BLOCK 1 Flashcards
prokaryotes
bacteria and archaea; lack a nucleus and internal membranes
eurkaryotes
multicellular animals; plants and fungi, unicellular protists; has nucleus and extensive internal membrane system
cell theory
- all living things are made of one or more cells
- the cell is the structural and functional unit of all living things
- all cells come from pre-existing cells by division
modern cell theory
- cells contain hereditary information which is passed from cell to cell during cell division
- all cells are basically the same in chemical composition
- all energy flow (metabolism and biochemistry) of life occurs within cells
the central dogma
activation
transcription
processing
translation
proteome
read gene sequences to predict the complement of proteins
differential gene expression
determines which genes are expressed and at what levels
transcription factors
proteins that interact with specific DNA regulatory sequences associated with genes to modulate transcription by recruiting RNA polymerases
transcriptome
genes being transcribed
epigenetics
heritable changes in the genetic potential of a cell without changes to the underlying DNA sequence
chromatin modifications
epigenetic changes that regulate the access to regulatory sequences and thus regulate transcription (methylating cytosines prevents accessibility to gene)
noncoding RNAs
regulate/ control mRNAs
microRNAs (miRNAs)
nonprotein coding; folded and cleaved into siRNAs
long non-coding RNAs
processed into siRNAs
small interfering RNAs (siRNAs)
destroys and inhibits complementary mRNAs by RNAi
RNA-induced silencing complex (RISC)
inhibits or destroys targeted RNA
primary cells
non-cancerous, non-transformed
some divide, some already differentiated
can be isolated or cultured
challenge of primary cells
not alive forever
transformed cells
cancerous cells
can be grown in culture; some model basic cell functions, others retain specialized functions
stem cells
can be isolated or induced
embryonic stem cells that have self-renew capacity, not differentiated but capacity to do so is there
light microscopy
limited in contrast, magnification, resolving power
why is contrast poor in light microscopy?
cells are transparent so they don’t absorb light and therefore contrast is poor
techniques for enhancing contrast in light microscopy
modulate phase of light using optical tools
modulate contrast of specimen
enhancing contrast: modulating phase of light using optical tools
phase contrast (strict contrast) differential interference contrast (DIC) --> 3D looking
magnification
the amount the initial image is blown up –> dependent on lenses used
resolution
how far apart two objects have to be to be seen as two separate objects
what determines resolution?
the wavelength of light used (shorter = better resolution)
the properties of lenses used (NA, width of light cone the objective gathers)
what is the resolution of a conventional light microscope?
1/2 the wavelength of light being used
airy patterns
when light interacts with a specimen, the light gets defracted into a pattern
airy disk diameter is determined by light WL (smaller WV = smaller diameter)
fluorescence microscopy
improves contrast and allows specific cellular structures to be labeled
fluorescence
when a molecule absorbs light of one wavelength and then re-emits it as a longer wavelength
fluorophores (fluors)
labels specific structures because is linked to various chemicals
fluorescence microscopy process
molecules are treated with light of a certain energy; the molecule will absorb it, kick out a photon with a certain WL, some energy will be lost; and a longer WL is transmitted (red shift)
fluorescent proteins
genetically encoded fluorescence markers that can be fused to proteins of interest at the DNA sequence level (allows live imaging)
CMV promoter
driving expression of a GFP-tubulin fusion protein in all cells
induces transfection, recruits transcription factors
tissue specific promoters
drive tissue-specific protein expression
immuocytochemistry
immunolabeling; visualizes proteins in cells
immunocytochemistry process
- fix: fixatives react, cross link, and freeze everything in the cell to nearby molecules
- permeabilize: detergent perforates membrane so antibodies can enter
- antibodies bind to target
- a fluorescent “secondary antibody” binds to primary antibody if the primary is not directly labeled
immunoblotting (western blotting)
allows us to take samples of a cell and figure out if a protein is present in the cells
immunoblotting process
electrophoresis and transfer; antibody detection; chromogenic detection
immunoisolation / immunoprecipitation
antibody specifically binds to a particular protein and the antigen is precipitated. process can be used to isolate and concentrate a particular protein from a sample
antibodies
immune proteins that bind to specific proteins; made by B cells (2 heavy and 2 light chains)
epitope
specific 8-12 amino acid sequence
monoclonal antibodies
made by isolating and cloning a single antibody-producing cell and thus recognize a single epitope; all antibodies produced are identical
polyclonal antibodies
mxiture of different antibodies produced by the host animals B-cells against various epitopes of the target protein and isolated from blood serum
transfection
to cause a foreign protein to be expressed in a cell
what kinds of things can you express in a cell with transfection?
