Cell Bio Exam 2

Molecular biology techniques, Cell membranes/junctions, Receptors, second messengers, Protein synth, mod, trafficking

Molecular Biology Techniques

A group of techniques designed to manipulate, analyze, expand, and express nucleotide sequences (RNA, DNA) so as to better understand genes and associated proteins

Genomics

the study of genes and expression

Nanopore

tiny pore used to sequence DNA or RNA by measuring changes in electrical current as nucleic acids pass through the pore

Daniel Branton

pioneered the concept of nanopore DNA sequencing.

Epigenetics

involves reversible chemical modifications to DNA or histones that regulate gene expression without changing the underlying DNA sequence

transcriptome

the full set of RNA molecules (mRNA, rRNA, and tRNA) produced in a cell at a specific time

proteome

the entire set of proteins produced in a cell or organism at a given time

metabolomics

• the study of small metabolic molecules in cells or tissues

• can reveal metabolic changes associated with processes such as cancer metastasis and cell migration

secretome

all substances secreted by a cell

exosome

• come from endosomes in the cytoplasm

• vesicles that carry proteins and RNA to mediate cell communication

• play roles in processes like tissue repair and disease

microvesicles

extracellular vesicles that form by budding directly from the plasma membrane

mutation

change in the DNA sequence that can alter gene function

DNA mutation techniques

UV light

• Causes thymine dimers

• Leads to errors during DNA replication

Chemical (ethyl methanesulfonate)

• Chemically modifies guanine

• Causes G–C → A–T mutations

L.H. Hartwell

used complementation analysis to study cell cycle control in yeast

complementation analysis

a genetic test used to determine whether two mutations are in the same gene or different genes

results of complementation analysis

• No complementation → same gene → no functional protein

• Complementation → different genes → function restored

underlying/foundational techniques for microbiology techniques

• Restriction Nucleases (Restriction Enzymes)

• Gene Splicing

• Nucleic Acid Hybridization

Microbiology Techniques

1. DNA Gel electrophoresis

2. Southern Blots

3. Single Base Change

4. Northern Blots

5. Molecular Beacons

6. cDNA microarray

7. RNA-Seq

8. Prokaryote Gene Expression

9. Eukaryotic cells

how DNA Gel electrophoresis works

• DNA is often cut using restriction nucleases

• DNA fragments are loaded into a gel

• An electric field is applied:

  • DNA (negatively charged) moves toward positive electrode

• Separation is based on size (molecular weight):

  • smaller fragments move faster/farther

  • larger fragments move slower

Two applications of DNA Gel Electrophoresis

1) DNA Binding Proteins (Band shift assay/EMSA)

• Control: DNA alone, Experiment: DNA + protein of interest (POI)

• DNA + protein complex moves slower → higher band (shifted)

• Indicates binding occurred

2) Apoptosis vs. Necrosis

• Run DNA from cells on a gel:

  • Control (healthy cells): Normal band pattern

  • Apoptosis (programmed cell death): DNA ladder pattern

    • Caused by non-random, specific cleavage

  • Necrosis (uncontrolled cell death): Smear pattern

    • Caused by random DNA degradation

how Southern Blotting works

• DNA is cut with restriction enzymes

• Run on gel electrophoresis

• Transfer DNA to a membrane (blot)

• Add labeled DNA probe → binds complementary sequence

• Detect bands

QUALITATIVE & QUANTITATIVE

Application of Southern Blotting

Transplants:

• Donor before transplant 

• Patient before transplant 

• Patient after transplant → want to see a match to donor electrophoresis bands; shows complete replacement match

Single Base Change (SNPs)

single base changes in DNA (Ex: A → G at one position in the genome)

• Panels of SNPs across multiple genes can be used to predict risk (e.g., autism, cancer, etc.)

How Northern Blot (RNA analysis) works

• Isolate RNA

• Run gel electrophoresis (separates by size)

• Transfer to membrane

• Add labeled probe → binds specific mRNA

• Detect bands

Can only probe one mRNA at a time

QUALITATIVE & QUANTITATIVE

how Molecular Beacons work

hairpin-shaped fluorescent probes that hybridize to specific DNA or RNA sequences, unfolding upon binding to separate a quencher from a fluorophore and produce a signal, enabling highly specific, real-time detection of nucleic acids in cells

how cDNA Microarray works

• use reverse transcriptase to convert mRNA into cDNA

• label control vs experimental with colors

• mix the samples together

• Apply to a microarray chip containing thousands of gene-specific DNA probes → labeled cDNA hybridizes (binds) to complementary probes

• Compare fluorescence signals to determine which genes are upregulated, downregulated, or equally expressed

how RNA Sequencing works

• Isolate RNA

• Convert to cDNA (reverse transcriptase)

• Sequence the cDNA

• Map sequences back to genome → determine which genes are expressed and how much

how Prokaryote Gene Expression works

recombinant proteins like insulin are produced in E. coli using the lac operon, where IPTG induces the promoter to drive continuous expression of the inserted gene

Eukaryotic cells

• ex: CHO cells

• Used to produce ~70% of biologic drugs (e.g., antibodies, therapeutic proteins)

