MCB 2210: FINAL EXAM
320 Cards in this Set
Front | Back |
---|---|
Functions of ABPs
|
Control of Polymerizationand Depolymerization
monomer sequestering proteins
nucleating factors
capping proteins
stabilizing proteins
depolymerizing proteins
severing proteins
Organization of filaments
cross-linking proteins
bundling proteins
membrane linker proteins
Movement of…
|
Capping Proteins
|
Stabilize filaments by binding to ends
CapZ - caps (+) ends
tropomodulin - caps (-) ends
|
Stabilizing Proteins
|
bind along lengths of filaments
stabilize polymerized state
|
Cross-linkng Proteins
|
two actin binding domains separated by spacer domains
filamin, dystrophin, spectrin
|
Bundling Proteins
|
Cross-linking proteins with short spacers
|
Membrane-actin Linker Proteins
|
attach the actin cytoskeleton to the plasma membrane
ERM Proteins: linker between F-actin and the integral membrane proteins
Spectrin: actin to membrane proteins
|
Functions of MAPs
|
Control of Polymerizationand Depolymerization
oligomer sequestering proteins
nucleating factors
end-binding (+TIP) proteins
stabilizing proteins
depolymerizing proteins
severing proteins
Organization of microtublues
cross-linking proteins
bundling proteins
membrane linker protei…
|
Tubulin oligomer binding proteins
|
Stathmin binds tubulin heterodimers or oligomers and promotes catastrophe and depolymerization
|
Nucleating Proteins
|
λ-TURC nucleates MT assembly at the centromere
|
End binding proteins
|
+TIPs bind to the (+) ends of of MTs
EB1 is a +TIP that can regulate (+) end dynamics
|
Severing Proteins
|
Katanin severs MTs and may promote depolymerization at (-) ends
|
Depolymerizing Proteins
|
Subset of kinesins promote MT depolymerization at the (+) end by binding and inducing protofilament curling
|
Polymer-binding MAPs
|
stabilize Mts by binding to sides
enhance assembly by stabilizing nuclei
organize MTs into bundles
mediate MT interactions with other proteins
two-major domains
microtubule binding
projection
MAP2 and Tau
organizing microtubules in neuronal axons and dendrites
|
Structural Features of Actin
|
binds ATP or ADP complexed with Mg2+
monomer: G-actin, filamentous: F-actin
F-actin: helical polymer of G-actin subunits held together by non-covalent interactions
polar
pointed end (-)
barbed end (+)
different dynamic properties of ends`
|
Study of Actin Assembly
|
In vitro: initiated by salts into a solution of G-actin
assembly and disassembly properties of pure actin are different from those for actin in the cell
|
Actin Assembly Kinetics
|
3 steps
nucleation: formation of a stable seed
enlongation: growth
steady state: no net increase or decrease in amount of polymerized actin
|
Critical Concentration
|
Concentration of free actin at the steady state of the system
if concentration is above Cc then subunits will add
|
Concept of Critical Concentration
|
ATP hydrolysis accompanies polymerization
two ends of actin filament have different critical concentrations
|
Treadmilling
|
flux of actin subunits through the filament
|
Steady state
|
active treadmilling, but no net growth or shrinkage
|
Actin Binding Proteins
|
control assembly and disassembly
|
Actin Sequestering Proteins
|
bind monomers of G-actin to prevent polymerization
Thymosin β4
profilin
|
Phalloidin
|
prevents filament depolymerization
|
Cytochalasin
|
Caps (+) end and prevents elongation; eventual depolymerization
|
Latrunculin
|
binds actin monomers and prevents them from polymerizing into filaments (sequestration); rapid disassembly of actin filaments
|
Formin (actin nucleator)
|
generate long unbranched filaments; facilitates (+) end growth while remaining attached to (+) end
|
Tandem actin monomer-binding proteins
|
nucleate unbranched filaments
|
Arp2/3 Complex
|
only actin nucleator to create branched filaments
works in conjunction with WASP family
WASP+Arp2/3 Complex have nucleating activity
(-) end capped by Arp2/3
|
Actin nucleators
|
control cell shape, movement, and division during health and disease
|
Carcinoma
|
cancer of epithelial cells
|
Sarcomas
|
Cancer of connective or muscle tissues
|
Leukemias and Lymphomas
|
Cancer of blood cells
|
Characteristicsof Cancer
|
uncontrolled cell growth
increased cell division
decreased cell death
enhanced cell migration
|
Cancer is caused by mutations in...
