a sorting signal found at the C-terminus of most soluble ER-resident proteins.

• If an ER-resident protein accidentally travels to the Golgi, its KDEL sequence binds to a KDEL receptor mainly in the cis-Golgi.

• The protein is packaged into a COPI vesicle and transported back to the ER.

• This prevents important ER proteins from being lost from the ER.

So KDEL acts like a: "Return to ER" label.

How are lysosomal enzymes targeted to the lysosome? What is the roleof the mannose-6-phosphate (M6P) tag?

• Newly synthesized lysosomal enzymes are tagged with mannose-6-phosphate (M6P) in the Golgi.

• M6P acts as a sorting signal that identifies the protein as a lysosomal enzyme.

• The M6P-tagged enzyme binds an M6P receptor in the trans-Golgi.

• It is packaged into a clathrin-coated vesicle.

• Pathway: Lysosomal enzyme → M6P added in Golgi → M6P receptor in trans-Golgi → clathrin-coated vesicle → endosome → lysosome

Compare and contrast COPI, COPII, and clathrin vesicles. Where do they go? What is their cargo?

• COPII

  • Direction: ER → cis-Golgi

    • Buds from the ER.

    • Carries newly synthesized proteins leaving the ER.

    • This is primarily anterograde/forward transport.

    • Think: COPII = OUT of ER

• COPI

  • Direction: Golgi → ER and later → earlier Golgi compartments (retrograde transport)

  • Returns escaped ER-resident proteins.

  • Returns certain membrane components and trafficking machinery.

  • Responsible for retrograde/retrieval transport.

  • Think: COPI = back IN toward ER

• Clathrin

  • Direction: plasma membrane → endosomes and trans-Golgi network → endosomes.

  • Cargo includes proteins entering through endocytosis and lysosomal enzymes being delivered toward the lysosome.

Explain how the trans-Golgi network (last part of the Golgi) is like an Amazon warehouse.

• Think about what happens at an Amazon distribution center:

  • Packages arrive.

  • They are identified.

  • Sorted based on their destination.

  • Packaged into the correct delivery route.

  • Sent to different locations.

• The trans-Golgi network (TGN) does basically the same thing with proteins.

• Proteins arrive after moving through the Golgi and are:

  • Identified by their sorting information.

  • Sorted.

  • Packaged into appropriate transport vesicles.

  • Sent to different destinations

    • Possible destinations include:

      • Plasma membrane

      • Secretory vesicles

      • Endosomes/lysosomes

  • Ex. Lysosomal enzyme → M6P added in Golgi → binds M6P receptor in trans-golgi clathrin-coated vesicle → endosome → lysosome

So:

Amazon warehouse: package → address → sort → truck → destination

TGN: protein → sorting signal → sort → vesicle → destination

Describe the role of dynamin in the final step of vesicle formation. What else is necessary for this process?

• Dynamin is a GTPase involved in pinching off certain budding vesicles, especially clathrin-coated vesicles.

  • As the coated pit forms, a narrow neck connects the budding vesicle to the donor membrane.

  • Dynamin assembles around this neck.

  • GTP hydrolysis causes dynamin-mediated constriction.

  • This helps complete membrane scission, separating the vesicle from the membrane.

• Other important components include:

  • Clathrin — forms the coat.

  • Adaptor proteins — connect clathrin to membrane cargo/receptors

  • GTP — required for dynamin's activity.

What is receptor mediated endocytosis (RME)? What coat protein is required?

• Receptor-mediated endocytosis (RME) is a selective method of bringing specific extracellular molecules into the cell.

  • General process

    • A molecule called a ligand binds to a specific receptor on the plasma membrane.

    • Receptor-ligand complexes cluster in specialized membrane regions.

    • Adaptor proteins recruit clathrin.

    • A clathrin-coated pit forms.

    • The membrane bends inward.

    • Dynamin helps pinch off the vesicle.

    • The vesicle enters the cytoplasm and sheds its coat.

    • Its contents are delivered into the endosomal pathway.

  • Required coat = CLATHRIN

    • Examples include uptake of:

      • LDL

      • Transferrin-bound iron

Where does LDL go once endocytosed? What happens to the LDL receptor?

Step 1

• LDL binds the LDL receptor at the cell surface.

Step 2

• Receptor-LDL complexes enter a clathrin-coated pit.

• Dynamin helps pinch off the vesicle.

Step 3

• Vesicle loses its coat and enters the endosomal pathway.

• The acidic environment causes LDL to separate from its receptor.

Step 4

• LDL continues toward the lysosome.

• Lysosomal enzymes break down:

  • ApoB protein → amino acids

  • Lipids → fatty acids

  • Cholesteryl esters → cholesterol

Step 5

• The LDL receptor is recycled back to the plasma membrane.

• It can then bind another LDL particle.

Important distinction:

LDL → lysosome

LDL receptor → recycled to plasma membrane

Discuss the importance of pH changes during RME of LDL and transferrin-iron.

• The endosomal pathway becomes increasingly acidic, and that change in pH helps separate cargo from its receptor.

  • LDL

    • LDL binds LDL receptor at the neutral pH outside the cell.

    • After endocytosis, the vesicle enters an acidic endosomal compartment.

    • Low pH causes a conformational change in the LDL receptor.

    • LDL dissociates from the receptor.

    • LDL continues toward the lysosome.

    • Receptor is recycled back to the plasma membrane.

    • At extracellular neutral pH, the receptor can bind LDL again.

  • Transferrin-iron

    • This system behaves differently.

      • Iron-loaded transferrin binds the transferrin receptor at neutral extracellular pH.

      • The complex is endocytosed.

      • Acidification of the endosome causes iron to be released from transferrin.

      • However, iron-free transferrin (apotransferrin) remains bound to its receptor at the acidic pH.

      • The receptor + apotransferrin complex is recycled back to the plasma membrane.

      • At neutral extracellular pH, apotransferrin dissociates from the receptor.

• So:

  • LDL system: low pH separates LDL from receptor.

  • Transferrin system: low pH releases iron, while transferrin remains with its receptor until they return to neutral extracellular pH.

How does autophagy differ from endocytosis? Why is autophagy so important for the health of the cell

Endocytosis

• Brings material from outside the cell into the cell.

• Begins at the plasma membrane.

• Material enters through endocytic vesicles and may eventually reach lysosomes.

Autophagy

• Deals primarily with material that is already inside the cell.

• Damaged or unnecessary cellular material is enclosed in a double-membrane structure called an autophagosome.

• The autophagosome eventually fuses with a lysosome.

• Lysosomal enzymes break down the material.

Autophagy can remove:

• Damaged organelles

• Abnormal/misfolded proteins

• Unnecessary cellular components

Why is it important?

• Prevents damaged components from accumulating.

• Recycles cellular building blocks.

• Helps cells respond to nutrient starvation.

• Maintains cellular homeostasis and quality control.

• Can remove damaged organelles before they interfere with normal cell function.

Easy distinction:

• Endocytosis = outside → inside

• Autophagy = damaged/old material already inside → lysosome