• Preferred because they can perform post-translational modifications

• Transient - protein expression only lasts for a day or so

  • Preferred type is long term stability

what does PCR do

Makes many copies of a specific DNA sequence

key features of Taq Polymerase

• Heat-stable → does NOT denature at 95°C

• Allows repeated PCR cycles

Antisense RNA/DNA

• Lowers gene expression

• Single-stranded nucleic acid complementary to a target mRNA

• Binding to mRNA:

  • blocks translation

  • can lead to mRNA degradation

siRNA vs shRNA

• both cleave target mRNA

• siRNA is transient, whereas shRNA is longer-lasting

• siRNA is easier to perform, shRNA is more difficult

CRISPR CAS-9

gene editing tool

Schleiden and Schwann

Recognized that cells have a boundary (“barrier”) separating inside from outside

Overton

• Found that lipid-soluble dyes entered cells more easily

• —> cell membrane must be lipid-like (lipid-based)

Gorter and Grendel

found the cell membrane was composed of 2 layers

Mudd & Mudd

• partition coefficient experiment

• while RBCs behave like lipids, WBCs interact more with water, providing evidence that cell membranes contain both lipids and proteins

Davson–Danielli Model

proposed that membrane was a lipid bilayer sandwiched between protein layers

J.D. Robertson

• Used electron microscopy with OsO₄ , revealing surface glycosylated proteins

Singer and Nicolson

freeze-fracture experiments showed that membranes split along the lipid bilayer and reveal embedded protein “bumps,” with more on the P face than the E face, supporting the fluid mosaic model; Proteins are embedded within the lipid bilayer, not just on the surface

Cell Fusion Experiment

• Two cells fused:

  • one labeled red mAbs

  • one labeled green mAbs

• Over time → colors mix

—> Conclusion: membrane proteins are fluid and move laterally

Patching / Capping Experiment

• Use one monoclonal antibody (mAb) to bind membrane proteins

• Proteins cluster (patching) → move to one side (capping)

—> Conclusion: proteins are mobile within membrane

Concanavalin A (“ConA”)

• binds to carbohydrates on glycoproteins

• restricts movement of those membrane proteins

FRAP

1. Label membrane proteins with fluorescence

2. Bleach a region (laser)

3. Watch fluorescence return

• Fluorescence recovers over time

• Means proteins move into bleached area

What percent of membrane proteins are freely fluid?

~50%

Why are Red Blood Cells (RBCs) commonly used in cell membrane research?

• plentiful and easy to isolate

• pure populations

• lack other membranes

• few membrane proteins

What are RBC “ghosts” and why are they useful?

• red blood cells with their internal contents removed, leaving only the membrane

• allows direct study of membrane structure and proteins.

Spectrin

• a dimer, fibrous cytoskeletal protein that forms a network beneath the membrane

• maintain cell shape and flexibility

Where are phospholipids made?

Endoplasmic Reticulum (ER)

2 kinds of phospholipids

Phosphoglycerides

• Based on glycerol backbone

Sphingolipids

• increase resistance across membrane

Sterols

ex: cholesterol

• Regulates membrane fluidity

• Acts as a structural component (“building block”)

Ectoplasmic (E) vs Protoplasmic (P) Face

Ectoplasmic face (E face)

• Faces the outside of the cell (extracellular side)

Protoplasmic face (P face)

• Faces the inside of the cell (cytoplasmic side)

What is the difference between integral and peripheral membrane proteins?

Integral proteins are embedded in the lipid bilayer (often spanning it), while peripheral proteins are loosely attached to the membrane surface.

Glycophorin

• integral membrane protein in RBCs

• Single-pass transmembrane protein

Three Cell Membrane Transport Proteins

Channels, Transporters, ATP-powered Pumps

Channels

• Fastest transport

• Form pores for ions or small molecules

• Gated and non-gated

• Transport is passive (down gradient, no ATP)

Transporters

• Bind substrate and change shape to move it across membrane

• Slower than channels

• Uniporter → one molecule

• Symporter → two molecules same direction

• Antiporter → two molecules opposite directions

ATP-Powered Pumps

• Use ATP directly

• Move substances against their gradient

• Type of active transport

Ex: Na⁺/K⁺ pump

5 Cell-Cell Junctions

Adherens Junction, Desmosome, Tight Junctions, Gap Junctions, Synapse

Adherens Junction

• Cell–cell adhesion

• encircles the cell

• Located near the apical domain

What protein is involved in adherens junctions and what is required for its function?

E-cadherin mediates adhesion; requires Ca²⁺

What cytoskeletal component is associated with adherens junctions?

Adherens junctions are physically linked to F-actin, providing structural support.

Desmosome

• Strong cell–cell adhesion

• localized spot-like junctions

• provide mechanical strength

What cytoskeletal element is associated with desmosomes?

Desmosomes are connected to intermediate filaments, such as keratin.