|
DNA repair
cell signaling,
cell cycle control
cell growth
programmed cell death
cytoskeletal rearrangements
tissue architecture
|
Cancer cells two heritable properties
|
reproduce in defiance of the normal restraints on cell growth and division
invade and colonize territories normally reserved for other cells
|
Tumor
|
new growth; normal cell that grows and proliferates out of control
|
Benign
|
non-invasive
|
malignant
|
cells have acquired the ability to invade other tissues
|
Metastasis
|
Spread of cancer cells
|
Caretaker genes
|
encode proteins involved in DNA repair
|
Caretake genes contribute to cancer via:
|
Loss of Function
|
Proto-onocogenes contribute to cancer via:
|
Gain of Function
|
Tumor-suppressor genes contribute to cancer via:
|
Loss of Function
|
Tumor-suppressor genes
|
encode proteins that inhibit cell proliferation
regulate or inhibit cell cycle progression
receptors or signal transducers for hormones or developmental signals
checkpoint control proteins that arrest the cell cycle
p53: "the guardian of the genome"
proteins that promote apoptosis
e…
|
Unphosphorylated Rb
|
binds to E2F transcription factors to prevent activation of many genes required fro DNA synthesis
|
Phosphorylation Rb
|
releases E2F, thereby activating transcription of genes required for S-phase
|
Transformation
|
rounder and less adherent; grow while normal cells have become quiescent
|
Ras°
|
oncogene
GTPase that controls signal transduction pathways that are activated by growth factors
capable of transforming cells in culture or inducing cancer in animals
called proto-onocgene
|
Multi-hit model for cancer induction
|
several events are required for carinogenesis
|
p53 "guardian of the genome"
|
cellular stress sensor and transcriptional regulator
as p53 levels rise in cells causes undergo cell cycle arrest, apoptosis, or replicative cell senescence
loss of function abolishes cell cycle (DNA damage) checkpoints
responsible for more than half of human cancer
|
ATM
|
ATM-mediated phophorylation of p53 directly stabilizes p53
|
Mdm2
|
Mdm2 complexes with p53 causing p53 ubiquitination and degradation
|
HPV
|
express proteins that inactivate RB and p53
|
angiogenesis
|
growth of new blood vessels
|
Cancer Cell migration/metastasis
|
driven by actin cytoskeleton
|
Stem cell contribution to cancer
|
may contribute to the replenishment of malignant cells
|
Cancer Stem Cells
|
generate additional malignant stem cells; generate rapidly-dividing transit-amplifying cells
|
Cancer: Nuclear size
|
enlarged nuclei represent more aggressive metastatic disease
|
Invadopodia
|
protrusions that degrade ECM; actin-dependent
|
Cell adhesion molecules
|
cells adhere directly to one another
|
adhesion receptors
|
cell-matrix adhesion
|
ECM
|
proteins and polysaccharides secreted by cells into extracellular spaces
|
CAMs
|
generate tight adhesion when weak interactions are compbined
|
Cadherins
|
cross-bridges between adjacent cells
|
Integrins
|
adhesion receptors bind to large multi-adhesive matrix proteins such as fibronectin
|
catenins
|
cytoskeletal adapter protein that links cadherins to intracellular actin fialmanets
|
desmosoms
|
cadherins are linked indirectly to intermediate filaments
|
Tight junction
|
forms barriers that seal off body cavities;
prevent diffusion of plasma membrane proteins between the apical and basolateral;
prevent diffusion of macromolecules in the spaces between cells
|
Gap Junctions
|
gaps through which ions and small molecules (<1kDa) can pass
|
proteoglycans
|
glycoproteins that cushion cells
|
collagens
|
insoluable sheet-or-fiber forming proteins that provide structural integrity and mechanical strength; most abundant protein in connective tissues
|
fibronectin and laminin
|
cross-link adhesion receptors
|
basal lamina
|
thin (60-120nm) sheet-like meshwork of ECM components that underlies or surrounds many epithelial and non-epithelial tissues
|
Type IV Collagen
|
trimeric protein that forms a 2D network of fibers
|
Laminin
|
fibrous 2D network; binds to adhesion receptors
|
Connective tissues
|
volume made up of extracellular matrix, rather than cells
|
fibroblasts
|
