BICD 110: midterm 1

nucleus

-separates transcription and translation
-2D structural support network composed of lamin intermediate filament proteins
-outer membrane connected to ER
-ER studded with ribosomes synthesizing membrane and secreted proteins

nucleolus

site of ribosomal RNA production and assembly of ribosome components

golgi and ER

-ER membrane is largest cellular membrane and forms network of cisternae
-smooth ER synthesizes lipids
-rough ER ribosomes synthesize membrane and secreted proteins
-vesicles from ER ->golgi -> proteins modified by glycosylation
-vesicles from golgi carry modified proteins

mitochondria

-25% of cytoplasmic volume
-outer membrane: large pores allow molecules to move from cytosol to intermediate space
- inner membrane: many cristae infoldings into central aqueous matrix compartment increases surface area as main site of ATP production
-has its own genome; most nuclear encoded

lysosomes

contains enzymes that degrade polymers into monomeric subunits

cytoplasm

microtubules, microfilaments, intermediate filaments

-control cell structure, cell motility, and intracellular motility of organelles and chromosomes during mitosis

growing cells for experiments

animal cells have to be grown in culture under conditions that mimic their natural environment: cell media supplemented with AA and growth factors

adherent cells

need to adhere to a solid surface to grow
- have cell-surface proteins called cell-adhesion molecules that cells use to bind to adjacent cells and components of the ECM

primary cells

cells isolated from a tissue; have a finite life span

transformed cells

cell derived from tumors; can go indefinitely

cell line

culture of cells with an indefinite life span that is considered immortal

stable cell line

cell line modified in the lab to express a specific gene
- GFP tag; selection on antibiotics

HeLa

Henrietta Lack's cells were the first human cell lines
-1952 obtained from uterine cervix tumor
-human cell lines are often derived from cancer
-others have been rendered immortal by transforming them to express oncogenes

how to culture cells

1. harvest cells
- Trypsin (enzyme) and EDTA (Ca2+ chelator) cause cells to detach
2. apply isolated cells in a culture dish with growth media
3. incubate
4. subculture every 3-5 days to obtain pure culture or to bypass problems

FACS

fluorescence-activated cell sorter separates cells having different levels of fluorescence

1. labeled cells mixed with sheath fluid buffer solution pass single file through laser light beam
2. both fl light emitted and light scattered from each cell are measured and used to determine cell shape and size
3. forcing the cell suspension through a nozzle forms tiny droplets containing at most a single cell; each has a negative charge proportional to fl of that cell
4. passage through an electric field differentially deflects the droplets into different bins

GFP protein tagging

used to observe localization and movement in a live cell
- need circular DNA bc its harder to detect; need to trick cell that there is no DNA in the cytoplasm

ectopical protein vs endogenous

ectopical proteins are introduced from the outside; transfection results in ectopical proteins

endogenous proteins exist in the genome; incorporated into the DNA

equilibrium density-gradient centrifugation

a cellular component floats at its equivalent density in a glycerol or sucrose density gradient
- proteomic analysis can identify all the protein components in a preparation of a purified organelle

monoclonal antibodies

bind one epitope on an antigen, can be produced by injecting a protein of interest into an animal, are important for basic cell biology research, and can be used as therapeutic agents

advantages of immunofluorescence

can do multiple tags, can use patient cell lines, can use tissue

membranes

formed by lipids (noncovalent association of their building blocks)
- primary component is phospholipids

why are membranes important and what are their requirements?

defines biochemical environment, establishes pH, allows biochemical processes to occur in parallel with our interfering

stable, fluid, impermeable, and self assembly

why are membranes fluid

lipids can move 10^7 times/sec so they are easily displaced by the lipids near them

lateral diffusion of lipids: FRAP

attach a dye to every lipid and then shine a laser into part of the lipid
- the lipids it goes through are labeled and a hole forms where fluorescence can be measured
-bleach the lipid and record the gradual recovery of fluorescence; the bleached lipid doesn't come back but the other lipids take its place
-can infer the diffusion constant of lipids by measuring the time of recovery

flipping

happens once a month
-caused by flippases
-important to maintain sidedness

homeoviscous adaptation

cells optimize the choice of phospholipids to maintain fluidity
-adding heat makes more fluid

shorter fatty acids are more fluid (less overall interaction)
desaturated fatty acids are more fluid (DB sterically hinder interactions)

saturated fatty acids

linear and pack closely together in crystalline layers
- hydrophobic and van der waals stabilize
-high melting point
-longer chain = more interactions = higher melting point

unsaturated fatty acids

desaturation causes a kink in the hydrocarbon chain that prevents close packing
- bend at each DB which inhibits tight packing through steric hindrance
- causes interactions to be weaker so MP is lower

cholesterol

lowers permeability (tightly packed)

moderates membrane fluidity:
- at low T increases fluidity (prevents right packing)
-at high T decreases fluidity (due to rigid structure)

