Terms Flashcards
erythrocytes
red blood cells
simplest type of blood cell
holds hemoglobin which transports O2 and CO2
lymphocytes
white blood cells
electron microscopy
uses electrons as source of illuminating radiation
wavelength of electrons goes down as velocity goes up
electrophoresis
SDS-polyacrylamide gel electrophoresis
gel is stained with dye that reacts w/ proteins
each band represents a protein w/ a different molecular weight
- higher MW = slower
- lower MW = faster
differential centrifugation
separated by size/density used when purity not important low speed centrifuge - nuclei/whole cells/cytoskeleton at bottom mid speed centrifuge - mitochondria/lysosomes/perioxisomes high speed - microsomes and small vesicles
gradient centrifugation-
slow heavy dense stuff at bottom. poke hole in bottom and collect
B cells
in bone marrow
produce antibodies
humoral immune response
T cells
thymus
cell immunity
antigens
invaders – recognized BY antibodies
antibodies
recognize antigens
epitopes
site where antibodies bind to antigens
many on a single molecule
complementarity
ability of molecules to combine w/ one another
like a lock and key
Immunoglobins (ig)
among most abundant protein components in blood
recognize foreign molecules and initiate events leading to their destruction
fluorescence microscopy
fluorescently label antibodies to detect binding and localizations of these antibodies w/ antigens
antibody - antigen bonding
NONCOVALENT
ionic, hydrogen, van der waals
association constant can range from 10^4 - 10^6 L/mol
extreme specificity
- complementarity. only works with each other
multivalence
- multiple sites for bonding
- (Ig has 2)
- leads to increase in binding strength (avidity)
- allows formation of lattices = precipitate in solution
papain
cuts Ig in 2, creates monovalent fragments w/ ONE binding site
Fab vs Fc fragments
membrane function
- forms boundaries to separate environments from one another
- helps maintain differences in compartments by selectivley allowing things to enter/exit
- receives signals from environment and transduces them to inside
- plays a major role in establishing cell migration and association
- hydrophobic interior is exclusive site for some reactions
- gives identity to cell
Fluid Mosaic Model
lipid bilayer w/ proteins sticking out
proteins floating like icebergs in sea of lipids
membrane fluidity
- diffusion of protein only within the plane of the membrane
- fatty tails can flex, rotate or move laterally BUT CANNOT FLIP FLOP
- fluidity depends on temp, composition (shorter tails = more fluid)
integral membrane proteins
proteins that require detergent to release them from the membrane
either SPAN the membrane or are HYDROPHOBICALLY embedded in a portion of the membrane
- transmembrane proteins extended across membrane as alpha helix of 20-30 AAs
- or extend across membrane as rolled up beta sheet. each strand in barrel = 10 AAs
peripheral membrane protesn
attached to the membrane by noncovalent interactions with other proteins
can be removed from the membrane by treatment w/ high or low ionic strength OR extreme pH
synthetic bilayers
can form lipid bilayer by dropping phospholipids into water
energetically favorable
asymmetry of membranes
membranes are composed equally of proteins and lipids (30-50% each) and 1-10% carbohydrates
electron spin resonance (ESR)
introduce nitroxide radical into head group of lipid
contains an unpaired electron which emits a paramagnetic signal that can be detected by ESR
use this to determine whether lipid is on inside or outside of bilayer
lipid vs diffusion coefficient
LIPIDS ARE FASTER THAN PROTEINS
FRAP
fluorescence revory after photobleaching
tight junctions
help confine membrane proteins to apical or basal side of cell (often in epithelial)
black membrane
artificial lipid bilayer usually in water
semipermeable
ion channels
formed by integral membrane proteins
selectivity for certain ions
Bacterial K+ channel
K+ must lose its bound water molecules to enter the filter and interact w/ carbonyl groups lining the filter that are rigidly spaced at the exact distance to interact with K+
Na+ is smaller and these carbonyl groups won’t interact with it
patch clamp method
current through individual channels can be measured
detach patch of membrane with a micropipette
electronic device called the clamp maintains the membrane potential at a set value while recording the ionic current through individual channels
ionophores
tools cell biologists use to increase the permeability of membranes to specific ions
mobile ion carriers
carry an ion across the lipid bilyaer
channel forming ionophores
