S3 Flashcards
dynamic instability
individual MTs do different things at different time (dont just grow at cc)
GTP cap
catastrophe
rescue
critical concentration for assembly
an amount at which there is enough in solution to polymerize
plus end has lower critical concentration than minus end - polarity
MTs also need GTP, 37 degrees, Mg
treadmilling
illusion of movement
one side grows while other side shrinks seen in vivo, not actual movement
selective stabilization
proteins can bind to lattice to prevent dissasembly - important for cell polariation
Tau, MAP. double cortin, STOP, plectin, etc
GTP hydrolysis and micrtotubules
all free tubulin is GTP-tubulin
adding to plus end forming GTP cap
then hydrolysis behind making GDP tubulin
catastrophe if GTP cap is lost,
rescue if cap comes back before dissasmbled
not technically necessary to form but without it cant dissassemble to do work
microtubule based molecular motor proteinsd
get more work done than that stored in MTs ATPase dynein is retrograde toward minus end kinesin is anterograde toward plus end motor and cargo domain processivity
tau
important for stabalizning MTs
absent in alzheimers
neruon specific
katanin
cuts microtubule by pulling tubulin through hexamer pore
microtubules
tubulin subunits hollow polar dynamic highly conserved railway for motor proteins
actin filaments
actin subunits non holow polar dynamic highly conserved railways for motor protieints
intermediate filaments
various kinds of intermediate filament subunits nonhollow non polar non dynamic diverse not a railway for motor proteins
MT subunits
alpha beta dimer
free tubulin dimers
MT polarity
alpha is minus end
beta is plus end
nothing to do with charge
mitotic vs interphase microtubule org
both have centrosomes with minus ends and plus ends out
but Interphase has one in the center going out
mitotic has 2 on oposite ends
‘centralized foci’
nucleation
getting new MTs started
has Lag without nucleation seed
less energetically favorable than elongation
needs MT nucleating elements to make favorable like centrosomes and basal bodies
nucleating sites
in pericentriole matrix PCM around centrioles
rings of gamma tubulin
centrioles themselves only source of extracellular MTs
cilia and flagella
short and long
9+2 arrangment
flagellar dyeinn
basal body is centriole that contacts cell membrane
gamma tubulin
gamma turc is ring for nucleation sites
unclear if it binds to alpha or beta but it helps make polar proto filament
cut and run
Mts break off centrosome then move for non centrosomal arrays like epithelial cells with apical and basal ends
coverslip movement
kinesisn walks toward plus end to MT moves in dirextion of minus
Dynein walks toward minus end so plus end leads
neuron MT patterns
dynien moves them into axon so plus end toward terminal
dendrite mixed because dynin moves in there then kinesins move it around
axon branch formation
katanin cuts and tau gets phosphorylated while it moves to new branch
spastin cuts and tau stays on MT chunks that move to new branch
actin filament growth and force on membranes
at cell concentrations actin is more stable in a filament so polymerization for brownian ratchet when it bends to allow another subunit then bends back working on the membrane
myosin force generation cycle
attached and nucleotide free
ATP binds mysosin so it releases actin
ATP hydrolysis to ADP.Pi bound cocks forward
Pi release ADP bound and bound to actin while cocked forward
ADP release so nucleotide free again and bends back moving actin
conservation of actin
highly conserved in eukaryotes
human actin isoforms
5
muscle - smooth, skeletal, cardiac
cytoplasmic - beta, gamma
actin monomer
unusual subdomain structure
ATP binding sites
hydrolyzes and exchanges nucleotides slowly
actin filament
polar
subdomain 2 into 1-3 cleft
ATP hydrolysis is fast ier in filament than in monomer
in vitro actin assembly kinetics
nucleation slow and highly concentration dependent
elongation is fast and aysmetric - barbed + end is 1-3 cleft
pointed - end
[actin]eq
a fixed value that is the balance of forward and revere reactions
the critical concentration
buffering free actin
