Week 8 + Module 7 Flashcards
(74 cards)
1
Q
The cytoskeleton
A
- A network of protein-based filaments that provide cell structure
- Dynamic; allows cell movement, growth, and differentiation
- Made up of three types of filaments (differ based on diameter and type of subunit used)
1. Actin
2. Microtubules
3. Intermediate filaments
2
Q
Actin filaments - labelling
A
labeled using fluorescently-tagged phalloidin
can also be labeled an antibody or with GFP
3
Q
Microtubules - labelling
A
Labeled using antibodies specific to one of the tubulin subunits
or tubulin GFP fusion protein
4
Q
Intermediate filaments - labelling
A
Can be labeled using an antibody specific to a monomeric subunit of the filament
or GFP fusion again
5
Q
Actin filaments - appearance and location
A
thinnest
- made of monomeric actin subunits
microvilli at cell surface (apical)
6
Q
Microtubules - appearance and location
A
thickest
- made of dimeric subunits (alpha+beta tubulin)
form networks for intracellular transport
7
Q
Intermediate filaments - appearance and location
A
many different filaments
- made of lamin proteins
span the cell (structural support)
8
Q
Motor proteins (3) - names
A
Myosin (actin)
Kinesin & dynein (microtubules)
None for IFs
9
Q
Motor proteins (3) - function
A
- Head domains bind to a cytoskeletal fibre (actin filaments or microtubules)
- Tail domain attaches to a cargo
- ATP hydrolysis provides energy for these motor proteins to “walk”
10
Q
Actin - functions / different structures
A
- Establishment of microvilli
- Formation of contractile bundles that form sarcomeres that power muscle cell contraction
- Formation of filipodia and lamellipodia needed for cell migration
- Contractile ring that directs cytokinesis or cell division
11
Q
G → F actin (general)
A
G-actins are monomeric subunits of F-actin
F-actin consists of 2 strands of helical polymers
- they behave differently
12
Q
Actin filament assembly and disassembly
A
Plus and minus end of actin depend on where the myosin head-protein binds
Like an arrow pointing towards the (-) side
(-) is pointed
(+) end grows more quickly
13
Q
G-actin monomers
A
G-actin stands for globular-actin
- Divided into 4 structural domains with large cleft between domains 2 and 4
- Cleft forms an ATP-nucleotide binding site/pocket
- Pointed to the minus end of the elongating polymer so the ATP pocket of each monomer is not exposed
Looks like a stack of “n” with (+) on the open end
14
Q
F-actin polymers
A
- F-actin constantly engages in polymerization and depolymerization and this can occur at both the plus and minus ends
- more growth at (+) end, shrinkage at (-) end
15
Q
ATP role in actin assembly/disassembly
A
- ATP-bound actin monomers in cytosol join the plus-end as long as [Actin-ATP] is high enough
- Actin has an intrinsic ATPase activity (ATP→ ADP) and releases Pi
- Happens within the polymer so usually most of the actin filament is made of actin-ADP
- ADP is not released because the nucleotide binding site ends up being covered in the actin filament
- Rate of polymerization is greater than the rate of depolymerization at the (+) end
- Opposite at the (-) end
16
Q
Critical Concentration
A
Concentration at which the rate of actin monomer addition is equal to the rate of removal
- different at +/- ends = different dynamics
→ If greater than critical condition: GROWTH
Rate of polymerization exceeds rate of depolymerization
and v.v. for shrinkage
17
Q
Chemicals impacting rate of reactions (poly/depoly)
A
Profilin:
- binds to actin-ATP = ATP binding + monomer activation
- dimer accumulates at (+) end = increases active actin monomers
POLY
Thymosin:
- binds to actin monomers and inhibits polymerization
- accumulates at (+) end = creating a buffer of stored actin monomers
INHIBITS POLY
Capping proteins:
- ends of F-actin filaments can also inhibit polymerization or depolymerization
INHIBITS POLY OR DEPOLY
18
Q
Treadmilling
A
When no net increase in length of filament
- just relative position moves forward
Because rate of polymerization (+) end = Rate of depolymerization (-) end
19
Q
Actin powering movement
A
- done through filament reorganization
- Formation of leading edge of cell (part of cell closest to direction of movement)
- Formation of fan like expansions of cell membrane (lamellipodia and filipodia)
- Movement of cell forward in direction of movement
20
Q
Myosin motor proteins: move along ______ and power _______
A
Able to move along actin filaments and power intracellular cargo trafficking
21
Q
Myosin I, II and V - common appearance
A
- All share a characteristic motor (head) domain at the N-terminus
- This has a site that binds actin filaments and a site that binds and hydrolyzes ATP to drive the motor
- However between the proteins, they differ in tail domains
- Carry cargo at different rates
22
Q
Myosin II - appearance
A
- Contains 2 heavy chains that form coiled-coil motif (2 wrapped alpha-helices)
- 4 light chains of 2 types (at the end)
- Does not carry any cargo but plays a role in generation of contractile forces needed for many cellular activities
23
Q
Myosin light chains phosphorylated by ____
A
MLCK
myosin light chain kinase