most abundant cells in connective tissues
|
Collagen Structure
|
triple-helical polypeptides
high abundance of glycine, proline, and hydroxyproline
|
Proteoglycans
|
secreted or surface-attached glycoproteins containing specialized polysaccharide chains called glyosaminoglycins (GAGs)
|
Glycosaminoglycans (GAGs)
|
long linear polymers of specific repeating disaccharides
|
Fibronectin
|
multiple domains for binding collagens, proteoglycans, and adhesion receptors
organizing other components of the ECM
regulating cell matrix adhesions
shape and movement of cells
organization of the actin cytoskeleton
binds to integrins (tripeptide sequence: RGD)
|
Hemi-desmosomes
|
cause the stable attachment of the ventral cell surfaces to the ECM
|
Integrins
|
most prominent adhesion receptors
|
Cell Cycle
|
ordered series of events that leads to cell duplication and division
|
G1-phase
|
period between completion of previous mitosis and initiation of DNA synthesis for the next mitosis
|
G2-Phase
|
the period between the completion of DNA replication and initiation of mitosis
|
G0
|
Phase that cells are in if they exit the cell cycle
|
Post-translational modifications
|
phosphorylation of tyrosines, series, and threonines
reversible
ubiquitination of lysines
protein degradations
irreversible
|
PI 3-kinase Pathways
|
receptor tyrosine kinases (RTKs) recruit and activate PI 3-kinasses (PI3K)
PI3K phosphorlyates the 3' position of inositols to generate PI(3,4,5)PS
Activation of serene/threonine kinase (Akt)
mTOR becomes activated
phosphorylation of targets involving proteins synthesis and metabolism…
|
Protein Degradation Pathaways
|
Lysosome
Proteasome
|
Proteasome
|
large macromolecular machine
degrade many cellular proteins
|
3 Functions of Protein Degradation:
|
It removes proteins that are misfiled, damaged, or potentially toxic
Controlled degradation of normal proteins provides a powerful mechanism to maintain appropriate levels of proteins
Regulated degradation also permits rapid responses to changing conditions
|
Ubiquitin
|
Highly conserved 76 aa polypeptide that marks proteins for degradation by the proteasome
|
Poly-ubiquniation
|
multiple molecules of ubiquitin are attached to a molecule; recognized for degradation by the proteasome
|
E3 Ub-ligases
|
enzyme that achieves poly-ubiquination
|
cyclin
|
regulatory subunit
|
cyclin-dependent kinase (CDK)
|
catalytic subunit
|
Protein phosphatases and ubiquitin ligases
|
key regulators of the cell cycle transitions
|
Regulation of CDK
|
Activation by cyclin-binding and T-loop phosphorylation
binding cyclin to CDK alters conformation of the T-loop
2. Inhibitory phosphorylation of the active site by Wee1 kinases - reduces activity of CDKs
3. De-phophorylation of these sites by Cdc25 phosphates
4. Physicial Inhibity by …
|
G1 cyclin-CDKs
|
transcription of genes required for DNA replication
|
SCF ubiquitin ligase
|
polyubiquitinates inhibitors of S-phase cyclin-CDKs
|
S-phase cyclin-CDKs
|
activate DNA replication origins
|
mitotic cyclin-CDKs
|
entry into mitosis
|
APC
|
polyubiquitinates securing and polyubiquitinates mitotic cyclins
|
Cdc14 phosphatase
|
promotes telophase
|
Cyclin B-CDK1
|
phosphorylates cellular proteins and causes changes for mitosis
|
Condensins
|
phosphorylization causes chromosome condensation
|
Nuclear lamins
|
phosphorylization causes nuclear breakdown
|
microtubule-associated proteins
|
phophorylization causes changes in microtubule dynamics
|
ER or Golgi associated proteins
|
phosphorylization causes reorganization of the ER and the Golgi
|
Checkpoint types
|
DNA-damage and spindle
|
Checkpoint purpose
|
ensure that one phase of the cell cycle is completed successfully before the cell is allowed to progress to the next phase
|
Checkpoint Mechanism
|
negative feedback mechanisms that block cell cycle progression
allow time for correction or repair
|
Sensor proteins
|
detect abnormalities
|
Transducer proteins
|
relay or amplify the damage signal
|
effector proteins
|
halt cell cycle in response to damage
|
Anaphase Promoting Complex (APC)
|
triggers degradation of the cohesins that connect sister chromatids by a separate enzyme
|
APC Activity Mechanism
