Flashcards in 9.2.16 Lecture Deck (99):
The cytoskeleton involves a dynamic array of what three interacting filaments?
1. Actin (smallest, 5-7nm diameter)
2. Microtubules (largest, 25 nm diameter)
3. Intermediate filaments (medium, 10 nm diameter)
What are the 6 major functions of actin?
1. Dictate cell shape
2. Mediate cell adhesion
5. Muscle contraction
6. Cell migration
Actin is localized below the ___.
What determine cell shape and surface area?
Cortical actin and actin arrays
What are the two types of cell adhesion mediated by actin?
1. Cell-cell, through adherens junctions
2. Cell-matrix through focal contacts
Actin arrays partition polarized cells into ___ and ___ compartments.
The ___ is an actin-based structure.
Bundles of actin filaments are used by ___ for muscle contraction.
What are the four general instances needing cell migration?
2. Wound healing
3. Immune system
4. Cancer metastasis
What is the soluble subunit of actin?
Describe the structure of the actin monomer.
Globular, contains ATP when in the cytosol, ADP when in the filament, has + and - end (polar)
What is the actin filament made of?
Flexible helix of 2 protofilaments
Actin monomers and filaments are ___. This gives it ___. Which end is more dynamic?
Polar; directionality; +
___ regulate actin filament assembly and disassembly.
Small soluble actin subunits are in ___ with large filamentous polymers.
A signal can lead to disassembly of filaments and rapid ___ of subunits, followed by reassembly at a ___ site.
What are the three phases to actin filament assembly?
1. Nucleation (lag phase)
2. Elongation (growth phase)
3. Steady state equilibrium (equilibrium phase)
What is the rate limiting step to actin filament assembly?
What is the critical concentration?
The concentration of actin monomers at steady state
What happens to the kinetics of actin formation if actin "nuclei" are added directly?
Removes the nucleation/lag phase
Actin is nucleated at the ___ end.
Nucleation occurs preferentially at the ___, which allows for cell surface structures to form.
Nucleation occurs via what protein(s)?
Actin related proteins (ARP) -> ARP2 and ARP3
ARP binds pre-existing filaments at a ___ angle.
What modulates filament growth and localization? What do each of these do?
End binding proteins (ARP and Cap Z) - ARP caps and nucleates the minus end, CapZ binds and stabilizes the + end.
___ modulate filament elongation. These are ___ (less or more) efficient than end binding proteins.
Subunit binding proteins; less
___ modulate filament stability and orientation. This includes what protein?
Filament binding proteins; cofilin (actin depolymerization factor that preferentially binds to ADP subunits in existing actin filaments.)
Filamentous actin contains enzymes to...
...hydrolyze ATP present in subunits.
ATP hydrolysis converts ___ form to ___ form.
T (stabilizes + end of filament); D
Nucleotide hydrolysis results in treadmilling. What is this?
+ end addition is fast and hydrolysis lags behind. - end addition is slow and hydrolysis catches up. This creates a flux of subunits.
A high critical concentration is favorable for ___; a low critical concentration is favorable for ___.
Critical concentration of ___ is less than that of ___.
Describe actin protrusion in lamellipodia.
Net filament diassembly occurs behind the leading edge via cofilin binding to the ADP form of actin to cause depolymerization and Cap Z binding. Free actin subunits can then reassemble at the leading edge, extending the cytoplasm of the migrating cell.
What are the 5 types of actin arrays in cells?
1. Stress fibers/focal contacts: contractile bundles of anti-parallel actin; link to extracellular matrix via integrins.
2. Cell cortex: actin gel-like network beneath plasma membrane
3. Filopodia and microvilli: tight parallel bundles of actin
4. Lamellipodia: gel-like network of actin
5. Adherens junctions: link to other cells via cadherins
___ determines the type of actin array generated.
The geometry of actin binding domains
What are the two types of proteins involved in actin filament cross-linking?
1. Bundling proteins
2. Gel forming proteins
What are the two types of bundling proteins?
Fimbrin (monomer, parallel bundle, tight packing, prevents mysoin II entrance) and Alpha-actinin (dimer, anti-parallel contractile bundle, loose packing, allows mysoin II to enter)
What are the two types of gel forming proteins?
Spectrin (tetramer) and filamin (dimer)
___ activation induces polymerization and bundling.
Rho (protein family of GTPases)
What are three types of Rho, where are they found, and how are they packed?
1. Cdc42: microspikes and filopodia, parallel, tightly packed
2. Rac1: lamellipodia, membrane ruffles, gel at cell cortex
3. Rho: stress fibers and focal contacts, anti-parallel, loosely packed
How do actin targeted drugs such as phalloidin, cytochalasin, and latrunculin work? Are they clinically useful?
They interfere with or promote depolymerization and polymerization through stabilization of that which they bind to; No
___ also target actin polymerization, inducing cell death and disrupting tight junctions.
___ binds actin filaments, reducing actin critical concentration to 0 and stabilizing existing filaments so they do not disassemble.
What is the actin molecular motor?
Myosins bind ___ filaments and use ___ to move in one direction.
Polarized; ATP hydrolysis
Myosin II moves to the ___ end of the filament and is found in ___.
What determines the polarity of movement and the cargo of myosin?