forms a channel in which other ions can get across lipid bilayer
passive transport
molecules flow in energetically favorable fashion down energy gradient
active transport
goes against concentration gradient and thus requires energy
- energy can be ATP driven, light driven, or a coupled carrier
primary active transport
dependent on direct hydrolysis of ATP
ATPases directly couple ATP hydrolysis to ion transport
P-type transport proteins
Na+ K+ ATPase is an example of this
maintains difference in sodium and potassium in cells
secondary active transport
energy stored in ion gradients can drive transport system
ex: Na+ - glucose transport
goes back and forth switching b/t Na+ gradient and glucose gradient. each facilitating the transfer of the other
uniport
just one molecule goes through carrier mediated transport
symport
transported molecule + co-transported ion
antiport
transported molecule + other molecule coming in from other side
hypothesis for evolutionary origin of nucleus
ancient procaryotic cell took up membrane which surrounded DNA. also took up ribosomes – formed ER
origin of mitochondria and plastids
pre-eukaryotic cell engulfed an prokaryotic cell which gained membranes and became mitochondria – have their own set of DNA
cytosol + nucleus + intermitochondrial matrices
REDUCING AGENTS
rest of cell + lumen
OXYDIZING ENVIRONMENT
microsome
lumen = inside = oxidizing environment
signal sequence
at amino-terminus of protein (side with N)
Signal Recognition Particle (SRP)
extracted from ER with salt
composed of 6 protein subunits and 1 RNA molecule
recognizes signal
arrests translation
binds to the signal and the ribosome and stops translation
SRP receptor is integral membrane protein
translocator
sec61 complex consists of 3-4 protein complexes, each composed of transmembrane proteins that can assemble into a donut like structure
transmembrane proteins
contain stop-transfer signals that anchor that portion of the protein in the membrane
can have signal sequences that are located a distance away from the amino terminus
can have several stop-start sequences
signals
ER: simple H2N —— COOH. signal at H2N side
Mitochondria: an amino-terminal amphipahtic alpha helix with positively charged residues on one face and hydrophobic residues on the other face
Peroxisomes: signal is at the carboxyl-terminus of protein - SKL
Nucleus: all contain 4-8 positively charged AAs. example = PPKKKRKV - can be anywhere in protein. not cleaved off after transport
chaperones
family of proteins that bind to protein in unfolded or denatured state
recognize hydrophobic patches on the protein
help proteins stay in unfolded state to promote correct folding of proteins that will remain in cytosol
need ATP hyrdolysis to relase chaperone from protein – can be bypassed with urea or denaturing agent that artificially unfolds protein
Hsp 70
family of molecular chaperones some of which are found in cytosol
gated transport
found in regulating traffic b/t nucleus and cytosol
nuclear transport
histones, gene regulatory proteins, DNA & RNA polymerase, RNA processing proteins must be able to get IN from cytosol
tRNAs, processed mRNAs, ribosomal subunits must be able to get OUT
nuclear lamina
supports inner membrane of the nucleus
meshwork of nuclear lamins
gives shape and stability – provides structural link b/t DNA and nuclear envelope
Co-and Posttranslational Modifications and Folding of proteins in ER
as proteins pass into the lumen of the ER, they get modified
- removal of signal sequence
- oxidation of sulfhydryl groups
- N-Glycosylation
- attachment of glycosylphosphatidyl-inositol (GPI) anchor (only in some proteins)
- protein folding (chaperones present in ER help proteins fold. include BiP)
N-Glycosylation
removes glucoses from the carbohydrate unit in he ER so that the protein is correctly folded and can move on to the secretory system.
secretory system
proteins first enter thi sytem by transiting to the lumen of ER
all proteins utilizing secretory patways must have signal for ER
golgi apparatus
has ordered series of compartments called cisternae
has cis Golgi network, cis cisterna, medial cisterna, trans cisterna, and trans Golgi network (5 parts)
proteosomes
digest incorrectly folded proteins that are tagged with polyubiquitin
KDEL
at the COOH terminal end of a protein = retention signal for soluble proteins that must remain in the ER
lysosomes
function as main site of digestion of intracellular nd extracellular debris including macromolecules and obsolete parts of the ell
vacuoles
= lysosomes in plants and fungi
signal patch
proteins destined for lysosomes undergo extra processing in cis-Golgi and get a signal patch.