|
APC ubiquitinates securin
causes the release of separase
activity of separatase degrades cohesion
|
Mitosis/Maturation Promoting Factor (MPF)
|
low in interphase
rises as cells enter mitosis
|
hfp://www.youtube.com/watch?v=6xMXKU7JnMQ&
|
Meiosis Self Study
|
Meiosis
|
one cycle of chromosome replication followed by two cycles of cell division to produce haploid germ cells
|
synapsis
|
pairing of chromosomes along lengths; allows for recombination between chromatids of homologous chromosomes occurs
|
Necrosis
|
premature death due to infection or injury
|
Apoptosis
|
programmed cell death
|
Autophagy
|
self-eating
|
Phagocytosis
|
involves one cell "eating" a dying cell
|
Telomeres
|
physical ends of linear chromosomes
|
Telomerase
|
reverse transcriptase enzyme that adds these telomere sequences to the ends of chromosomes and maintain 3-20kb of DNA repeats in humans
|
Senescence
|
withdrawal from the cell cycle and stop dividing
|
p53
|
becomes activated by telomere shortening and can trigger apoptosis
|
Apoptotic Cell Features
|
chromosome condensation at the nuclear periphery
shrinking of the cell body
blabbing of the cell membrane
endonuclease cleavage
flipping of phophatidylserine from the inner leaflet of the plasma membrane to the outer leaflet
|
Apoptosis Benefit
|
eliminating abnormal or misplaced cells during development in a quality control process
elimination of cells that could be potentially dangerous, as self-reactive immune cells
elimination of cells with the potential to cause cancer
the killing of virus-infected cells by cytotoxic T cel…
|
caspases
|
intracellular machinery responsible for apoptosis is comprise of proteases
|
Intrinsic pathways to apoptosis
|
triggered from within a cell
DNA damage
hypoxia
low nutrients
lack of proper extracellular survival signal
|
Cytochrome C
|
Binds to proteins that activate procaspases
|
Extrinsic Pathways to Apoptosis
|
binding of extracellular signaling proteins to cell surface 'death' receptors
|
Autophagy
|
conserved intracellular pathway that controls protein and organelle degradation, and has key roles in development, survival and homeostasis
|
Autophagosome
|
double-membrane vacuole
degradation of cytoplasmic components after fusion with lysosome
|
Integrins
|
physical link between the ECM and actin filaments
|
Stress Fibers
|
anchored to focal adhesions
|
Actin polymerization at the leading edge
|
coupled to membrane protrusion
|
Retrograde flow
|
network of filaments is thought to flux backwards
|
Steps of Membrane Mobility
|
first step involves the forward protrusion of the lamellipodium which is driven by the force of actin polymerization
adhesion: lamellipodium interacts with the substrate; forms structures such as focal contacts
cell body translocation: been postulated to occur via a dynamic network cont…
|
Dominant-negative (GDP-locked) versions of Cdc43, Rac, and Rho
|
inhibit cell migration
|
Signal Transduction
|
How signals are transmitted from the outside of the cell to the inside, allowing cells to modify behavior
|
Molecules that can cross the membrane directly
|
Hydrophobic signaling molecules
steroid hormones
receptor is in the cytoplasm or nucleus
Nitric Oxide/Carbon Monoxide
dissolved gas regulates many pathways
|
Molecules that cannot directly cross the membrane
|
hydrophilic
|
Signaling via chemical messengers
|
Autocrine, paracrine, and endocrine signaling
cell surface receptor binds a molecule secreted by itself (autocrine) a nearby cell (paracrine) or a distant cell (endocrine)
|
Contact-dependent signaling
|
cell surface receptor binds a signal on the surface of another cell or on the extracellular matrix the cell is in contact with
|
Mechanism of signal transduction via membrane receptors
|
same signal can bind to multiple types of the receptor
receptor can be coupled to multiple types of transducer
second messengers can provide high signal amplification
second messenger can be coupled to multiple effector types
|
Ligand gated channels
|
signal opens a channel - (ion pore)
|
G-protein coupled receptors
|
signal activates a G-protein
|
Enzyme-linked receptors
|
signal activates the enzymatic activity of the receptor (Usually a kinase)
