Exam 3 Flashcards
Actin Monomer and Structure/Polarity
Globular Actin Monomer (G-Actin); made of two lobes separated by a nucleotide binding cleft (ADP or ATP) - Cleft opening is at minus end and closed end is plus end —> POLAR
Actin Filament Formation
Multiple G-actin monomers bind in the same direction (one’s plus ends interact with another’s minus ends) to form the helical, polar F-actin (Cleft is exposed at one end and not exposed at the other)
Inducing F-Actin Formation
Add Mg2+ –> Associates with Nucleotide bound in Cleft
3 Steps for F-Actin Formation
- Nucleation - involves trimer assembly as a seed for growth; slow step because actin dimers are unstable
- Elongation - subunits add to the trimer; growth
- Steady State - rate of elongation = rate of subunit loss; occurs when G-actin is at critical concentration
Critical Concentration for Actin
If G-Actin exists below critical concentration, no polymerization will be occurring yet
If G-actin exists above critical concentration, polymerization will occur
Always trends toward making [G-actin] = [Critical]
Rate of Polymerization at each end of F-Actin and why?
Critical Concentration at (+) end is lower and thus growth occurs much faster = 0.12 um
Critical Concentration at (-) end is higher and thus growth occurs much slower = 0.6 um
This is due to polymerizing actin monomers containing ATP. As these monomers exist in the filament, said ATP is hydrolyzed and the affinity of the actin monomers for each other decreases. Because it will be typical that subunits will “move down” F-actin towards minus end, the affinity for a free G-actin for the (-) end (ADP-containing monomer) will be lower than for the (+) end (ATP-containing monomer)
Real Cell Critical Concentration for F-Actin Formation and what occurs when this value is reached?
0.2 um; Actin Filaments will exist in the Steady State. (+) ends are adding G-actin and (-) ends are releasing monomers such that there is no net growth or shrinkage – known as Treadmilling!!
Reality is G-actin does not exactly exist at critical concentration, in fact a concentration much higher…what then prevents a great extent of F-actin formation?
Actin-Binding Proteins and Actin Sequestering Proteins
Examples of ABPs and ASPs that prevent spontaneous filamentation
Thymosin Beta-4: binds to free ATP-Actin to prevent polymerization
Profilin: promotes ADP to ATP exchange in cleft while remaining bound to (+) end; promotes polymerization while preventing nucleation
Cofilin: sequesters considerable segment of ADP-Actin from F-actin; promotes depolymerization by “creating more (-) ends”
What exists to catalyze nucleation? 3 Classes
Actin Nucleating Proteins (3 General Classes)
1. Formin Family - attaches to (+) end and uses its FH2 domain to preform multiple (+) end dimerizations, leading to long unbranched filament formation
- Tandem Actin Monomer-Binding Proteins: caps (-) end to promote nucleation and (+) [open] end growth
- ARP2/3 Complex: binds to existing F-actin to create new branch and stays bound to said new branch’s (-) end; MUST pair with WASP protein to get function/nucleation
Chemical Interference with Actin Cytoskeleton: 3 Compounds
Phalloidin: binds along actin filaments to stabilize and prevent depolymerization
Cytochalasin: caps (+) ends to prevent elongation and eventually cause complete depolymerization of said F-actin
Latrunculin: Binds actin monomers to gatekeep them out of polymerizing
Microtubule Subunit and Structure/Polarity
Alpha- Beta- Tubulin Heterodimer; alpha subunit will bind GTP and never hydrolyze it while beta subunit will hydrolyze it
Microtubule Formation
Multiple heterodimers align to form linear structures - protofilaments.