- GFP-tagged versions of molecules
- proteins that aren’t normally present in the cell
- mutant proteins that are constitutively active (enzymes; mutate to make always active)
- mutant proteins that are dominant negative (proteins that don’t function right and block the function of the cell’s own version of the molecule
transient transfection
expression from plasmid
stable transfection
DNA integrates into genome; heritable
RNA interference (RNAi) purposes
- protects against viral RNA
- regulates stability of cell’s own mRNAs via miRNAs or siRNAs
RNAi process
miRNA and siRNA direct enzyme complexes to degrade mRNA molecules and prevent translation when transfected into cells
laser-scanning confocal microscopy
uses pinholes to deblur by eliminating light from upper and lower planes (thin, focused plane of light)
digital deconvolution
uses computational methods to deblur; predicts peak intensity of brightness
super resolution fluorescence
PALM
STORM
allows images to be taken with a higher resolution than the one imposed by the diffraction limit
STORM (stochastic optical reconstruction microscopy)
lasers are used to photo-activate fluorphores that quickly switch back off; repeating allows the center of the light spot to be calculated and mapped onto a digital image
electron microscopy
shorter wavelength than light - higher resolution (.004nm instead of 400-500nm)
TEM
SEM
TEM
transmission electron microscope
images electrons that pass through a specimen
TEM process
shining beam of electrons at a thin stained sample; electrons get deflected by metal bound to the structures of cell and a detector picks up the deflected electrons
resolution of TEM
.1-.2nm with magnetic lenses
.002nm with optical lens
SEM
scanning electron microscope
images electrons scattered by an intact object; depth of focus gives 3D image quality
SEM resolution
5 nm
SEM process
coat sample in metal stain; electrons bounce off to deflectors and image is created from that
SEM use
used to look at surfaces of structures
SEM use
used to look at surfaces of structures
electron microscopy process
samples must be dehydrated; imaging is done in a vacuum and water creates noise - must be fixed and stained with heavy metals which leaves a scaffold of what was the cellular material
immunoelectron microscopy
labeling structures in electron microscopy
attach dense particles (gold beads) to antibodies to make them visible; gold beads are electron dense, antibody sticks to protein of interest in the cell –> electrons will scatter upon hitting the bead and show where proteins are
centrifugation
differential centrifugation
gradient centrifugation
differential centrifugation
components separate depending on their densities; the less dense a material, the longer or faster it needs to spin to separate
lipid rich structures are not dense so it is difficult to separate them
gradient centrifugation
fine tunes differential centrifugation
many tubes of solution (let’s say sucrose) are made with different concentrations which have different (known) densities. A concentration gradient with the sucrose solutions is made –> sample from differential centrifugation is added and components will separate and settle out on the level where their density matches the density of the sucrose
membrane functions
separate compartments/ selectively permeable
provide scaffold for biochem activities (energy transduction)
mediate some kinds of cell-cell interactions
key element of signal transduction pathways
lipids
phospholipids, glycolipids, sterols
amphipathic
phospholipids
all have phosphate linkage to a head group and 2 fatty acid tails
phosphyglycerides, sphingomyelin
phosphoglycerides
major component of most membranes
two fatty acid chains linked to a glycerol with glycerol phosphate on head group
one saturated (straight) chain and one unsaturated (kinked)
sphingomyelin
sphingosine amino group (instead of glycerol) links to phosphate head
two saturated fatty acid chains
saturated chain
straight
unsaturated chain
kinked
glycolipids