|
Transmembrane Receptors
|
7 membrane spanning helices
extracellular domain binds molecule signal
transmit signal to cyptoplasmic domain
TM receptors act as a GEF
|
Heterotrimeric G protein receives signal
|
Three different subunits
two are tethered to the membrane by covalently linked lipids
transmits signal to effector protein
|
Adenyl cyclase
|
Gα binds to adenyl cyclase activating it
makes cAMP from ATP
cAMP acts as second messenger
cAMP binds to PKA causing inhibitory subunit to fall off
kinase is now active and goes to nucleus
phosphorylates nuclear proteins to change expression
|
Receptor Tyrosine Kinases
|
Usually signal pass transmembrane proteins
binds ligand on the outside of the cell
causes receptor to dimerize
autophosphorylation of part of the cytoplasmic tail of the receptor
phosphorylizaiton is always on tyrosine
Both a ligand binding protein and a kinase that phosphorylates it…
|
G Proteins
|
control cellular processes like cell division and motility
binds GTP or GDP
GTPase activity that cleaves GTP to GDP
Normally bound to GDP (off)
Controlled by GEF, GAP, and GDI
|
GEF
|
causes GDP to be released and allows GTP to bind
|
GAP
|
stimulates the GTPase activity so that GTP is cleaved and activity turned off
|
GDI
|
prevents GDP from coming off keeping in the offs state
|
Metaphase
|
point when chromosomes become aligned in one plane halfway between two spindle poles
|
Anaphase
|
sister chromatids separate and are pulled forward by spindle poles by the kinteochore MTs
|
Anaphase A
|
the kinetochore MTs shorten and the chromosomes move toward the poles
|
Anaphase B
|
polar MTs elongate and the poles move farther apart
|
Spindle Assembly Checkpoint activation
|
kinetochores that are not attached to MTs
kinetochores that are not under tension
|
CENP-E
|
Kin-N
protein at the kinetochore that binds to the MT
movement can be driven by depolymerization of the (+) end
|
Dynein Motors
|
located at the cell cortex participate in pulling the poles apart
|
Telophase
|
daughter chromosomes arrive at the poles
nuclear envelope reformes
chromatin begins to de-condense
|
Cytokinesis
|
cytoplasmic division by cleavage
|
contractile ring
|
draws plasma membrane in
|
midbody
|
remnant of the spindle
|
Contractile Ring in amoebae, fungi, and animals
|
centrally-positioned contractile ring of actin filaments and myosin II
|
Successful cytokinesis depends on:
|
placement of cleavage furrow
contractile ring dynamics
separation of the plasma membrane
|
Rho GTPases
|
active; mark the cleavage site around the equator
|
Intermediate Filaments basic Characteristics
|
10 nm in diameter
very stable
do not bind nucleotides
non-polar
withstand stretching forces
|
Intermediate Filament Structure
|
helical coiled rod domain
form parallel dimer
assemble in to antiparallel tetramer
tetramer is basic building block
no polarity
|
Types of Intermediate Filaments
|
Type I (acidic) and Type II (basic) keratins
heteropolymers
epithelial cells
2. Type III Vimentin and related filaments
homopolymers
most widely distributed intermediate filament protein
3. Type IV Neurofilaments
heteropolymers
found in axons
4. Nuclear Lamins
meshwork of Ifs th…
|
Septins Characteristics
|
10 nm diameter
bind and hydrolyze GTP
cells with mutations arrest at cytokinesis
important for contractile ring
|
Septin Structure
|
GTP-binding domain
coiled coil
|
Functions of Septin Filaments
|
Cytokinesis: cleavage furrow and contractile ring
Scaffolding
Membrane trafficking: movement and fusion of membrane vesicles
|
Fibroblasts
|
cell of connective tissue
|
Lamellipodia
|
thin sheet-like structures
|
Filopodia
|
thin needle-like projections or spikes
|
Stress fibers
|
long cables of actin that function in cell adhesion and contraction
|
focal adhesion
|
attach the cell to the underlying substrates
|
Cell Theory
|
all living creatures are made of one or more cells
the cell is the basic structural unit of living things
cells can only arise by division from pre-existing cells
|
Basic Properties of Cells
|
composed of proteins, lipids, carbohydrates and nucleic acids
high complexity
encoding information in genes
membranes