Active state (phosphorylate)
- Initiates extension of myosin tails
- Activates actin-binding domains on motor heads
24
Q
Myosin II bipolar thick fragment
A
- assembly of 15-20 myosin II proteins
- Motor heads to left and right
- Bare patch/zone of myosin tails in middle
- Motor heads are exposed so they can be associated with actin filaments
25
Contractile skeletal muscle fibers diagram + explanation
Z disk, cap Z, plus end of filament, actin thin + myosin thick, minus end, m-line, ... repeat
- Associated with thin actin filaments into structures called “sarcomeres” and gives the striated/striped muscle appearance
- Plus-ends of actin filaments are fixed to Z-discs within the sarcomere
- Capping proteins cap ends:
a. Tropomodulin at minus-ends
b. Cap-Z protein at plus end
- Nebulin binds parallel actin filaments
- Between parallel actin fibers
- There are myosin thick filaments attached to Z-discs by a molecular spring (titin protein)
26
Skeletal muscle fiber contraction - process
- Myosin heads associate with actin filaments and results in actin filaments being pulled past the myosin filaments towards the middle of the sarcomere
- Occurs by the cyclical association of the actin filaments with the myosin motor heads
- Causes shortening of sarcomere without change in myosin thick or actin thin filament length
→ Each myosin head cycles through ATP binding and hydrolysis to track/move along actin filaments
- Causes shortening of sarcomere and muscle contraction
- Also a calcium-dependent process to allow exposure of myosin binding sites along actin filaments:
- Calcium dissociates from actin filaments and myosin heads along the thick filament releases actin thin filaments
- Thick and thin filaments slide past each other to allow sarcomere elongation and muscle relaxation
27
Chemical process of muscle contraction
1. Myosin is attached to actin
2. ATP binding to myosin releases actin
3. ATP → ADP and Pi, facilitated by myosin head, changed myosin conformation to the relaxed conformation
4. Release of Pi increases affinity of myosin head for actin and allows binding
5. Release of ADP from myosin head changes myosin conformation
- Since myosin is attached to actin, this pulls the actin filament to the left
28
Myosin V
Powers intracellular trafficking of cargo along actin filaments
29
Melanosomes in melanocytes - myosin V
- Membrane-enclosed organelles containing pigment granules (melanin)
- Each melanocyte in epidermis of skin has dendrites that stretch to connect it with keratinocytes
- Melanin in the keratinocytes and distribution of pigment protects skin cell DNA from UV damage (process called tanning)
- Myosin V distributes melanosomes to cell membrane along actin filaments
30
What determines rate of myosin motor protein movement
- Ranges from 0.2 - 60 um/sec
Based on:
- Rate of ATP hydrolysis by ATPase in each myosin head
- Proportion of time myosin is bound to actin (affinity)
- Myosin V moves more slowly than myosin II
31
What determines step size
- length and angle of arm
Lever arm length: distance by which the power stroke properly myosin forward
- Myosin V is 3 times longer than that of myosin II
- V: 36 nm, II: 7 nm
32
Microtubule fibers - composition
- 13 protofilaments each (like strands in a pull and peel twizzler)
- arranged in a circular pattern
- made of dimers of alpha and beta tubulin proteins
- protofilaments are staggered so that they spiral up (like a spring)
33
Alpha and beta tubulin relation to GTP
- 1 alpha + 1 beta bound to 2 units GTP
Alpha subunit is tightly bound to GTP
- GTP is never hydrolyzed and does not exchange with nucleotides in solution
Beta subunit is less tightly bound
- GTP is cyclically hydrolyzed between GDP and GTP
(+ end) GTPb / B-tubulin / GTPa / A-tubulin (- end)
34
Microtubule end characteristics - polarity
(+) end is the fast-growing end
- Beta-subunit is closer to the plus-end
- Where alpha-beta-GTP dimers are added (rescue phase)
(-) end is the slow-growing end
- Alpha-subunit closer to minus-end
- Where alpha-beta-GDP dimers are released from shrinking filament (catastrophe)
35
EB1 and microtubule growth
EB1-GFP is a plus-end binding protein which prevents premature catastrophes and acts as a positive regulator of microtubule growth
36
MAPs - what are they and what do they do
Microtubule Associated Proteins
Control assembly and disassembly of microtubules
- Interconnect microtubules to help form bundles/cross-bridges
- Increase stability
- Alter rigidity
- Influence assembly rate of microtubules
37
MAP classifications (2)
1. Those that stabilize the filament
Ex. Tau and EB1
Result in longer, less dynamic microtubules
2. Those that destabilize the filament
Ex. catastrophin
38
Nucleation by gamma-tubulin
Nucleation: starting off the growth of microtubules
- Y-tubulin is present in much smaller amounts compared to alpha/beta-tubulin
- Forms the y-tubulin ring complex (y-TuRC)
- Ring nucleates at the minus-end of a new microtubule by forming a template for the growing, plus-end
- y-TuRC acts as a cap of the minus-end while microtubule growth and dynamics occur at the plus-end
39
Where do microtubules start forming in cells?