13 protofilaments laterally interact (in parallel fashion) to create a cylindrical shaped structure with a (+)/beta end and (-)/alpha end
3 Steps of Microtubule Formation
- Nucleation - involves several heterodimers associated to create seed for growth (slow step)
- Elongation - more heterodimers add to the seed; growth
- Steady State - rate of elongation = rate of subunit loss; occurs when free/available tubulin is at critical concentration
In-Vitro versus In-Vivo Microtubule Assembly
In Vitro: Subunit Addition or Loss is observed at both ends
In Vivo: (-) end is capped/anchored in MT Organizing Center/Centrosome to help with initial nucleation – only (+) end growth is observed
Free Microtubule Dynamics - Treadmilling
Undergoes treadmilling can experience addition from both ends
GTP bound heterodimers will add and create a GTP cap; as these subunits “progress down” the microtubule, the beta subunit undergoes hydrolysis and “leaves” the GTP cap
If [free tubulin] > [critical], rapid addition and thus rapid GTP cap growth occurs
If [free tubulin] ≈ [critical], there is a stochastic chance that GTP cap could be lost if addition is too slow; this loss is destabilizing
Behavior of Microtubule Growth about Critical Concentration
If [free tubulin] > [critical], rapid addition and thus rapid GTP cap growth occurs
If [free tubulin] ≈ [critical], there is a stochastic chance that GTP cap could be lost if addition is too slow
Loss of GTP cap is destabilizing as it causes a conformational change that induces curving and weakens lateral protofilament interactions
Free Microtubule Dynamics - Dynamic Instability
Based on rates of addition, microtubules randomly alternate between two growth states due to nucleotide conversions at the (+) end
Sudden Growth to Shrinkage: Catastrophe
Sudden Shrinkage to Growth: Rescue
When in the steady state, these effects cooccur such to keep total amount of polymerized heterodimers constant
Where are microtubules typically anchored? What is the purpose?
The Microtubule Organizing Center/Centrosome
Contains two centrioles surrounded by pericetriolar material, inclusive of the gamma-tubulin ring complex (γ-TURC)
γ-TURC is a nucleation site where the (-) ends anchor; treadmilling is no longer possible but dynamic instability at the (+) ends is
Chemical Interference with Microtubule Cytoskeleton: 3 Compounds
Taxol: Binds along filaments to stabilize polymer and prevent catastrophe
Colchicine: Caps filament ends to prevent addition/polymerization, causing formation of a GDP cap [destabilizing, promoting depolymerization]
Nocodazole: Binds to tubulin subunits to prevent them from adding, causing inevitable formation of the GDP cap and subsequent depolymerization/catastrophe
Actin Capping Proteins
Stabilizes F-actin by binding to ends to prevent addition/loss of subunits
CapZ for (+) ends
Tropomodulin for (-) ends
Actin Stabilizing Proteins
Stabilizes F-actin by binding length-wise to stabilize filament
Tropomyosin
Actin Severing/Depolymerizing Proteins
Does not have a consistent manner of binding but always leads to disassembly
Cofilin: severs filaments to create more (-) ends, more sites for depolymerization
Gelsolin: breaks filaments and binds to (+) ends
Actin Cross-Linking Proteins (2 Results)
Produces different assemblies of F-Actin based on built-in spacer domain
Typically has two binding domains to bind multiple filaments and create the assembly
With Flexible, Longer Spacer Domains, we see Gel-Like Networks
Caused by Filamin, Spectrin, ARP2/3
With Rigid, Shorter Spacer Domains, we see Aligned Bundles
Caused by Fimbrin, Villin, Fascin, and Alpha-Actinin [slightly longer]
Membrane-Actin Linker Proteins
Create connections between Actin Cytoskeleton and Plasma Membrane, usually Peripheral Membrane Proteins that serve as the linkage between cytoskeleton and integral membrane protein
ERM Family (Ezrin, Radixin, Moesin)
Spectrin
Dystrophin
Spectrin-Based Cytoskeleton (Example)
Spectrin binds to Actin Filaments and Ankyrin
Ankyrin binds to Band 3 (Integral Membrane Protein)
What happens consequence to mutations in Membrane-Actin Linker Proteins?