Structure (most are directed to the + end)
Myosin forms a ___ of two alpha helices; at the N-terminus, there are two ___ that can bind to myosin. Ultimately, this forms ___ to which 2 actin filaments bind.
coiled-coil; head groups; myosin II bipolar thick filament
In the myosin II bipolar thick filament, where are the myosin heads located?
Myosin ___ on actin at a high speed.
What is the myosin motor mechanism?
Rigor state: myosin bound, no ATP, ATP binds to myosin, which detaches, ATP hydrolysis occurs (cocked), myosin binds to actin, myosin releases ADP, the power stoke occurs.
Is myosin attached or detached for most of the hydrolysis cycle?
What disease is caused by mutations in the myosin beta heavy chains and leads to enlarged heart, cardiac arrhythmia, and sudden death in young athletes?
Familial hypertrophic cardiomyopathy
What are the 3 functions of microtubules?
1. Positioning of organelles
2. Intracellular transport
3. Cell motility (flagella and cilia)
The Golgi and the ER are localized via ___.
Intracellular transport via microtubules occurs in what three instances?
1. Mitotic spindles
2. Vesicular transport on ER via MT tracks
3. Organelles move bidirectionally along microtubules
What is the soluble subunit of microtubules?
Alpha-tubulin and beta-tubulin heterodimers
Both alpha-tubulin and beta-tubulin can bind ___.
13 ___ form the microtubule.
___ GTP can be hydrolyzed.
Are microtubules polar? If so, which end is more dynamic?
Yes; + end more dynamic
The - end of microtubules is buried in the ___.
Microtubule Organizing Center (MTOC)
MTOCs organize different types of MT arrays. What are 3 of these?
1. Gamma-tubulin ring complex: embedded component of MTOC
2. Centrosomes: spherical MTOCs, contain a pair of centrioles (help duplicate centrosomes, found near nucleus)
3. Basal bodies: organize cilia and flagella MT (found near plasma membrane)
MT grow from the ___ in the ___ direction.
Gamma-tubulin ring complex; negative to positive
MT assembly and disassembly results from ___.
Describe the process of dynamic instability in MT.
Only the + end of MTs are free. The - ends are buried in centrosomes. The GTP-form favors assembly. Accidental loss of the GTP cap occurs (catastrophe). Rapid depolymerization occurs, as the GDP-form favors disassembly. Regaining the GTP cap (rescue) begins the process over again.
Which form favors MT assembly? Disassembly?
T form (GTP bound); D form (GDP bound)
___ organize MTs into astral arrays.
What are MAPs?
Microtubule Associated Proteins; filament cross-linking proteins involved in spacing and stabilizing MT.
What are the two major types of MAPs?
1. MAP2 - has a long arm
2. Tau - has a short arm
What determines MT spacing?
Spacer arms in MAPs
What protein aggregates in Alzheimer's?
What makes MT targeted drugs such as taxol, colchicine, vinblistine, and nocodazole effective cancer drugs?
Disrupt mitotic spindle formation selectively in rapidly growing cells.
MT molecular motors bind MT and use ATP hydrolysis to move cargo in ___ direction(s).
What are the two types of MT molecular motors?
Kinesin and dyneins
What do kinesin and its related proteins (KRPs) do?
Move cargo to the + end
What do dyneins do?
Move cargo to the - end
Kinesin ___ hand-over-hand in a processive fashion on the MT as a ___.
Kinesin is in contact with the MT ___% of the time.
Kinesin moves cargo slowly but ___.
What are two specialized MT-based structure known as axonemal dyneins?
Flagella and cilia
Where are flagella found and what type of motion do they utilize?
Protozoa and sperm, wave-like motion
Where are cilia found and what type of motion do they utilize?
Respiratory epithelium, whip-like motion
What is an axoneme?
9 doublets + 2 singlets, configuration of MT core inside cilia and flagella; also contains many connector proteins
What are basal bodies?
MTOCs that anchor cilia and flagella at the cell surface, similar to a centriole, always by plasma membrane
Basal bodies involve ___ MT triplets.
What is Kartagener's syndrome?
Primary ciliary dyskinesia (defect in ciliary dynein), leads to infertility in both sexes, respiratory issues (sinus infections), and situs inversus (organ position inverted)
What are the 4 functions of intermediate filaments (IF)?
1. Mechanical strength - give cells and tissues their integrity.
2. Cell adhesion - promote cell-cell adhesion at the desmosome and cell-matrix adhesion at hemidesmosome
3. Neuron axon diameter and strength
4. Nuclear lamins - contribute to nucleus mechaniacal integrity
Mutant IF causes ___.
Epidermolysis Bullosa simplex (EBS)
Laminopathies are associated with mutations in ___.
What causes the high physical strength of IF?
Staggered and lateral subunit interactions
Are IFs polar?
No - they are symmetrical
What is the soluble subunit of IFs?
Staggered tetramer of 2 coiled-coil dimers
___ tetramers associate laterally to form an IF.
In IF cross-linking, the ___ domain binds to neighboring filaments.
What are tonofilaments?
Bundled keratin IFs
Accessory proteins organize IFs. What are two accessory proteins?
1. Filaggrin - bundles keratin IF
2. Plectin - bundles vimentin IF, binds to actin filaments, motor ptoeins, plasma membrane
IF proteins involve a heterogenous gene family with ___+ members.