MTOC - microtubule organizing center
MTOC is where microtubule nucleation occurs
the center of the starburst
40
Centrosomes in MTOCs
- 2 cylindrical structures called centrioles
- Cloud or pericentriolar material (PCM)
- Contains multiple y-TuRC complexes
- Minus-ends are nucleated at y-TuRC complexes
- Microtubule plus ends are directed towards the periphery of the cell
41
MTOC importance during mitosis
- Microtubules of mitotic spindle attach to the chromosomes and help replicate sister-chromatid separation
- Mitotic spindles are dynamic and are assembled and disassembled during the cell cycle (depends on dynamic instability of microtubules)
- Centrosomes are duplicated in mitosis to create 2 MTOC’s
- As replicated MTOC’s separate, microtubules are nucleated at y-TuRC complexes and plus-ends grow outwards
42
Microtubules during mitosis
Interphase:
Network of microtubules fills the cell
Metaphase:
- Compact replicated chromosomes are aligned at the equator of the metaphase spindle via attachments between microtubules stretching from poles of spindle to attachment sites at centromeres of the chromosomes
Anaphase:
- Spindle poles move apart and microtubules attached to chromosomes shorten
43
Microtubule toxins - what are they + examples
Small molecules can inhibit microtubule’s dynamic characteristics; lethal to the cell
Colchicine and taxol
44
Microtubule toxin; Colchicine
- Derived from plants: meadow saffron or autumn crocus
- Act as microtubule toxin by inhibiting polymerization
- Colchicine binds and stabilizes free aB-tubulin dimers
- While bound dimers can be incorporated into the growing microtubule, they prevent the subsequent addition or loss of other tubulin dimer subunits
- Cells in mitosis will be arrested in metaphase without chromatid separation
45
Microtubule toxin; taxol
- Binds to B-tubulin and increases affinity of dimer for the plus-end, preventing depolymerization and stabilizing the microtubules
- Prevents assembly of mitotic spindle and thus inhibits mitosis
- Effective cancer treatment (known as paclitaxel) derived from Pacific Yew Tree
46
Kinesin - structure
- Tetrameric Complex made of 2 identical heavy chains and 2 identical light chains
- Globular head at N-terminus of the heavy chain = motor domains
47
Kinesin - what determines the specificity of cargo binding
Tails determine specificity of cargo binding (highly variable)
Kinesin motor proteins are highly conserved across family members but tails are highly variable
48
Kinesin cargo movement direction
- Towards the plus ends of microtubules
- Move cargo away from the cell MTOC (towards cell periphery)
49
Mechanochemical cycle of kinesin
Hand-over-Hand motion
- There are 2 motor domains in the kinesin dimer and one is always attached to the microtubule
- 2 heads to kinesin are coordinated - at any one time they are present in the complementary stage of the chemical cycle
50
Kinesin leading/lagging explanation
1. Lagging head bound to ATP and leading head is bound to ADP
- Since the ATP-bound kinesin has a higher affinity for the microtubule so that is why its lagging
- In the lagging head, the ATPase motor head hydrolyzes ATP → ADP and Pi
2. ATP → ADP causes lagging head > leading head due to increasing affinity for microtubule
3. Binding of ATP causes conformational change in neck region causing lagging head to swing in front of the leading head
51
Dynein - structure
Contains 2 identical heavy chains and a variety of intermediate and light chains
52
Dynein cargo movement direction + 2 forms
- Minus-end directed motor (move away from cell periphery towards MTOC of cell)
Comes in 2 forms:
1. Cytoplasmic (associated with microtubules and direct movement of organelles and vesicles in the cytoplasm)
2. Axonemal (found in structures that power the movement of whole cells (cilia, flagella))
53
Ability of dynein to move cargo is driven by __________
alternate power-strokes of the linker
linker = arm near cargo attachment site
54
Dynein movement steps
Step 1) ATP binding releases the motor head group from the microtubule
Step 2) ATP hydrolysis (dynein + ADP + Pi) can now attach to the microtubule
Step 3) Release of Pi power the power stroke of the linker, pulling the cargo to the left
→ Each power stroke = cargo moves towards minus end by 8 nm
55
If there is bidirectional movement, what determines the overall direction of movement?