protein in cis-Golgi acts as receptor for this signal patch and will bind to all proteins that have it. phosphorolates mannose
Glycoprotein goes through medial and trans Golgi app where GlcNAc is removed to expose the phosphoylated mannose
mannose acts as a signal for lysosomes
regulated secretion
some cells are specialized to carry out the regulated secretion of secretory proteins only in response to a specific signal
the cell stores these proteins in special secretory vesicles in the cytosol until needed
default pathway = constitutive secretory pathway
non-selective pathway that targets proteins and lipids in the ER that are not destined for other locations
ER –> Golgi –> cell exterior
endocrytic pathway
leads inward from the secretory pathway
vesicular transport carries glycoproteins through this system and they can undergo various modiications as they proceed through diff compartments
receptor mediated endocytosis
process by which an external ligand binds to a receptor on the cell surface and is then internalized to endosomes and then passed on to lysosomes
vesicular transport
budding - vesicle formation
docking -vesicle recognition
fusion - fusion of vesicle with target compartment
budding
involves accumulation of coat on cytosolic side of membrane
ex: clathrin binds, squeezes forms vesicle, breaks apart
docking
complementarity is important
Rab proteins guide the targeting of the vesicles (on surface of vesicles) bind with Rab receptors on the target membrane
SNARES mediate docking and membrane fusion
v- SNARES on vesicle bind to t-SNARES on target membrane
SNARES mediate membrane fusion. lipid bilayers must be within 1.5 nanometers of each other + water must be displaced from the hydrophillic surface of the membrane
monomeric GTPases
proteins act as molecular switches to insure the direction and fidelity of transport through the secretory system
flip between active w/ GTP attached and inactive w/ GDP bound
w/ GTP, GTPase protrudes a tail and is inserted into budding membrane.
controlled by regulatory proteins
- GEFs -catalyze exhange of GDP for GTP
- GAPs - hydrolyse GTP to GDP
organelle biogenesis
organelles are unique each w/ their own complement of lipids and proteins
must be inherited during cell division
EPIGENETIC in that at least one distinct protein that exists in the organelle membrane is also required along with info thats not exclusively in DNA
cytoskeleton
scaffolding that supports the cytosol. up to 30% of cellular material composed of actin (microfilaments) 7 nm diameter intermediate filaments 8-10 tubulin (microtubules) 25 nm dynamic and flexible
intermediate filaments
role is primarily to resist stress (mechanical stress)
contains heptad repeat motifs. two coiled coil dimers associate antiparallel
then stagger into tetramers then two tetramers packed together and so on
microtubules
long stiff polymers extend throughotu the cytosol
hollow formed from tubulin
tubulin heterodimer = microtubule subunit
- beta on top, alpha on bottom
these dimers polymerize to form a protofilament
13 protofilaments form a hollow cylinder which is the microtubule
have polarity. beta = plus end, capable of rapid growth
negative end attached to centrosome – lies next to the nucleus and serves as the nucleation center of the microtubule
microtubule dynamics
microtubules continually assemble and disassemble. GTP binding to beta subunit is important!!
with GTP bound, protofilaments are strong and straight, with GDP, curve and weak
growth with GTP capped end. with loss of GTP cap, shrinks but can regain GTP cap and regrow
microtubule associated proteins (MAPs)
some stabilize against disassembly, others link to other cell components
results in longer, less dynamic microtubules
can also organize growth in specific directions
organelle movement
organelles can move along microtubules sometimes
actin
actin filaments are polymers of G-actin
most abudant protein in many cells
polymerizes into filaments (F-actin)
ATP-actin adds on to form helican filament
polymerization of actin stimulates hydrolyis of ATP undergoes conformational change.