Mutations in genes encoding any part of Spectrin-Based Cytoskeleton leads to Hereditary Spherocytic Anemia or conditions where RBC rupture easily
Mutations in Dystrophins lead to Duchenne Muscular Dystrophies (progressive weakening of skeletal muscle)
Microtubule Tubulin Oligamer Binding Proteins
Sequester heterodimers or oligamers to prevent rebinding/rescue and thus promote catastrophe/depolymerization
Stathmin
Microtubule Nucleating Proteins
Assist with Nucleation at Centrosome
γ-TURC
Microtubule End Binding Proteins
Regulate end dynamics and linkages to other cellular structures
EB1: (+) tip - specifically for linkages to organelles
CLIP-170: (+) tip - specifically for interactions between the end and chromosomes or membrane
Microtubule Severing Protein
Break MTs to create more (-) ends and promote catastrophe/depolymerization
Katanin
Microtubule Depolymerizing Proteins
Promote (+) end depolymerization via binding protofilaments and promoting a destabilizing curling conformation
Depolymerizes GTP-Tubulin; IS NOT destabilizing by hydrolysis
Kinesin 13 + Related Subset
Polymer Binding Microtubule Associated Proteins (4 Functions)
i. Stabilizes by binding to sides
ii. Enhance assembly by stabilizing multi-dimer nuclei
iii. Organize MTs into Bundles
iv. Mediate interactions between MTs and Proteins, other Filaments, or Actin
Typically have two domains
1. Microtubule Binding: binds several dimers at once
2. Projection: stems off of Microtubule Binding Domain to interact with other structures
Plectin: used to link different types of filaments
Tau and MAP2: MT organization in neuronal axons and dendrites (MAP2 provides more spacing than Tau)
Loss of Tau PB-MAP Function
Results from rapid phosphorylation of Tau and its consequent sequestration into neurofibrillary tangles; can no longer stabilize microtubules
Associated with Alzheimer’s Disease and Taupathies
Actin Motor Protein
Myosin – mechanochemical enzyme that uses ATP hydrolysis for conformational change/movement
All except Myosin VI undergo (+) end movement
Composition of Actin Motor Protein
Myosins create complexes using heavy chains and light chains
Heavy chains contain
Head (for actin binding and ATPase activities)
Neck (binds Calmodulin: binds Ca2+ and/or the light chain)
and Tail (determines what the myosin binds, whether it’s a monomer/dimer, it polymerizes to create thick filaments domains, and/or whether it binds to other proteins/membranes)
Light Chains are used for structural and regulatory function
Studying Actin Motor Protein Movement
Microscopy of Filament Binding Assays
- Myosin sticks to glass with bound tails and free heads
- Label Actin with Phalloidin (stabilizing)
- Observe binding between Myosins and Actin
- Add ATP, Myosins now push Actin Filaments in a certain direction
Mechanism of Actin Motor Protein Movement (5 Stages)
Myosin goes through a Cross-Bridge Cycle
1) Rigor State: Due to lack of ATP, myosin is tightly bound to actin – stiffness/rigor mortis
2) ATP Binding: ATP binds, ATP Cleft Closes and Actin Binding Cleft Opens – weakens interaction between myosin and filament
3) ATP Hydrolysis: When myosin releases from actin, ATP hydrolysis occurs, causing head to undergo conformational change analogous to forward movement. Rebinds actin after this
4) Inorganic Phosphate Release: Pi release induces a 2nd conformation change (aka the Power Stroke) to exert force on actin, causing movement. Myosin now readopts Rigor State
5) ADP Release: simple a nucleotide release; remains in rigor state (potential to repeat process now)
Myosin I Description
Exists as monomer that either
has an Actin-Binding site to help filaments move past each other
OR
has membrane-binding sites and is involved with movement of vesicles and organelles
Myosin II Description
Well known isoform is involved with muscle contraction
Exists as dimer with 2 heavy chains and 2 coiled alpha-helices in tail region which creates a bipolar filament/polymer organization for itself [head region on outsides, tails on interior]
Myosin V Description
Exists as a dimer that undergoes processive movement (head-over-head movement along F-actin)
Also thought to be involved in vesicle/organelle transport
Mutations in Myosin II and Myosin VII genes leads to what?
Myosin II: bleeding problems, hearing loss, kidney disease, cataracts
Myosin VII: deafness and blindness in humans
Disappearance of Myosin VI leads to what?