One model suggests proteins are engaged in a molecular tug of war
- Kinesin to (+ = anterograde), Dynein to (- = retrograde)
- Final direction of movement is decided by the winner of the battle
56
Transport of melanosomes in fish skin cells
- The movement of the organelles changes the colour of the skin cells in response to behavioural signalling, made possible by molecular motors
Dynein:
concentrates melanosomes at the center (move melanosomes along microtubule tracts towards (-) ends at the MTOC near the center of the cell)
- Cells near center cause cell to appear lighter
Kinesin motors:
are responsible for dispersing melanosomes and carry melanosomes to the periphery
- Dispersion of melanosomes cause cells to appear darker
57
What are lectins
Carbohydrate-binding macromolecule proteins that are highly specific to sugar groups that are part of other molecules
58
What are CAMs - how many families
Cell adhesion molecules
4 families
1. cadherins
2. immunoglobins
3. integrins
4. selectins = lectins
59
5 types of lectins
1. Calnexin (protein folding chaperone)
2. Selectins (mediate cell-cell interactions)
3. Coronavirus spike protein (viral infections)
4. FimH (bacterial infections)
5. Plant lectins (often toxic, grains, legumes, dairy)
60
Covid (virus) adherence
Spike protein (lectin) receptor binding domain on SARS
- recognizes oligosaccharides containing sialic acid
- SARS bound by ACE2 and glycans
61
Bacteria (pathogen) adherence
E. coli
- fimH lectin adheres to D-mannose
- on uroepithelial cells
H. pylori
- BabA, SabA, LabA
bind to
LeB, sLeX, LacdiNAc
- on gastric epithelial cells
GAS (group A strep)
- M protein has a high affinity for blood group H antigens
- on oral epithelial cells
62
What is UPEC
uropathogenic E. Coli
- causative agent for most UTIs
63
UTI treatment + issue with this treatment
antibiotics
- antibiotic resistance (can be passed on to other bacteria)
- disturbs beneficial gut microbiome
64
UTI adhesion
- required so that organisms are not swept away by natural cleaning mechanisms of the host
- need to stay long enough to multiply and cause damage
65
What happens after adhesion during a UTI
- FimH on E. coli invades cells
- can cause apoptosis
- bursts and spreads via exfoliation
- exist undetected in a niche
66
What is FimH
FimH adhesin is a lectin on the ends of pilli
Type 1 Fimbriae (pilli) on UPEC attach to mannose oligosaccharide groups on the surface of urothelial cells
67
Two domains of the FimH adhesin
i) COOH-terminal [pilin domain] incorporates FimH into Type 1 pili
ii) NH3-terminal [adhesin domain] contains a carb binding pocket that can bind to D-mannose residue
68
Is FimH necessary and/or sufficient for infection
Yes necessary (exp removed fibriae)
Yes sufficient (exp added to latex beads)
69
If UPEC adhesion blocked
- bacteria will be eliminated by the flow of urine before they can start an infection
70
Anti-adhesion drugs for UPEC
Carbs
- bind to fibH and compete with the binding of mannose
71
Anti-adhesion drugs for FimH - experiment+results
FimH = bacterial lectin
Mannan definitely blocks binding of a-delta-mannose
Experiment to see if man, glc, and gal (mannose derivatives) would also block
Result:
- weak competitive effect
- bc many pilli
- each can bind to a host receptor
= multivalent pathogen-host interactions
72
Cranberries preventing UTIs
- contain polyphenol molecules called pro-anthocyanidins
- reduce adherence of UPEC
- reduced infectivity (to primary bladder epithelial cells)
- act through FimH
73
Anti-adhesion drugs: remaining challenges
Many pathogens carry a VARIETY of adhesive structures
- need cocktails of anti-adhesive agents
- and/or combine aa agents with conventional antimicrobials
74
Water purification (anti-adhesion)
- reduction of bacterial colony forming units by mannose-linked nanodiamonds
- grab impurities