sphingosine amino group links directly to a sugar head group (no phosphate)
two saturated tails
sterols
four ring hydrocarbons, cholesterol can increase or decrease membrane fluidity depending on conditions
structures formed from lipids in water
micelles
bilayers
micelles
small spheres with tails pointed in
bilayers
two layers of lipids with tails pointed toward each other
spontaneously forms; close upon themselves to make a continuous surface interacting with water
phosphatidylcholine
unsaturated tails provide a thinner membrane
sphingomyelin
saturated tails provide a thicker membrane
cholesterol
inserts itself into membranes and can affect properties
cholesterol + phosphatidylcholine
straightens kinked tails, increases thickness
cholesterol + sphingomyelin
doesn’t affect thickness
properties that can affect a membrane structure
size of head groups, tail shapes
lateral shifting / lateral diffusion (membrane)
a lipid’s ability to drift within a leaflet on the same plane
flexion / rotation
bending of tails from thermal energy (common)
transverse flip-flop
when lipids on opposite planes of a leaflet switch places (rare, not natural since hydrophobic heads need to come in contact with water –> requires energy)
how to determine lateral mobility of lipids
microscopy –> fluorescence recovery after photobleaching (FRAP)
FRAP
determines lateral mobility of lipids; phospholipids are labeled with a fluorescent probe; a bright laser is shined on a small spot of membrane to bleach the fluorescence on those lipids; the time it takes for other fluorescent lipids to diffuse into the bleached region demonstrates lateral mobility
leaflet
lipids are synthesized in the ER and inserted into one or the other faces of the bilayer
not randomly distributed into the plane
flipases
membrane proteins that flip-flop lipids back to their normal sides to maintain asymmetry
sphingolipids in the membrane
tend to cluster relative to other membrane lipids
microdomains
created from clustering of lipids in membrane –> lipid rafts
lipid rafts
regions of membrane with clustered lipids that serve structural and signaling properties
membrane protein functions
selective permeability signal transduction biochemical reactions cell-cell interactions membrane properties
roles of proteins
enzymatic
structural
regulatory
protein structure: primary
sequence of amino acids, determined directly from RNA sequence
linked by peptide bonds
side chain drives protein structure and function
N-terminus
start of amino acid sequence; amino group is exposed
C-terminus
end of amino acids sequence; carboxyl group is exposed
protein structure: secondary
emerges from amino acid sequence (primary structure)
hydrogen bonding of peptide backbone causes AAs to fold into a pattern
alpha helix; beta pleated sheet
protein structure: tertiary
3D FOLDING due to side chain interactions (hydrogen bonds, hydrophobic interactions, ionic interactions etc)
covalent disulfide bonds
translation
ribosomes translate mRNAs into proteins by driving sequential formation of peptide bonds between carboxyl of one AA and amino of another
N-terminal exits ribosome first
protein folding
can occur co-translationally
many ways to fold a protein but only one right way for a specific function
chaperons and chaperonins
proteins that bind during or after synthesis to aid in proper folding
HSP70
major chaperone
take proteins and hydrolyzes ATP to conformationally change chaperone protein and in turn folds the protein –> upon rebinding to ATP, chaperone releases folded protein
chaperonins
help protein folding more than chaperone – larger complexes
how to test if a protein needs a chaperone
isolate protein, apply heat to denature protein, if protein reforms and regains function upon cooling, protein doesn’t need chaperone
disulfide bonds
covalent bonds between two cysteine amino acids in tert structure
cysteine amino acid
side chains have sulfur in them –> under oxidizing conditions, will form disulfide bond which stabilize structures
disulfide bonds: do they form in the cytoplasm?
