Cell Bio Exam 2

Langmuir Trough

• experiment w/ pressure → phospholipids can form a bilayer

Ernest Overton

• discovered lipid-soluble dyes enter cells → membrane is lipid-like

Gorter and Grendel

• cell membrane is a phospholipid bilayer

Schleiden and Schwann

• since their cell theory stated that all living things are made of cells, there must be something separating them

gene transfection

• introducing a functional gene into cells to study gene expression

• can include reporters like GFP

gene editing

• adding/removing DNA sequences

• can modify single bases or entire genes

oxford nanopore sequencing

• DNA passes through a pore and disrupts electrical current

• each of the four bases has a unique signal

transcriptome

• the set of all RNA

• “what’s being expressed”

proteomics

• the set of all proteins

• “functional output”

secretome

• protein, cfDNA, exosomes

exosomes

• vesicles secreted by cells

• contain proteins and RNA

• can act as therapies (replace stem cells) or biomarkers (cancer)

• Delivered IV or aerosol

epigenetics

• reversible changes in gene expression without DNA sequence change

• influenced by environment

• Ex) DNA methylation, histone modification

Yeast cells

• easy to grow

• haploid and diploid

• can create mutants

temperature-sensitive mutants

• function normally at a "permissive" low temperature but become inactive or unstable at a higher "restrictive" temperature

complementation analysis

• determines if mutations are in same gene

• growth → different genes

• no growth → same gene

restriction nucleases

• enzymes that cut DNA at specific sequences

• found in bacteria, used in cloning

hybridization

• binding of complementary nucleic acids

• Ex) FISH, antisense RNA/DNA, southern/northern blots, cDNA microarrays, and molecular beacons

DNA gel electrophoresis

• separates DNA by size

• band shift assay → detects protein-DNA binding

  • shift upward = binding

• cell death analysis

  • apoptosis → DNA appears as a ladder

  • necrosis → DNA appears as a continuous smear

variable number tandem repeats (VNTRs)

• repeated DNA sequences

• used in forensics

single nucleotide polymorphisms (SNPs)

• single base differences

• used for personalized medicine

southern blots

• run gel → transfer to membrane → add probe

• measures size and abundance (qualitative and quantitative) of DNA

northern blot

• run gel → transfer to membrane → add probe

• measure gene expression (RNA)

• measures one transcript at a time

molecular beacons

• fluorescent probe with quencher → fluoresces when bound

• detects and locates specific DNA sequences

complementary DNA (cDNA) microarrays

• shows what genes are being expressed (up to 8,600)

• green → control group

• red → experimental group

• yellow → both expressed

• gray → neither expressed

single cell RNA sequencing

• sequences RNA to determine gene expression of a cell

• used in tissue engineering

single cell genome sequencing

• compares genome to reference

• requires cell sorting

genomic cloning

• all of the DNA (genome) of one organism is placed into a vector and placed into bacteria which undergoes mitosis

cDNA cloning

• mRNA → cDNA → vector

• only expressed genes

• used for protein production (Humulin)

transfection

• introducing DNA into cells

• methods: electroporation

transient transfection

temporary

stable transfection

permanent

polymerase chain reaction (PCR)

• used to expand/amplify a DNA sample

• uses Taq polymerase

• heat cycles

transgenic mice

• inject DNA into embryo → implant in mother rat

• 10-30% chance that the offspring will have foreign DNA

antisense RNA/DNA

• uses DNA or RNA to block mRNA coding for a specific protein

siRNA

• degrades mRNA/ blocks transcription

• used for transient transfection

• Huntington’s disease

shRNA

• used for transient or stable transfection

• hard to make → mechanism not fully known

Mudd

• RBCs → prefer oil

• WBCs → prefer water → membrane contains proteins

Davson-Danielli

• sandwich model → protein-lipid-protein

• incorrect

JD Robertson

• used OsO4 stain

• supported Davson-Danielli model

• proposed glycoprotein coat

freeze fracture

• revealed that membrane has integral proteins → not Davson-Danielli model

• “bumps” = proteins not phospholipids

cell fusion

• shows lateral movement of proteins → revealed that membrane proteins are FLUID (can move within membrane)

cell patching & clamping

• antibodies cluster specific proteins

• revealed that membrane proteins are fluid

FRAP

• uses concanavalin A

• revealed some proteins are mobile and some are anchored

Singer and Nicholson

• fluid mosaic model

• fluid lipids + mosaic of proteins

red blood cells

• no organelles

• easy to isolate

• very few cell proteins

ghost cells

• RBC membranes after hypotonic lysis → internal contents removed, leaving just the plasma membrane

phosphoglycerides

• Ex) phosphatidylserine → moves to outer membrane in apoptosis

sphigolipids

• Ex) sphingomyelin → insulates neurons (myelin)