reproduction
acquire and utilize energy
carry out chemical reactions
carry out mechanical activities
respond to stimuli
self-regulation
evolving
|
Central Dogma
|
DNA
mRNA
protein
|
Prokaryotes
|
lack a nucleus and internal membranes
|
Eukaryotes
|
have a nucleus and and extensive internal membrane system
|
Plasma Membrane
|
lipid bilayer
|
Cytoplasm
|
soluble internal contents of the cell
|
Nucleoid
|
area in which DNA is concentrated
|
Plasma Membrane Properties
|
selective permeability
diffusion rates
transporters
|
Passive Diffusion
|
molecules diffuse directly through the lipid bilayer
|
Passive Transport
|
down a concentration gradient;
no energy beyond thermal motion
|
Active Transport
|
extra energy input for transport
|
Passive Transport 1
|
passage in either direction
multipass transmembrane proteins
ion specific
regulated
|
K+ Channel
|
large channel
selectivity filter at one end
Fits K+ perfectly
Na+ is too small in interact properly and sticks
|
Passive Transport 2: Carriers
|
no direct energy input
mulipass membrane
bidirectional
reversible conformation change
|
Carrier: GLUT
|
12 transmembrane domains
N and C intracellular termini
affinity for glucose
responds to insulin
|
Active Transporters
|
active transporters move against a chemical, electrical or electrochemical gradient
|
Primary active transporters
|
ATP-dependent pumps
light driven pumps
|
Secondary active transporters
|
Symport
moves 2 molecules in same direction
Antiport
moves 2 molecules in the opposite direction
|
ATP-dependent Pumps
|
Uses ATP energy to move one or more molecules
v-type: does not become phosphorylated
p-type: becomes phosphorylated from ATP during transport
|
Na+/K+ ATPase
|
3 Na+/2 K+
|
K+/K+ ATPases
|
Stomach acidification
P-type
|
Ca++ ATPases
|
pump Ca out of the cell
p-type
|
ABC Transporters
|
v-type
isoforms important in cancer
|
Vesicular H+ ATPases
|
Acidification of lysosomes, golgi
v-types
|
Secondary Active Transport
|
energy input needed
moves one molecule against a gradient
uses energy from an existing gradient
Carrier Mediated: Protein changes conformation
|
Antiporters/Exchangers
|
two ions are moving in opposite directions
molecule is moving down a concentration gradient
one moving up a concentration gradient
exchangers
|
Action Potentials Characteristics
|
all-or-none
encode information by the frequency of action potential
|
Steps of Action Potential
|
Na+ channel in dendrite senses depolarization
Na+ channel opens
membrane depolarizes
K+ channels open
Na+ channels inactivate
Membrane returns to -70mV
|
Membrane Functions
|
separate compartments
provide scaffold
mediate interactions
signal transduction
|
Components of biological Membranes:
|
Lipids
cholesterol
proteins
|
Miscelles
|
small sphere with tails pointed in
|
Bilayers
|
Two layers of lipid with tails pointed toward each other
|
Flourescence recovery after photobleaching (FRAP)
|
label phospholipids with a fluorescent probe
shine a bright laser on a small spot of membrane to bleach it
measure how long it takes for fluorescent molecules to diffuse back into the region
|
Flipases
|
membrane proteins flip-flop the lipids back to normal sides
|
Lipid Rafts
|
Areas with high concentrations of certain lipids and proteins
|
Protein Primary Structure
|
sequence of amino acids
|
Protein Secondary Structure
|
local shape
|
Protein Tertiary Structure
|
3D shape
|
Protein Quaternary Structure
|
multi-subunit assemblies
|
Functional Domain
|
acts as a unit but is part of a protein
|
Kd
|
Higher Kd: lower affinity
Lower Kd: higher affinity
|
Integral membrane protien
|
interact directly with the lipid portion of the bilayer
|
Lipid anchored proteins
|
covalent addition of a lipid to a protein targets the lipoprotein to the membrane
|
Peripheral membrane proteins
|
attached to the membrane via interactions with other membrane proteins, not lipids
|
Structure of Transmembrane region of proteins
|
hydrophobic/ amphipathic alpha helix or amphipathic beta sheet
hydrophilic regions face inwards of the transporter
|
Beta-barrels
|
hydrophobic are outward
polar are inside
forms a pore through the membrane that is hydrophilic
|
Cell Fusion
|