No - cytoplasm is not an oxidizing environment
oligosaccharides
large chain of sugars covalently attached to some amino acids that influence function
common in secreted proteins and membrane proteins (with ECM portion)
used as tags to mark the state of protein folding
membrane glycoproteins
oriented so that the carbohydrate chains face the EX domain
glycosylated proteins
processing in the ER and golgi does this to membrane and secreted proteins
functional domains
combination of helices and sheets can fold into a functional domain that acts as a unit but is still only part of a protein
functions of functional domains
ATP binding sites (myosin motor)
calcium binding sites
enzyme activity of a particular sort
regulation via interactions with another protein
domain shuffling
new proteins can be formed by putting together new combinations of domains
mammalian PLC
composed of functional domains found in other proteins (PH domains, EF-hand domains, X,Y domains, C2 domains)
PH domains
lipids
EF hand domains
Calcium
XY domains
cleaving
protein structure: quaternary
formed by the interaction between two or more proteins (subunits) that form a protein complex
homodimer
quat structure; complex is composed of two identical subunits
heterodimer
quat structure; if complex is composed of two different polypeptides
quaternary structure is determined by…
hydrogen bonds, hydrophobic interactions, ionic interactions, polar interactions, van der Waals interactions, covalent-disulfide bonds
affinity
protein binding - proteins with longer lasting associations, due to more non-covalent interactions have a higher affinity for each other
on rate
dependent on starting material concentrations; determines how often they are likely to encounter each other in the first place
on rate is dependent on
rate of diffusion, size of molecules, whether there is a favored orientation required for binding
off rate
dependent on the Koff rate constant
Koff rate constant
depends on the sum of the forces that will hold A and B together
what truly determines the affinity of a reaction?
Koff rate constant
higher affinity reaction
higher concentration of complexes at equilibrium
Kd
equilibrium dissociation constant
smaller Kd
higher affinity, smaller Koff
larger Kd
lower affinity, larger Koff
classes of membrane proteins
integral membrane proteins, lipid anchored proteins, peripheral membrane proteins
integral membrane proteins
tightly associated with lipid bilayer
amino acids interact directly with the lipid portion
transmembrane proteins span the bilayer one or more times while others associate with only one leaflet
lipid anchored proteins
covalent addition of a lipid to a protein anchors the protein to the membrane
some can cycle between membrane-bound and soluble forms
lipid anchored proteins: fatty acids
added to attach proteins to the inner leaflet
lipid anchored proteins: glycophosphatidylinositol (GPI)
are added to attach proteins to the outer leaflet
peripheral membrane proteins
indirectly attached to the membrane via interactions with other membrane proteins, not lipids – localized
immunolocalization
using immunofluorescence or immuno-EM to immunilocalize the protein to the membrane vs. the cytosol
(antibodies with fluorescent tag)
process of immunolocalization
A) purify membrane and determine which proteins are present
- break cells by homogenization
- membranes have different density than other molecules so can be separated with sucrose gradient centrifugation
- different membranes of the cell have different compositions so can be separated from each other
- special detergents can be used to dissolve the membranes but keep the membrane protein active for immunoblotting or biochem. assay
B) purify the protein and show that association with membrane lipids is required for its function (proteases)
proteases
cleave or digest accessible protein regions and can be used to deduce the topology of a protein in the membrane
digestive proteases
break down the protein into many small, non-functional, peptide fragments and amino acids
trypsin
cleaving proteins
cleave proteins at a specific sequence, thus generating larger, intact, potentially functional fragments
transmembrane protein region structure
proteins are hydrophobic or amphipathic alpha helices
single pass transmembrane protein
cross the bilayer once; one helix
multipass transmembrane protein
crosses the bilayer 2 or more times; 2+ helices
what is the length of an alpha helix needed to cross the membrane?
20-30 amino acids (~4nm)
different lipid composition gives rise to different bilayer thickness, so the length of a membrane protein’s hydrophobic alpha helix will influence what kind of lipid composition the protein “wants” to be in
hydrophobic alpha helices
favorably interact with the inner hydrophobic region of the membrane
typical of single pass transmembrane proteins or isolated transmembrane domains
multiple amphipathic alpha helices
can associate in the membrane to form water filled channels or pores
what can associate in the membrane to form water filled channels or pores?
amphipathic alpha helices
single amphipathic alpha helices
can also allow a protein to associate with one leaflet of the bilayer
beta barrel
beta sheets can interact with membranes –> nonpolar on one side, polar on the other and rolled into a barrel –> forms a pore through the membrane
can a protein leave the membrane once inserted?
no - too much energy is required to tear the hydrophobic region of the hydrophobic layer
can the topology of a protein change once inserted into the membrane?
no - if it is made with X transmembrane domains, it will stay that way
can the conformation of a protein change once inserted into the membrane?