Approximately how long does it take a lipid to go from one pole of an E. coli cell to the other? D~10^-8 cm^2/sec=1 um^2/sec

t=x^2/2D

flippases

IMP that play a key role in maintaining membrane asymmetry

use ATP hydrolysis to facilitate movement of lipids

recognize specific head groups

phospholipids are in charge of the _____ barrier

permeability

transmembrane transport: simple diffusion

gases and small hydrophobic molecules can diffuse freely
rate of diffusion is slow, can be accelerated by proteins

transmembrane transport: passive transport

uncharged polar molecules are passively transported down a concentration gradient

charged polar molecules are transported based on concentration and charge (electrochemical gradient)

Donnan Effect

describes the behavior of charged particles in solutions separated by a semipermeable membrane that doesn't allow some of the particles to pass

cells have a negative net charge in the cytoplasm

membrane proteins

integral membrane proteins: span the bilayer and form dimers and higher order oligomers

lipid-anchored proteins: tethered to one leaflet by a covalently attached hydrocarbon chain

peripheral proteins: associate primarily by specific noncovalent interactions with integral membrane proteins or membrane lipids

membrane proteins in the transmembrane domain

20-30 aa
nonpolar
alpha helix

membrane transport proteins

form a protein-lined pathway across the membrane that enables hydrophilic substances to move through the membrane without contacting its hydrophobic interior

channels, transporters, ATP-powered pumps

channels

10^7-10^8 ions/sec
facilitate movement of specific ions down their electrochemical gradient

nongated: open all the time
gated: open only in response to specific chemical or electrical signals

transporters

10^2-10^4 molecules/sec
uniporter: transport a single type of molecule down its concentration gradient

symporters and antiporters: energy available from movement of one or more ions down an electrochemical gradient drives movement of one molecule against its concentration gradient (secondary active transport)

features that distinguish uniport from simple diffusion

1. rate of substrate movement by uniporters is higher than simple diffusion
2. the transported molecule partition coefficient K (energy needed to go into the hydrophobic core) is irrelevent because the molecule never enters the phospholipid bilayer hydrophobic
3. the maximum rate depends on the number of uniporters
4. transport is reversible but always down a concentration gradient
5. transport is specific

ATP-powered pumps

10^0-10^3 ions/sec

energy released by ATP hydrolysis drives movement of specific ions or small molecules against their electrochemical gradient

4 classes of transmembrane proteins

couple energy released by ATP hydrolysis with energy-requiring transport of substances against their concentration gradients

P-class, V-class, F-class, ABC superfamily

P-class pumps

generate ion gradients across membranes

two catalytic alpha subunits- phosphorylated as part of transport cycle
two beta subunits- present in some pumps, may regulate transport

Ca2+ ATPase

GLUT1

catalyzes movement of glucose into erythrocytes at 50,000x the rate of uncatalyzed diffusion
- transports D-glucose into erythrocytes with the gradient (passive)
- speeds up the rate of equilibration

P type ATPase: SERCA Ca2+ pump

exports two Ca2+ per cycle to maintain homeostasis
1. Ca2+ and ATP bind the transporter on the cytoplasmic side
2. The N domain phosphorylates the P domain (on Asp351)
3. Phosphorylation triggers a large conformational change
4. Ca2+ is delivered to the extracellular side of the membrane
5. A domain dephosphorylates the P domain, reversing the conformational change
6. Pump is ready to begin another transport cycle

Na+K+ATPase

antiporter that regulates sodium and potassium homeostasis
- both are transported against their gradient
- charge difference makes the inner leaf of the cell membrane more electronegative (membrane potential) which is critical to cellular function

V-class pumps

most transport only H+
no phosphorylation intermediate
couple ATP hydrolysis to transport of H+ against a concentration gradient
generate low pH of plant vacuoles, lysosomes, and other acidic vesicles in animal cells by pumping protons from the cytosol into the organelle

F-class pumps

most transport only H+
no phosph. intermediate
work in reverse to V-class pumps; use energy in a proton concentration or voltage gradient to drive ATP synthesis in mitochondria and bacteria

ABC superfamily

-made of transmembrane domain and ATP binding domains
-couple ATP hydrolysis to solute movement
- ABC proteins are specific for a single substrate or group of related substrates

ABCB1

aka MDR1
- first eukaryotic ABC protein found in drug resistant cancer cells
-uses ATP hydrolysis to export a variety of related rugs from the cytosol

transcellular transport of glucose from the intestinal lumen into the blood

*look at and understand picture on slide 11 of lecture 5

tight junctions in polarized cells:
- establish biochemically distinct apical and basolateral plasma membrane regions that contain different transport proteins
- limit glucose transport through the paracellular pathway between the cells

transcellular transport of glucose from the intestinal lumen into the blood mechanism