Potential Deafness (observed in mice)
Microtubule Motor Proteins
Kinesins and Dyneins - both mechanochemical ATPases
3 Major Classes of Kinesins
- Kin-Ns (Conventional): motor domain at N-terminus – (+) end movement
- Kin-Cs: motor domain at C-terminus – (-) end movement
- Kin-Is: motor domain is internal; used for protofilament peeling
ex) Kin 13
Kinesin Movement Mechanism and Purpose
Can study using same setup as Myosin Binding Assay
Undergoes Processive Movement to travel long distances without disassociating
1) Forward head binds beta-tubulin and releases its ADP
2) Allows said forward head to bind ATP, causing conformational change that swings rear head around
3) Previously forward head hydrolyzes its ATP; New forward head binds beta-tubulin and releases its ADP (REPEAT)
Has light chains that bind to tail domains to mediate interactions with membrane vesicles and organelles that it will be transporting
Structural Description of Dynein + Movement Pattern
Heavier MW-wise compared to Kinesins; KNOWN FOR (-) END MOVEMENT
Mainly concerned with Cytoplasmic Dyneins
- Dynactin Binding Domain Tail
- 2 ATPase Domains
- 2 Microtubule Binding Domains
Power Stroke for movement produced by a change in the angle between the DBDT (stem) and MBD (stalk)
What is Dynactin Complex?
The thing that mediates the attachment of Dyneins to its vesicles and organelles it is transporting using an inherent ARP1 (Actin-Related Protein) that binds Spectrin + Ankyrin to create its own membrane connection
What is caused by defects in Kinesins, Dyneins, and Dynactin?
Kinesin defects lead to Charcot-Marie Tooth Disease or Kidney-Related Problems
Dynein defects lead to Chronic Respiratory Tract Infections
Dynactin defects lead to ALS
Considering Cell Locomotion?
Consider Fibroblasts
Fibroblast Structure and Behaviors: Lamellipodia
Thin sheets contained in a branched network; protrudes cell to give a little force to encourage movement (driven by ATP hydrolysis)
Membrane is known to exhibit some resistance to the protrusion of lamellipodia and thus pushes back
Depolymerization at negative ends is occurring, so treadmilling is observed
Fibroblast Structure and Behaviors: Filopodia
Thin projections or spikes arranged in parallel bundles; protrudes cell to give a little force to encourage movement (driven by ATP hydrolysis)
Membrane is known to exhibit some resistance to the protrusion of filopodia and thus pushes back; said to experience “retrograde flow,” being pushed by membrane towards where its (-) end resides; into the cell
Depolymerization at negative ends is occurring, so treadmilling is observed
Fibroblast Structure and Behaviors: Stress Fibers
Supports movement encouraged by Filopodia and Lamellipodia, located behind the other fibroblast structures to exist as a contractile unit
Made of long bundles of anti-parallel actin filaments with Myosin II inbetween each layer (both heads try to move to (+) end; contraction!) and orthogonally attached alpha-actinin (spacer protein)
End of fiber contains Integrin-Containing Focal Adhesions for binding to extracellular substrates/matrix
Formation of Lamellipodia
Uses Rac-GTP (Rho Family GTPase) for activation
This activates WAVE (analogous to WASP) which activates the ARP2/3 Complex –> Polymerizes Branched Actin (Lamellipodia)
Formation of Filopodia
Uses Cdc-42 (Rho Family GTPase) for activation
This activates
i) Par6 to generate polarity
ii) Formin
iii) WASP which activates ARP2/3
Therefore, ii and iii lead to the formation of Parallel Actin Polymerization and thus Filopodia formation
Regulation of Stress Fiber Activity
Contractile Behavior is regulated by Phosphorylation
- Myosin Light Chain Kinase phosphorylates; activates; contraction
- Myosin Light Chain Phosphatase dephosphorylates; deactivates; unbound fibers
Regulation of Myosin Light Chain Phosphorylation Activity
Uses Rho-GTP (Rho Family GTPase)
This activates Rho Kinase which interacts with Myosin LC Phosphatase to determine how much Myosin LC is phosphorylated –> regulates contraction and expansion