Tight Junctions

• Form a seal between adjacent cells

• Prevent movement of liquids and solutes between cells

• Found in tissues where leakage must be controlled:

  • Kidney, Intestinal, Bladder

Gap Junctions

• Form channels between adjacent cells

• Allow passage of ions & small molecules

• Enable cell–cell communication

• Two connexons (one from each cell) align → form a channel

Rectifying vs Non-rectifying Gap Junctions

• Non-rectifying

  • ions/molecules move both directions

• Rectifying

  • movement is one direction only

Neurochemical Synapse

• Uses neurotransmitters

• Neurotransmitter is released from presynaptic neuron

• Binds to receptors on postsynaptic cell

• Has a ~2 ms delay

What is the difference between chemical and electrical synapses?

Chemical synapses —> neurotransmitters; have a delay

Electrical synapses —> direct ion flow; are instantaneous

Receptors

• Proteins that bind a ligand to trigger a cellular response

• Can cause short-term cytoplasmic changes or long-term changes in gene expression

ligand

• Molecules that bind to receptors

• ex: hormones, neurotransmitters, drugs

3 Types of Ligands

Agonist, Antagonist, Modulator

Agonist

• Activates the receptor

• Mimics the natural ligand

• Produces a biological response

Antagonist

• Binds receptor but does NOT activate it

• Blocks the receptor from being activated by the normal ligand

Modulator

• Alters receptor activity

• Can increase or decrease response

• Does not necessarily activate receptor on its own

What factor determines the different types of ligand signaling?

the distance ligands travel to reach their receptors

Plasma membrane-attached ligands

• Distance: Å (very tiny, direct contact)

• Ligand stays attached to the cell membrane

• Requires cell–cell contact

Synaptic signaling

• Distance: few nanometers

• Neurotransmitter released across a synapse

• Very fast and localized

Autocrine signaling

• Cell releases ligand that acts on itself

• Ex: Cancer (glioblastoma)

  • releases growth factor, stimulating its own growth

Paracrine signaling

• Acts on nearby (adjacent) cells

• Short-range diffusion

• Ex: tissues, local signaling

Endocrine signaling

• Distance: long-range (~meters in body)

• Ligands travel through bloodstream

• Ex: hormones (insulin, etc.)

Hydrophobic ligands

• Ex: Vitamin D, Testosterone, steroids

• Can diffuse through the membrane —> Do NOT need membrane receptors

Hydrophilic ligands

• Cannot cross the membrane

• Must bind receptors on the cell surface

What are the main types of cell surface receptors?

• Ion channel receptors

• Receptor-mediated endocytosis receptors

• Receptor-associated kinases

• Receptor tyrosine kinases (RTKs)

• GPCRs

Steroid Receptors

• Receptors for hydrophobic ligands

• Located in the cytoplasm

Explain Primary & Secondary Responses

• Set of genes initially activated

• —> Primary proteins shut down the primary response & activate the secondary response

Receptor-Mediated Endocytosis (RME) & its key feature

the selective uptake of ligands via receptors, characterized by clathrin-coated pits and vesicles

Clathrin

• Forms coated vesicles

• Structure = triskelion (3-legged)

• Can self-assemble with little ATP

RME Pathway example with transferrin receptor

Step-by-step

1. Ferrotransferrin (iron-bound transferrin) binds receptor

   • Occurs at pH ~7

   • No ATP required

2. Receptor-ligand complex moves into clathrin-coated pit

3. Forms clathrin-coated vesicle

4. Vesicle → early endosome

5. Endosome acidifies (pH 7 → ~5)
   👉 Iron released

6. Apotransferrin + receptor recycled back to membrane

7. At surface (pH ~7):
   👉 Apotransferrin is released

Megalin receptor

• Large, multi-ligand receptor

• Found in kidney

• Binds over 60 ligands vitamins, growth factors, toxins, drugs 

Why is acidification important in receptor-mediated endocytosis?

Acidification of endosomes (pH ~7 to ~5) enables ligand dissociation, receptor recycling, and proper sorting, and blocking this process disrupts RME

LDL

• carries cholesterol

• “Particle” includes:

  • Phospholipids

  • Cholesterol

  • Apolipoprotein, which acts as the ligand for the LDL receptor

• is taken up by cells via RME

GPCRs (G Protein-Coupled Receptors)

• 800+ GPCRs in humans

• Called “serpentine receptors” because they span the membrane 7 times

• 7-transmembrane receptors that activate trimeric G proteins by exchanging GDP for GTP on the α subunit

What happens when a G protein is activated?

The α-GTP subunit and βγ complex interact with effectors to initiate signaling pathways such as cAMP production

GPCRs in Visual Transduction (Phototransduction)

Light activates rhodopsin (GPCR), which activates a G protein (transducin), leading to PDE activation, cGMP breakdown, closure of Na⁺ channels, and decreased neurotransmitter release

Why must visual signaling be rapidly shut off?

Because a single photon produces a highly amplified response, and without shutoff, it would cause over-amplification and visual noise

Cytokines

Small signaling proteins released by cells

kinases

Enzymes that add phosphate groups (PO₄³⁻) to proteins

• a.k.a. phosphorylation

Cytokine receptors vs RTKs

• two different receptor systems that BOTH lead to gene activation and cell responses

• Same goal (turn on cell responses), different mechanisms (“two doors to same facility”)

• RTKs have own kinase activity; cytokine receptors do not