Step 1: Generation of Na+ gradient and electrical potential - Basolateral Na+/K+ ATPase generates/maintains Na+ and K+ concentration gradients. Outward movement of K+ ions through nongated K+ channels generates an inside-negative
membrane potential across the entire plasma membrane.
Step 2: The Na+ concentration gradient and the membrane potential both drive the uptake of glucose from the intestinal lumen by the apical two-Na+/one-glucose symporter.
Step 3: Glucose transported out of the cell into the blood by the basolateral GLUT2, a glucose uniporter

receptor-mediated endocytosis

used to import selected extracellular molecules
-specific receptors recognize and bind to an extracellular ligand
-clathrin-coated pits and vesicles provide an efficient pathway for taking up molecules

ex: cholesterol, iron carrying proteins, hormones, glycoproteins, viruses

dynamin

GTPase that polymerizes around the neck and hydrolyzes GTP into GDP to drive the pinching off od CCVs
- in presence of non-hydrolyzable GTP, buds form but cannot pinch off
- in presence of mutant dynamin that cannot bind GTP, cells do not form CCVs

uncoating and fusion to endosomes

mediated by HSP70 which is an ATPase that uses energy to peel off the clathrin coat
-uncoated vesicles fuse with early endosomes
-acidic pH in endosomes (H+ ATPase pump) causes detachment of ligand from receptor

low density lipoprotein

cells take up lipids from the blood in the form of large, well-defined lipoprotein complexes

classes:
- shells composed of apolipoprotein and a phospholipid monolayer containing cholesterol
- hydrophobic core composed mostly of cholesteryl esters/triglycerides

LDL particle- contains only a single molecule of one type of apolipoprotein wrapped around the outside of the particle

endocytic pathway for internalizing low-density lipoprotein

cholesterol molecules are packed in LDLs which is surrounded by a protein and phospholipid layer => recognized by LDL receptors on the surface of cells

adaptin binds to tail of LDL receptor and recruits clathrin molecules which coats the membrane and causes membrane to bend and form a vesicle inside the cell

once inside, the vesicle uncoats and fuses with the endosome

the endosome has low internal pH which causes LDL receptors to release their cargo => LDL receptors are recycled

LDL particles need to be disassembled; endosomal content is transferred to lysosome which contains hydrolytic enzymes that digest the particles; free cholesterol is released into cytosol to be used in the synthesis of new membranes

imaging subcellular details: fixed cells

fixed with a common fixative such as formaldehyde or gluteraldehyde that crosslinks AA on adjacent molecules that makes cells stable; tissue is usually embedded in paraffin for sectioning (cells are thin enough for direct visualization and don't need to be sectioned)

imaging subcellular details: permeabilized

with non-ionic detergent that pokes holes in cell membranes, making plasma membrane permeable to reagents (antibodies for fluorescence or EM, fluorescent dyes)

imaging subcellular details: stained

with fluorescent dyes or gold particles that are covalently attached to specific antibodies, with heavy metals that stain different biomolecules to gain contrast, or with small fluorescent dyes that bind to membranes, DNA, or other structures

light microscopy: phase-contrast

enhances contrast in unstained cells by amplifying variations in density

light microscopy: fluorescence

shows locations of specific molecules tagged with fluorescent dyes or antibodies

electron microscopy: scanning electron microscopy

shows a 3D image of the surface

electron microscopy: transmission electron microscopy

shows ultrastructure of organelles in thin section

light microscopy: pros cons

*live or fixed cells

pros: good for visualizing size, morphology of live cells or stained fixed structures

cons: bad for viewing specific proteins/molecules or viewing subcellular structures

fluorescence microscope: pros cons

pro: good contrast between stained and unstained region, quantitatively measure protein/target molecules , can be used with living cell

con: photobleaching, photon toxicity, not the best resolution, blurry for thick specimen

confocal fluorescence microscopy

-optical sectioning

modified version of FM
-photon toxicity/photobleaching occur
-pinhole aperture blocks scatter light that might blur the image

deconvolution microscopy

computer image processing improves contrast/reoslution of digital image
- used with image received from FM
- could be used as an alternative to confocal microscope

total internal reflection fluorescence microscopy

- evanescent waves used to minimize background noise which is a pro and a con because its limited to samples that are immediately adjacent to the coverslip
- good for studying membrane dynamics and proteins involved in cell-cell interactions

super resolution light microscopy

as two points come close to each other, the interference makes it difficult to distinguish them
- fluorescence microscopies spatial resolution is limited by the diffraction barrier. SRLM overcome this

-have a small number of molecules fluoresce at one time with a short pulse of activating light => few molecules get activated

tomography

get images of specimens tilted at different angles to make a 3D density map

generally use unstained, vitrified biological specimens

FRET

fluorescence resonance energy transfer

when 2 fluorescent proteins are close enough, en excited donor transfers energy to an acceptor group

FACS sorting

fluorescent data:
- cells labeled with fluorescent ab specific for a certain cell marker
- give +/- charge to cells based on labeling
- cells sorted into collection tube by their charge