Yes - shape changes allow proteins to pass signals from outside or inside
can proteins move laterally within the membrane?
some, but not all
fluid mosaic model of membranes
the lipid bilayer is a flexible 2D sheet in which membranes float in
membrane domains
proteins that are restricted in their location to a particular region of the membrane; may be evenly distributed while others are restricted
can be restricted in one type of membrane and not another
mechanisms to non-randomly distribute proteins
link them to other membrane proteins
link them to outside molecules
link them to inside molecules
prevent their diffusion to parts of the membrane
methods to measure membrane protein mobility
Cell fusion
FRAP of fluorescently labeled protein
single particle tracking
cell fusion
proteins of one cell labeled with red dye
proteins of a second labeled with green
fuse the membrane of two cells to form heterokaryon
watch two dyes –> overtime will mix
FRAP of fluorescently labeled protein
connect protein to fluorescent tag and transfect it into cell with bleach
if fluorescence recovers, proteins are mobile
single particle tracking
attach visible tracker to desired membrane protein (gold beads attached to antibodies) to trace movement of protein
lipid rafts
made of cholesterol, sphingolipids, proteins
includes different proteins than non-raft areas
longer helix not happy in thinner bilayer
ex: GPI linked proteins
transport proteins
enzymes that catalyze movement of substances across the membrane
passive transport
facilitated diffusion
allows net movement down a gradient with ion channels and uniporters
active transport
moves molecules against a gradient requiring energy with ATP pumps, symporters, and antiporters
carriers
specific, initially rapid and maxes out, largely conformational, high affinity interaction
passive transport domains
carriers, channels
channels
small conformational changes, low affinity interaction
kinetics of transport: carrier-mediated
transport peaks on carrier-mediated transport when binding sites are saturated due to high affinity interactions
hypotonic
net water in, swelling cell
hypertonic
net water out, cell shrinks
isotonic
no net flow; when the total number of particles in and out are equal, the osmolarity is the same on both sides
uncharged molecular movement
move on concentration gradients
charged molecule movement
move on electrochemical gradient
what determines Vm?
conductances for multiple ions; will be closer to Eion for the ion with greatest conductance
ion channels
multipass transmembrane proteins form a pore
bidirectional down a gradient
ion specific, fast
regulated by gating mechanisms
voltage gated K+ channel
tetramer with multiple membrane domains
N + C termini are intracellular
opened by depolarization, selective for K+
ion selectivity filter for K+ channel
ions have hydration shell - Na+ is too small for this selectivity filter; opening the channel requires a small change in conformation
patch clamping
flux through ion channels is so fast that currents flowing through a single channel can be recorded
uniporters
multipass transmembrane domains that act as an enzyme
substrate binding induces reversible conformational change
functionally bidirectional but down an electrochemical gradient
slower than channels
GLUT transporters
uniporter; 12 transmembrane domains (N+C intracellular)
mM affinity for glucose
GLUT4 is stored in vesicles and inserted into muscle or fat cell membrane in response to insulin; regulated secretion to put protein on membrane surface
ATP pumps
hydrolyze ATP and use energy to move 1 or more molecules across the membrane
P type pumps
become phosphorylated (Na+/K+ ATPase, K+/H+ ATPase)
V type pumps
vesicular H+ ATPases
no phosphorylation, pumps H+ into membrane compartments
acidification of endocytic vessels, lysosomes, golgi
Na+/K+ ATPase
3Na+ in / 2K+ out both against their gradients
binding of Na+ changes shape allowing for ATP to bind (autocatalytic reaction), phosphorylation changes shape and allows Na+ to be released; K+ binding allows release of Pi group
ouabain
plant compound that blocks Na+/K+ ATPase and prevents reestablishment of gradients
K+/H+ ATPase