Formation of Stress Fibers
Uses Rho-GTP (Rho Family GTPase)
This activates Formin that polymerizes the Actin to form Stress Fibers
Mechanism for Endocytosis + Endosome Trafficking in Interphase
Actin is typically used for transport near membrane and then transfers cargo to a microtubule-based network
Actin is known to potentially polymerize on only one side of an endosome and encourage “rocketing”
Can also polymerize on membranes to mark certain structures for segregation
General Role of Microtubule Cytoskeleton
Used for Organelle Positioning and Membrane Transport
Most especially, the ER and Golgi localization is consequence of Microtubule Motor Proteins
- Dynein used to keep Golgi close to Centrosome
- Both Dynein and Kinesin used to spread ER all around the cell
General Overview of the Cell Cycle
- G1 (Gap 1) - period between completion of previous mitosis and next DNA replication
- S - DNA/Chromosome replication
- G2 (Gap 2) - period between DNA replication and initiation of mitosis
- M (Mitosis) - includes mitosis (nuclear division) and cytokinesis (cytoplasmic division)
Characterized by Chromosome Condensation, Mitotic Spindle Assembly, and Formation of Contractile Ring
1-3 are all considered Interphase
Importance of S/G2 (4 Things)
(Replication) 2 Identical Sister Chromatids result from Replication –> In long linear form
(Cohesion) “Glued” together length-wise by cohesins
(Condensation) Linear form is supercoiled and compacted by condensins (ATPases) and topoisomerases; left attached at centromere
- The kinetochores assemble on each side of said centromere
At some point, centrosome is duplicated but the 2 remain closely associated
First Step of M Phase (What signifies its beginning + 3 Things)
Prophase
begins when condensed chromosomes are seen in microscopy
i) Interphase microtubules gone, centrosomes disassociate
ii) Each centrosome approaches opposite sides of nucleus to form aster array of microtubules (more arrays and more dynamic than interphase microtubules)
iii) Beginning of Mitotic Spindle Formation (proper organization of centrosomes and chromosomes)
Second Step of M Phase (What signifies its beginning + what does it enable?)
Prometaphase
Starts when the Nuclear Envelope begins to breakdown as cytoplasmic dynein binds to nuclear envelope and pulls membrane towards (-) ends at centrosomes
- also includes phosphorylation of nuclear lamin to promote breakdown
Now spindle fiber microtubules can enter nuclear region
Types of Spindle Fiber Microtubules in 2nd Step of Mitosis and Beyond
Prometaphase
1) Kinetochore MTs: connect chromasomal kinetochore to spindle pole at centrosome
2) Polar MTs: stem from both poles and overlap each other in antiparallel fashion; connected by Kin-Ns
3) Astral MTs: grow towards plasma membrane and interact with cell cortex
Organization/Forces of the 3 Types of Spindle Fiber Microtubules in 2nd Step of M Phase
Prometaphase
For kinetochore MTs, a single kinetochore is captured and the accompanying chromosome is pushed towards to the opposite spindle pole where it can be more easily captured by the second microtubule
For polar MTs, 2 of them are cross-linked by Kin-N Kinesin 5 –> pushes MTs back to their source and thus the centrosomes apart
Also cross-linked by Kin-C Kinesin 14 –> pushes MTs toward opposite pole and thus brings the centrosomes closer to each other
For astral MTs, there exist membrane-bound dynein motors –> pull MTs and thus centrosomes toward membrane
Can Spindle Fiber Organization be achieved without Centrosomes?
What is this process driven by?
Yes, MTs will be assembled about chromatin; it is thought that RanGTP will be concentrated near cells and create a “gradient,” attracting importins like in nuclear transport and then the consequent binding of RanGTP releases cargo (which is throught to be MT nucleating and stabilizing proteins)
How do Kinetochore MTs bind to Kinetochores in the proper orientation? What do they have to ensure this?