polarized just like microtubule. adds on to plus end
NO DYNAMIC INSTABILITY
treadmilling
ATP actin adds on plus end at same rate as ADP actin dissociates at neg end. therefore, length of filament stays constant while still getting raped exchange of subunits in cell
requires 3 actins to form nucleated structure, catalyzed by actin-related proteins (ARPs)
regulation of polymerization
50% actin as monomer. needs special mechanisms to prevent its actin from polymerizing (since it has higher conc of monomers vs polymers)
ATP-ADP exchange is slow whereas GTP-GDP with tubulin is fast so long delay before cell can reuse actin
specific small proteins bind to actin monomers
ex: thymosin - binds to actin molecules and prevents polymerization
ex: profilin - binds to monomeric actin and accelerates ATP-ADP exchange
organization of actin filaments
bundles
web-like networks
actin movement
actin polymerization can lead to movement if on an appropriate substratum
kinesins
microtubule dependent motors
move toward PLUS end
two heavy chains plus several light chains
heavy = conserved globular ATP bound head and lgight chains at tail
dyneins
move toward minus end
microtubule dependent
signal transduction
must occur accross the membrane. converts an extracellular binding event into intracellular signals that change the behavior of the target cell
G-protein coupled receptors
includes number of proteins that have similarities in sequence and mech but react w diff ligands
belong to large superfamily of homologous proteins that all make SEVEN transmembrane passes rhough the plasma membrane
G protein is the transducer which relays that the signal has bound to the receptor across the membrane to the primary intracellular target
primary intracellular target
protein that can change the concentration of a second messenger or an act as an ion channel
Gs subunit
activates adenlyate cyclase which is also primary target. cyclic AMP is produced
Golf
converts chemical signal of smells to electrical signal
activates adenylyl cyclase, increase in cAMP = cAMP gated channels to open and allows influx of Na+
Gt
functions in sight
cyclic GMP phosphodiesterase –> Na+ channels
Gq
activates Phospholipase C-beta
produces 2 messengers
- IP3. causes release of Ca++ which functions as second messenger and also activates protein kinase C. Ca++ binds CALMODULIN. which activates CaM -kinases
- Diacylglycerol activates protein kinase C.
enzyme linked receptors-
single pass transmembrane proteins
prtoein acts as the recepor, transducer, and pimary target.
inactive –> becomes active catalytic domain upon binding with signal
autophosphorylation
binding of signal causes two cytosolic domains to come together and cross phosphorylate one another on multiple tyrosine residues
SH2 and SH3 domains
SH2 domain binds to phosphorylated tyrosines
SH3 domain binds to other proteins in the cell
results in activation of Ras proteins
Ras proteins
members of family of monomeric GTPases
lipid tail that anchors them to cytosolic side of plasma membrane
relays signals from receptor kinases to the nucleus to stimulate cell proliferation and division
FRET (Fluorescence Resonance Energy Transfer)
distance dependent interaction b/t the electronic excited states of two dye molecules in which excitation is transferred from a donor molecule to an acceptor molecule (must be in close prox)
each of the two interacting components is labelled with a different colored fluorescent dye.
while they interact with one another, the emission wavelength of the fluores is changed
Ras + GTP
when Ras is bound to GTP it activates a serine/threonine phosphorylation cascade that results in activation of MAP kinases
cell cycle
cell reproduction cycle
M phase - mitosis
INTERPHASE
G1 - interval b/t end of mitosis and beginning of DNA rep
S - DNA replication phase
G2 - interval bt end of DNA rep phase and beg of mitosis
cyclin-dependent kinase (cdk)
must be bound to cyclins to have protein kinase activity
G1
cdk combines with cyclin to form start kinase
induces transcription of genes
G2
Wee1 kinase - hosphoylates a tyrosine close to the active site
MO15 kinase - activating kinase that phosphorylates a threonine in the molecule that is needed eventually for activity
CDC25 phosphatase removes phosphate from tyrosine
kinase is activated and stimulates further activation by stimulating the phosphatase and inhibiting Wee1 inase POSITIVE FEEDBACK EFFECT
gets gradual accumulatino of cyclin until rises above critical point –> drives cell into mitosis
G2 cyclins degraded at metaphase - anaphase transition
checkpoints
placces in the cell cylce at which it the cell cycle can be stopped if previous events are not completed
G0
quiet state where cells can stay.
largely varies length of cell cycle
growth factors
made by target cells which neurons take up and become larger. if neurons dont get enough GF they die
Rb gene
encodes Rb protein which helps regulate the passage of cells from G0 to G1 and past the G1 checkpoint
Cancer
due to proliferation of cells in defiance of normal controls
- mutations can make an inhibitory gene inactive
- usually recesive.