P type; stomach acidification
Ca2+ ATPase
P type; pumps Ca2+ out of cell or into ER
ABC
ATP binding cassette
no phosphorylation; cancer cells overproduce this channel type and pump drugs out of cell –> become resistant
cystic fibrosis gene product is an ABC for Cl- in lungs, sweat glands, kidneys
symporter / cotransporters
both molecules move in the same direction; uses energy from molecule moving down its existing gradient to move a second up its gradient
large conformational changes
Na/glucose symporter
2 Na out, 1 glucose in; concentrates glucose from intestine into epithelial cells; works against glucose gradient using the Na+ gradient
can accumulate glucose 30k fold
antiporters
two ions moving in two directions, one along its gradient
carrier mediated and pronounced conformational changes
glucose absorption in the intestine
Na+/glucose transporter
facilitated glucose transporter
Na+/K+ ATPase
nerve cell signaling
action potentials ya ya ya ya ya ya info is in frequency of it ya know
initiation AP
nerves, mechanical, sensory
gating mechanisms
voltage, ligand, mechanical
cardiac
u kno dis
signal transduction: hydrophobic signals
can cross membrane directly
steroid hormones, NO, CO
steroid hormones
receptors in cytoplasm or nucleus
NO/ CO
dissolved gas regulates many pathways and can enter cell easily
signal transduction: hydrophilic signals
cannot cross the membrane; instead needs to indirectly signal across the membrane by binding to transmembrane proteins
types of signals
chemical messenger signaling, contact-dependent signaling
chemical messenger signaling
autocrine
paracrine
endocrine
autocrine signaling
cell surface receptor binds a molecule secreted by itself
paracrine signaling
nearby cell secretion and binding
endocrine signaling
hormone secretion into blood onto distant target cells
contact-dependent signaling
cell surface receptor binds a signal on the surface of another or to ECM
signal transduction scheme
signal (first messenger) –> membrane receptor -=-> transducer –> second messengers –> effectors
types of plasma membrane receptors
ligand gated channels
GPCR
enzyme-linked receptors
GPCR
signal activates enzymatic activity of a G protein
GPCR pathway
ECM domain of a transmembrane receptor (often 7 spanning helices) binds molecule and transmits signal to cytoplasmic domain which changes conformation as a result and changes gene expression / has other effects
GEF
guanine nucleotide exchange factor (transmembrane receptor acts as this)
allows GTP binding on Galpha
G protein
guanosine nucleotide binding protein
tethered to membrane by covalently linked lipid
transmits signal to effector protein as a result of GTP-GDP exchange
adenylyl cyclase
signal binds receptor on membrane
receptor binds G protein
G protein undergoes ADP-ATP exchange
makes cAMP with ATP
cAMP is second messenger and binds to PKA
PKA releases from catalytic subunit
catalytic subunit goes to nucleus and phosphorylates nuclear proteins to change gene expression
kinases
enzymes that phosphorylate target proteins
serine/threonine kinases
phosphorylate serine/threonine in target proteins (PKA, PKC)
tyrosine kinases
phosphorylate tyrosine residues (RTKs, Src)
what do phosphorylated proteins do?
can act as an adaptor or enzyme
Receptor tyrosine kinases
single pass transmembrane proteins
dimerization and cross phosphorylation
activated RTKs –> IP3 and PKA
–> activate phospholipase C –> IP3 / Ca2+ signaling and PKA activation
activated RTKs –> proliferation
–> stimulates SH2 –> GEF (ras activating) –> + GTP, -GDP –> activated Ras –> MAP kinases –> proliferation
activated RTKs –> cell growth + survival
–> PI3 kinase –> PK1 and PK2 –> cell growth + survival
GEF
allows GTP binding
GAP
GTPase stimulating
GTPase
cleaves GTP –> turns off
GDI
keeps GDP on; keeps protein off
proteosome
degradation pathway; 50 protein subunits to degrade proteins by ATP hydrolysis
functions of proteosome
removes misfolded or damaged proteins
maintains appropriate protein levels with controlled degradation
permits rapid responses to changing conditions
ubiquitin
marks proteins for degradation (ubiquitination)
E3 UB-ligases
degrade 1Kb allowing NFkB into nucleus
Ikb
sequesters NFkB