measures protein mobility
label proteins of one cell with red dye
label proteins of second cell with green dye
fuse cells to form a heterokaryon
watch what happens
|
Primary Cells
|
non-cancerous, non-transformed cells
|
Transformed Cells
|
cancerous cells
|
Light microscopy limitations
|
magnification, contrast, and resolving power
|
Magnification
|
amount you blow up the initial image
dependent on lens
|
Resolution
|
how far apart tow objects have to be to be seen as two separate objects
not related to magnification
determined by
wavelength of light
properties of lens
|
Fluoresence
|
molecule absorbs one wavelength of light and admits alone
|
Immunocytochemistry
|
Immunofluorescence
|
Techniques involving antibodies
|
immunoblotting
immunoprecipitation
immunoisolation
|
Transfection
|
infecting cells with foreign DNA to cause a foreign protein to be expressed in a cell
|
Transmission electron microscope (TEM)
|
images electrons that pass through a thin specimen
|
Scanning Electron Microscope (SEM)
|
images electrons scattered by an intact object
|
Microtubule Structure
|
polymer of tubulin subunits
form a cylindrical filaments
formed from α and β tubulin heterodimers
both bind GTP
α - irreversably
β - hydrolyzes GTP to GDP
13 protofilaments
polar
(-) embedded in the MT organizing center (MTOC or centrosome)
|
GTP Cap
|
stabilizes (+) end of MT to prevent depolymerization
|
Taxol
|
binds to MTs and stabilizes the polymer preventing disassembly
|
Cholchicine
|
prevents further depolymerization not the MT end
|
Nocodazole
|
binds to tubulin subunits and triggers MT depolymerization
|
Pericentrioloar Material (PCM)
|
microtubules emerge from PCM near the centrioles of the centrosome
|
γ-TURC
|
anchors MTs at the MTOC/centrosome
|
γ-tubulin
|
microtubule nucleator
|
Mitosis
|
process of chromosome segregation
|
M Phase Subcellular changes
|
chromosome condensation: organized by histones, compacted by condensing
mitotic spindle assembly: aligns the replicated chromosomes in a plane that bisects the cell, moved by spindle microtubules to opposite spindle poles
Formation of the contractile ring: actin filaments and myosin II …
|
Prophase
|
Two centrosomes separate
each centrosome forms radial MTs called an aster
asters move apart
|
Pro-metaphse
|
nuclear envelope breakdown
MTs are captured by kinetochores
chromosomes move back and for the between the pole
|
Nuclear envelope breakdown
|
disassembly of the nuclear lamina cytoskeleton
|
congression
|
bi-directional movement of chromosomes near the equator of the spindle during pro-metaphase
|
Metaphase
|
point when chromosomes become aligned in one plane halfway between the two spindle poles
|
Myosin motor proteins
|
couple energy from ATP hydrolysis to conformational changes; myosins are (+) ended motors
|
Myosin Structure
|
Head: motor, actin binding ATPase activities
neck: regulatory
tail: determine properties
|
Myosin-Actin Cross-bridge Cycle
|
rigor state
ATP binding
ATP hydrolysis
Pi release
power stroke
ADP release
|
Kin-N Kinesins
|
motor domain at the n-terminus; move to the (+) end
|
Kin-C Kinesins
|
motor domain at the c-terminus; move to (-) ends
|
Kin-I Kinesins
|
Internal motor domain; promote protofilament peeling
|
Conventional Kinesin
|
two heavy chains
two light chains
head
neck
tail
light chains mediate interaction of Kinesin with membrane vesicles
|
Processive movement
|
movement over long distances without dissociating
|
Dyneins
|
mechanochemical enzymes that couple the energy from ATP hydrolysis to (-) end directed movements along MTs
|
Dynactin Complex
|
mediates the attachment of Dynein to vesicles and organelles
critical for the function of cytoplasmic Dynein
|
Methods of Viral Entry
|
fusing with the plasma membrane
endocytic-based mechanisms
both require microtubules after entry
|
normal flora
|
commensal microbes
|
pathogens
|
organisms that can caused overt disease in healthy people
|
virulence factors
|
proteins that contribute to the ability of an organism to cause disease
|
Pathogenic Strategies and Virulence Factors
|
colonize
multiply
evade host response
cause damage
spread
|
Types of Virulence Factors:
|
Toxins: released, alter or disrupt normal processes