very little exists in cells
binds to beta-thymosin blocking both ends
most bins profilin which is a nucelotide exchange factor blocks elongation only on pointed end
growth on barbed end
beta thymosin blocks both ends
prolifin allows barbed end addition
almost all elongation this way
nucleation of actin
blocked by beta thymosin and or prolifin so nucleation is prevented in cells
nucleation factors for actin
needed to allow filaments to grow in the right place at the right time
actin elongation control
capping proteins to prevent elongation and depolarization like barbed end CapZ
elongation factors like formins, ENA/VASP control actin delivery and are capping protiens
actin disassembly
ATP hydrolysis destabalizes the filament meaning that ADP actin dominates away from barbed end
severing factors generate new pointed ends
capping proteins will arrest growth at barbed end while the unpcapped pointy end depolymerizes
lamellipodia
branched arrray
broad flat rapidly polymerizying protuisions in 2d environments
densely branched actin
lamellipodia assembly and disassembly
nucleated by Arp2/3 complex at membrane
WASp/Scar proteins activate Arp2/3
growing filaments pus mesh away from membrane into cell
agin filaments are enriched by ADP actin and targetted for recycling by coflin
depolarized gets grabbed and recharged by profilin
filopodia
parrallel bundled arrays
fast growing
nundling protein like fascin and a TIP complex
come from branched arrays
actin crosslinking
different bundling proteins result in different structures
alpha-actin - anti-parallel bundling factor
usually told apart by distance btwn filaments
myosin
actin binding motor
walks toward barbed end
different myosin functions
same idea but differneent strucutre can be used for different cargo
stress fibers
have focal adhesions of actin but look similar to muscle
sarcomere
repeated in Skeletal muscle making it look striated
like a stress fiber with overlapping actin and myosin and caps instead of focal adhesions
skeletal muscle regulation
troponin is bound to tropomyosin which blocks myosin from binding to actin but Ca comes and removes troponin so tropomyosin moves so myosins can do its thing
skeletal mucle triggering
t tubules send action potential
sarcoplasmic reticulume then releases Ca
cell polarity
differences in shape and structure of cells
asymmetry
regulated polarization
like lymphocyte becoming macrophage which is a direected homoestatic or immune response for migration and contact with other cells
T cells
2 poles and an axisl of polarity
migration
competition
asymmetric division
epitheilai cells
polarized protein diestribution between basal and apical ends
apical has actin and cell cell junction
Par6, Par3, APKC on apical side of junction
uses gradents and transporters to have nutrients flow through
cell cell junction dilineate transporter types
distribution of PAR proteins
Par3/Par6/aPKC
anterior side of asymmetric cell division
distal end of neve axon
leading end of cell migration
apical side of cell cell junction in epithelial cells
3 steps of cell migration
extension of leading edge
nuclear movement
tail contraction
migrating cell adhesion
extend adhese translocate de adhese focal adhesion kinase
distribution in C elegans
Mex5/mex6
Par3/6/aPKC
all anterior
symmetric vs asymmetrci
cadherin complex zonular protein apical marker/cadherin hole if even amounts of above three then symmetrical side with cadherin hole becomes NPC
2 axis of polarity
apical-basal
planar cell
PCP mutants
disorginization
division of neural progentior cells
vertically = 2 NPC
horizontal or angled = 1 NPC 1 neuron
Pro axon factors
PI3K
AKT
tau
PKB
pro dendrite
PTEN
GSK-3 beta
planar cell polarity
2 axis: circunferential and radia
strucutres organs
downstream of wnt
posterior c elegants
pie1
par1,2
p-granules
wnt signaling
frizzled
disheveled
knock out experiments
gene transcription (canonical has beta catinin or calcium)
cytoskeleton remodeling for PCP has rho gtpase and JNK
why are there size limits in microscopy
visible wavelength, resolution
importance of tissue processing like fixation embedding sectioning
just to better visualize