• Demyelinating diseases: multiple sclerosis and dementia

steroids (cholesterol)

• membrane stability

• precursor to hormones and vitamin D

flippase (ABCB4)

• enzyme that flips phospholipids across membrane

• requires ATP

movement of phospholipids and cholesterol from ER → membrane

1) vesicles transport them

2) proteins pinch the membrane inwards to let the phospholipids/cholesterol go there

3) proteins in cytosol transport them

integral proteins

• embedded in membrane

• hard to remove

• types: single-pass, multi-pass, barrel

peripheral proteins

• sits on surface, often lipid-anchored

glycophorin

• a single pass integral protein in RBCs

• glycosylated

bacteriorhodopsin

• 7 pass integral protein

• pumps H+ across membrane → makes ATP

porin

• barrel-shaped integral protein

• outside is hydrophobic, inside is hydrophilic

• passive diffusion channel for small molecules, water, and ions

channels

• fast, usually ions

• ligand-gated → ACh receptors

• voltage gated

transporters

• DOES NOT REQUIRE ATP

• membrane proteins that move specific molecules or ions across the cell membrane

uniporter

• transports 1 ion in 1 direction

symporter

• transports two different ions in one direction

antiporter

• transports 2 different ions in two different directions

ATP pumps

• use ATP to move ions against their gradient

hydropathy plots

• graph showing hydrophobic regions

• each spike (above x-axis) = 1 membrane pass

MDCK cells

• used to study junctions; have 4/5 types

adherens

• cell-cell adhesion → sticks cells together

• uses E-cadherin → requires Ca2+

• links to actin

desmosomes

• strong “spot welds” → act as rivets or bolts

• uses desmoglein and desmocollin

• linked to intermediate filaments (keratin)

hemidesmosome

• anchor to basement membrane (“floor”)

• cell → ECM

tight junction

• seals the space between cells → blocks movement in between cells

• occlusion → prevents leakage → found in kidney, intestine, bladder

gap junction

• channels between cells → communication

• passes ions and small molecules

• functional unit: connexons

neurochemical junction

• not found in MDCK cells

• neuron communication

• uses Ca2+ → neurotransmitter release

receptor

• protein that bind ligands → change cell behavior

• outcomes are short or long term

ligand

molecule that binds receptor

hydrophobic ligands

• ligands that can pass through the membrane

hydrophilic ligands

• ligands that can’t pass the membrane → bind membrane receptors

agonist

• activates a receptor

antagonist

• inhibits a receptor

ligand-gated ion channels

• fastest

• Ex) nicotinic ACh receptor

• opens → Na+/K+ flow

receptor mediated endocytosis (RME)

• slowest (60-90 minutes)

• internalization pathway (taking things from outside the cell and internalizing it)

• transferrin receptor

• low pH required for release

contact signaling

• ligand interacts with receptor through plasma cell membrane attached proteins

• very small → measured in Angstroms

synaptic signaling

• neurochemical signaling

• measured in nm

autocrine

• signaling from one part of a cell to another part of the same cell

paracrine

• cell to cell signaling

• measured in um

endocrine

• signaling all around the body

• can be up to meters

pheromones

• chemical signals between organisms

steroids

• hydrophobic → intracellular receptors

• two domains: hormone-binding, transcription activating

bafilomycin

• blocks proton pump

chloroquine

• raises pH

low-density lipoprotein (LDL)

• serves as a ligand

• made up of cholesterol center, phospholipids, and apolipoprotein B

apolipoprotein B

• protein that binds the LDL receptor

familial hypercholesterolemia (FH)

• genetic disease that causes LDL to precipitate around the body → fat pockets around the body

• LDL receptors are under produced → so LDL is not broken down properly

G-Protein Coupled Receptors (GPCRs)

• 7-pass (serpentine) integral proteins that translate external signals into intracellular actions

• 35% of drugs target them

x-ray crystallography

• used to analyze GPCR structure

radiological binding assays

• used to determine competitive agonists or antagonists of GPCRs

competitive binding assays

• used to measure receptor function of GPCRs

GEF

• activates GTPase proteins

GAP

• deactivates the proteins

muscarinic receptors

• GPCRs that will open up K+ channels and slow heart rate

visual transduction

• Dark: -30 mV

• Light: hyperpolarization

second messengers

• small intracellular molecules or ions that relay and amplify signals received by cell-surface receptors to effector proteins within the cell

• cAMP, cGMP, Ca²+, DAG/IP3

cytokine receptor

• single ligand

• undergoes dimerization

• JAK → phosphorylates tyrosine