- lost gene = tumor reporessor gene - mutations that would make a stimulatory gene hyperactive
- dominant
oncogenes
hyperactive stimulatory genes that cause cancer. normally active ones = protooncogenes
apoptosis
programmed cell death.
cells die tidily - nucleus condenses, cell shrivels and changes surface chemistry so it can be recognized by macrophages. engulfed with no leakage of cystolic components
extracellular matrix (ECM)
intricate network of molecules constituting the extraceulluar space
composed of proteins and polysaccharides that are secreted by local cells and assembled into an organized meshwork in close association with the cells that produce them
- ground substance - provides tensile strength and resistance
- molecules embedded in ground substance that help promote adhesion, communication, and migration
highly organized meshwork of macromolecules. composition and orientation of macromolecules largely determined by the cells embedded in it and can be highly specialized depending on the needs of the particular cells
ground substance
compare to building. need strength such as rods and filler material such as concrete but also want flexibility.
plants use carboyhydrate fiber (cellulose)
animals use proteins (collagen)
cellulose
in plant cell walls
long unbranched chains of about 500 glucose reidues
high tensile strength
arranged in layers
take up enormous volume
cells make it outside cell or make it in small pieces and assemble them outside
collagen
animal’s cellulose
filler material
Animal ECM
- Glycosaminoglycans (GAGs) - unbrached polysacchardie chains composed of repeated disacchardie units
Plant ECM (cell walls)
- pectins - branched polysaccharides with many galcturonic acid units
- cross linking glycans
fibronectin
first bound in fibroblasts
allows them to flatten and produce actin bands
helps to organize the ECM and helps cells attach to it
also improtant for guiding cell migrations in vertebrate embryos
Integrins
transmembrane cell adhesion poteins that act as ECM component receptos and help link the matrix to the cell’s cytoskeleton
velcro principle
bind simultaneously but weakly to large number of matrix molecules, allowing cells to explore the environment w/o losing all attachment to it. thus keeps cells from being irreversibly glued ot the matrix
ECM-cytoskelton linkages
can be small and transiet or large and durable depending on the cell type
local adhesions
found in cells that hold onto ECM through their integrins that link intracellularly to actin filaments
ex: fibroblasts which grip the ECM at focal adhesions where bundles of actin filaments terminate
cell-cell adhesion
can dissociate invertebrates such as sponges into their indiv cells by treating them with an EDTA solution (common chelating agent often using to sequester calcium and magnesium from solution) and could get the cells to resassociate by removing the EDTA and adding calcium
they would sor tthemselves out and resassociate with cells of the same kind/color
cell-cell adhesion molecules (CAMs)
homophillic binding - binding of identical moecules on sperate cells to one another
heterophillic binding - binding of a molecule on one cell with a different molecule on the other cell
Cadherins
a superfam of proteins that mediate cell-cell ahesion in all animals
Mediate Ca++ dependent cell-cell adhesision
uses homophillic bidning mechanism
Neural cell adhesion molecules (N-CAM)
members of the immunoglobulin superfamily
all contain one or more Ig like domains
bind by homophilic mechanism
selectins
heterophillic binding mech
proteins that bind to carbs on surface of other cells
innate immunity
dependent on proteins and phagocytic cells that recognize conserved features of pathogens that are absent in the host
found in verts, inverts, and plants
pathogen-associated stimulants
often occur on pathogen surface in repeating patterns
pattern recognition receptors
recognize pathogen-associated immunostimulants
complemetn
a series of about 20 interacting soluble proteins that are made in the liver and circulate in the blood and extracellular fluid
phagocytosis
engulfing a solid particle
internal endocytosis
lymphocytes
develop from hemopoeitic stem cells in fetal liver and adult bone marrow (b-cells)
2x10^12 lympcytes in human body
recirulate between blood and secondary lymphoid tissues
Humoral Immune Response
production of antibodies
antibodies function by recognizing antigen and initiating events leading to their destrcuturion
Fc regions of antibodies are involved in destruction
immune response
primary response - occurs on animal’s first encounter to antigen A
secondary response - if animal is reexposed to antigen A. shows there is a memory
naive cell
will express its antibody on the surface as a receptor
antibody diversity
- combinatorial joining of gene segments. V region joins a J region
- junctional diversification - during joining of the antibody gene segments a variable number of nucleotides are often lost from the ends of the recombining gene segments. one or more randomly chosen nucleotides may also be inserted
- allelic exclusion - during B cell development get activation of only one light chain allele and one heavy chain allele.thus ony one type of antibody will be produced by each cell.
MHC cells
presents antigens to T cells