importance of specialized illumination
better visualize
how to follow protein dynamics in living cells
fluroscence
light vs electron vs atomic force microscopy
biggest to smallest
compare images from atomic force microscopy, scanning electron microscopy, and transmission electron microscopy
d
refractive index
ratio of light in a vaccuum to light through medium
used to match numerical aperture of a lens
limits of resolution
minimum that can be achieved given the limitations of the medium used for microscopy and what is used to produce the image
defined by distinguishing btwn two objects
photobleaching
overdoing the flourescence and destroying the flurophore
photoprotective scavengars help
phototoxicity
hurting cells/samples by shining laser on i t
deconvolution microscopy
math enhances images
two photon microscopy
better than confocal
fluorescence resononance energy transfer FRET
multiple dyes used to map interactions
photoactivation
caged molecules remain unseen until catalytically freed
total internal reflection fluorescence
TIRF
used to amplify from 2D coverslip
light bounces at an angle
transmission electron m
shoots electrons through sampe to acheive 2 milllion x mag
wavelenght of electron is smaller than light
v inverse to w
ultra thin slicing
scannning electron m
cover sample with metal and shoot with electrons
image based on scatter
atomic force microscopy
bounce laser off of tip of probe
measure deviations
can see individual atoms
visible light
0.4um to 0.7um
meiosis vs mitosis
meisosis has two stages ending in 4 haploid cells
mitosis has its steps leading to 2 diploid cells
meiosis I vs mitosis
homologus chromosomes connect and exchange parts
then pairs move to opposite sides
APC/C
something to do with metaphase anaphase checkpoint
gets activated by cdc3- to help ubiquinate m cyclin-cdk and end metaphase
Cdk activating kinase
and cyclin
get turned on by Cdc25 and off by wee1 kinase
different versions G,s, M m is for m phase
phases of meisosis
pro meta1 ana 1 telo 1 pro 2 meta 2 ana 2 telo 2 basically 2 division: first pairs of homolougs then sister chromatids
prophase I
homologuous recombination
crossing over synaptonemal complexl
synaptonemal complex
combines two homologs with cohesisn and transverse filaments
cohesin
4 units making a ring around sister chromatids
scc3-scc1 hook gets cleaved in anaphase
homologous recombination
homologous dna exchange parts
for repair
accurate separation because chiasmata between correct homo pairs
genetic diversity
chiasmata
happens after cross over when physically linked still
HR deficient
fail to form SC and dont get cross overs
separase in meiosis I
releases cohesin along the arms but cohesin near centromere is intact
nondisjunction
no crossover - unstable and random
distal cohesion - unstable and random
proximal crossover - reductional division in meisosi II (stuck)
so crossovers cant be random but must be medial
cross over interference
good thing?
two or more crossovers happen, so adjacnet events are located furthera apart than expected from random
but protects chiasmata
When can DSB happen
prophase I
spo11
catalyzes double strand breaks
part of large complex
so phosphorylation of Mer2 upregulates it
double strand break recombination model
resection strand invation (Rad51) synthesis capture second end synthesis double holliday junction intermediate cut both for resolution and either have crossover or non crossover
synthesisi dependent strand annealilng
syn
dissociat
anneal
get non crossover
Non crossover resultes
DSBR fits a lot but non crossovers appear at wrong time so maybe SDSA makes those
mitotci spindle
astral MTs
kinetochore MTs
interpolart MTs
astral MTs
stabalize centrosome
kinetochore
connect centrosome to homo
interpolar
stabalize entresomes to each other
3 stages of apoptotici cell removal
find me
eat me
anti inflammatory cytokines
procaspase activation
inactive procaspases have 2 celaveage sites
cleaveage activated by other active caspase so cascade
then dimerize
fas ligand model
extrinsic apoptotic pathway