Adhesins and/or invasins: on surface; promote attachment or entry into cells
Secretion systems: translocate (inject proteins from bacteria into host cells
Effectors: transolcated proteins that alter or disrupt normal processes within …
|
AB toxins
|
(A) Subunit: enzymatic
(B) Subunit: cell-binding
|
Invasion
|
process of induced-cellular uptake
|
invasin
|
strucural similarites to fibronectin and binds to β1-integrins to promote actin cytoskeletal rearragements and enter cells
|
internalin
|
binds to E-cadherin to promote actin cytoskeletal rearrangements and internalization
|
effector proteins
|
trigger actin cytoskeletal rearrangements that result in bacterial invasion
|
Zipper and Trigger
|
mechanisms for pathogen-induced phagocytosis both require the polymerization of actin
|
vacuole escape
|
Listeria and Shigella
|
Prevention of Lysomal Maturation
|
Mycobacterium tuberculosis, salmonella, Legionella pneumophila, and Chlamydia trachomatis
|
Growth within lysosomes
|
Coxiella Burnetii
|
ER Functions
|
site of membrane and secretory protein synthesis
Ca++ storage
site of lipid synthesis
detoxifying enzymes in liver cells
|
ER Structure
|
Rough
smooth
|
Protein insertion into ER
|
co-translationally
|
Protein Targeting to the ER
|
proteins destined for membrane compartments or secretion are targeted initially to the ER
|
Chaperone Proteins
|
in ER lumen aid in folding
|
Fibroblasts
|
migration (actin)
|
osteoblasts
|
collagen secretion
|
neuronal cells
|
axonal trafficking (MTs, motors)
|
Muscle cells
|
sarcomeres and contraction (Actin and myosin)
|
Macrophages
|
extravasation
|
T-cells, B-cells, Antigen presenting cells
|
immunologicalsynapses (actin)
|
Respiratory epithelia, sperm
|
motile cilia and flagella (MTs, motors)
|
sensory cells
|
non-motile cilia - primary cilia (MTs, motors)
|
Sarcomere
|
thin and thick filaments
actin filaments and actin-binding proteins
myosin II motors
|
leukocytes
|
cross from the blood into tissues in response to chemical signals called chemoattractants that are released by other cells or by pathogens
|
selectin receptors
|
leukocytes can attach loosely to endothelial cells
|
extvasation
|
leukocyte's shape changes dramatically and it migrates between endothelial cells into the underlying tissue
|
Immunologicaly synapse (IS)
|
focal point for exocytosis, endocytosis, and signaling at a physical junction between lymphocytes
|
cilia
|
tiny hair like appendages with an arrangement of MTs at their core
|
axoneme
|
core consists of an array of microtubules and associated proteins
|
totipotency
|
ability of a single cell to divide and produce all the differentiated cells in an organism
|
somatic cells
|
cell that forms the body of a multicelluar organism
|
differentiation
|
process by which a less-specialized cell becomes a more-specialized cell
|
stem cells
|
undifferentiated cells that can reproduce themselves and also give rise to diverse specialized cell types
|
Pluripotent stem cell
|
can differentiate into any adult cell type
|
Multipotent stem cells
|
limited number of lineages
|
terminally differentiated cells
|
cells that are considered to be permanently committed to a specific function
|
Stem cells
|
characterized by the property of self-renwal and the capacity for differentiation; indefinite supply of fresh differentiated cells where these are lost, discarded, or needed in greater numbers
|
epidermis
|
epithelium that is self-repairing and continually renewed
|
Hematopoetic stem cells (HSCs)
|
found in bone marrow, peripheral blood, and umbilical cord blood
|
autologous transplants
|
patients receive their own stem cells
|
allogeneic transplants
|
patients receive stem cells from their brother, sister, or parent
|
nuclear reprogramming
|
switch in nuclear gene expression from one kind of somatic cell to that of an embryo or other cell type
|
Induced Pluripotency
|
expression of the four transcription factors Oct4, Sox2, Klf4, and c-Myc can reprogram somatic cells to a state that is similar to that of embryonic stem cells
|
Nuclear transfer to eggs
|
somatic cell nucleus is transplanted to an enucleated X.laevis (or mammalian) egg
|