fas ligand on killer lymphocyte binds to Fas death receptor leading to association of fas-associated death domain FADD and assembly of Death inducing signaling complex DISC which activates procaspase 8,10
death effector domain DED
Caspase activating and recruiting domain CARD
cytocrhome c
apoptotic stimulus relases from mitochondria
cc activates apaf1 (apoptotic protease activating factor)which has CARD domain and multimerizes to apoptosome
recruites procaspase 9 which gets executioner caspases
activation of intrinsic pathway
apoptotic stimuli like BH3 inactivates the anti-apoptotic Bcl2 protein allowing BH123 to dimerize on outermembrane of mito so cc can get out
scheme of mitochondria
basically bax/bak open a pore casuing cc and then mDNA to fall out
increased production of Bcl2 protein
after a survivial factor activates a receptor, transcription is upregulated making more bcl2 to block apoptosis
inactivation of proapoptotic BH3 only bcl2 protein
survival activates receptor activating AKt kinase whichactivates Bcl2 and inactivates Bad
blocking apoptosis
inactivation of IAPs
survival factor and receptor
MAP kinase inactivates Hid which activates IAPs which i think block cc
inflammosome
causes inflammation kinda like DISC in apoptosome
ASC or apoptis asssociated speck like protein containing a caspase recruitment
activated by toxins to mobilize immune system, caspase 1 releases cytokines to recruit the A team
NALP3 and IPF3 are variants
necroptosis
causes immune response
necrosome with RIP3 kinase, caspase 8 activates
death from membrane osmolysis, energetic catastrophe, lipid peroxidation
start/restriction point
when cells decide to go from G1 to S
cyclin dependent kinase
depending on type of cyclin, will activate CDK for M or S phase
G1-CDK
cyclin D
cdk4, cdk6
G1/S-CDK
cyclin E
cdk2
S-Cdk
Cyclin A
Cdk2,cdk1
M-CDK
cyclin B
Cdk1
regulation of CDK
transcription
+/-p
Cdk inhibitors
ubiquitind dependent proteolysis
+/-P of Cdk
CAK cdk activating kinase
Wee1 +P inhibits
Cdc25 -P activates
Cdk inhibitors
p16, p21, p27
bind to an inactivate the active cyclin-cdk complexes
ubiquitin dependent proteolysis
APC/C: Anaphase-promoting complex/cyclosome: ubiquitin ligase complex
gets turned on by Cdc20 which tags cyclin B for degredation to make meta to ana transition
SCF
Skp1-cullin-f-box ubiquitin ligase complex
tags a Cdk inhibitor to promote S phase entry
S phase
DNA replication
sister chromatid cohesion
M phase
mitosis
cytokinesis
licensing factors
Cdc6
Cdt1
make sure chromosome duplication happens once only
prophase
chromosome condensation
kinetochore
two centrosomes already
prometaphase
nuclear envelope breaks down
mitotic spindle forms
centrosome at spindle poles
metaphase
chromosomes are attached to spindle MTs at kinetochore
MTs line up signaling Met-anaphase transition
anaphase
sister chromatid separation
Telophase
chromosomes arrive at poles
chromosome decondensation
nuclear envelope reassembles
contractile ring starts to contract
cytokinesis
contractile ring creates cleavage
telophase steps complete
which Cdk starts mitosis
M-CDK
Cdk 1 and m-cyclin
condensin
protein for prophase condensation of chromosomes
breakdown of nuclear envelope
M-Cdk
parts of mitotic spindle
spindle poles
astral microtubules
kinetochore microtubules
interpolar microtubules
spindle poles
centers of microtubule nucleation with minus ends
astral MTs
radiate outward from the poles and contact the cell cortex to help position MS
kinetochore MTs
attach sisters at kinetochores
interpolar MTs
+ ends overlap making antiparallel array
centrosome duplication
G1/S-CDK
centrioles separate
act as templates
dont spread and duplicate until M phase
tension check
auroro B kinase phosphorylates Ndc80 when low tension and increases affinity for MT plus end so it can get pulled away
APC/C and separation
ubiquinates securin freeing separaste to cleave cohesin to separate sister chromatids
if choromosomes not properly attached to MS
Mad2 binds APC/C and inhibits so no separation
RhoA GTPase
assembly and contraction of contractile ring which uses actin and myosin II
extracellular promoters
mitogens - mitogen receptors
growth factors - RTKs and mTOR
survival factors
extracellular suppressors
Stress
apoptotic signals