Encounter and attachment is stochastic
Kinetochores contain a Kin-N (CENP-E), a dynein, a Kin-I (Kin 13), and the Ndc80 complex
If MT is bound incorrectly, CENP-E will walk chromosome to (+) end
The MT is held in place by Ndc80, which leaves enough room for dynamic growth and shrinkage without disconnecting from MT
- Becomes bi-oriented when both spindle poles are connected properly
What type of movement occurs after chromosomes become bi-oriented?
Congression (Oscillatory movement that decreases in distance covered OT) until they reach a still state at the equator between the two spindle poles
Assisted by (de)polymerization of the (+) ends on each side – one grows while the other shrinks –> MVMT TO MIDDLE
2 Characteristic Kinetochore MT Motions
1) Poleward MT Flux
There are length changes at both the (+) and (-) ends
(+) end: grows with Kinesin 7 and shrinks with Kinesin 13
(-) end: only see shrinkage with Kinesin 13
Therefore, treadmilling is possible!!
2) Polar Ejection Force
Kinesin 4 (Kin-N) is located at the top of the chromosome arms and can attach to polar MTs, bringing to equator
3rd Step of M Phase (What characterizes it and when does that happen?)
Metaphase
Characterized by all chromosomes becoming aligned in a single plane halfway between spindle poles (named the Metaphase Plate) – held in tension state
This occurs when all (+) end polymerization, (-) end depolymerization, and chromokinesin promote movement equilibriates
4th Step of M Phase (What must be checked?)
Anaphase
Must pass Spindle Assembly Checkpoint before anything can continue
Kinetochores must be properly attached to MTs and under proper tension; if not, SAC signals are produced that inhibit Anaphase Promoting Complex
4th Step of M Phase (What happens after passage of checkpoint?)
Anaphase’s 2 Simultaneous Movements
Movements are dependent on activation of Anaphase Promoting Complex by M-Cdk and Cdc20 – this allow polyubiquitination of securin protein that inhibits a separase
- This separase degrades the cohesins and allows chromosome separation
Anaphase A: Kinetochores shorten using Kinesin 13, bringing chromosomes towards poles (chromosomes remain attached using CENP-E and Ndc80)
Anaphase B: Polar MTs elongate while Kinesin 5 walks to force spindle poles further apart; also includes dynein motors pulling astral MTs toward membrane
5th Step of M Phase (What signifies it? What happen?)
Telophase
Chromosomes arrive at poles, Kinetochore MTs disappear, Nuclear Lamins dephosphorylate to reform
Nuclear envelope begins to reform; chromatin decondenses
6th and Final Step of M Phase (What happens?)
Cytokinesis
Contractile Ring forms that draws plasma membrane in to create cleavage furrow that closes until it meets midbody (a spindle remnant)
When this midbody disappears, the cell divides completely and the division is complete!
How does cleavage furrow end up in the proper orientation/alignment in the 6th and Final Step of M Phase?
Cytokinesis
Info comes in the form of signals from astral spindles and central spindle to direct contractile ring precursors (called NODES) to the center of the cell, position is marked by the first arrivals using RhoA
Formation of cleavage furrow in the 6th and Final Step of M Phase?
What is the final step that does the separation?
Cytokinesis
The already existent nodes accumulate formin (for nucleation and elongation) and myosin II (ring organization and contraction of filaments)
This closes to midbody and eventually proteins accumulate (most importantly ESCRT) and membrane abscission occurs!
Intermediate Filaments - Simile and Structure Description
Act like a strong rope for a cell
Important for cell-cell attachments and mechanical integrity/support
Does not bind any nucleotides
Nonpolar, symmetrical structure
Able to withstand stretching forces/tension
Intermediate Filaments - Structure/Assembly + How do Units Add?
Basic building block is alpha-helical coiled rod that creates parallel dimer
These assemble into antiparallel tetramers, creating symmetrical and nonpolar structure
The tetramers assemble end to end to create the protofilaments, which group laterally to create protofibrils (sheet-like)
Protofibrils then further assemble into intermediate filaments
Tetramers substitute in structure (pop in and out) – overall less dynamic
Types of Intermediate Filaments
1) Keratins: epithelial cells, hair, nails, skin
2) Vimentin: fibroblasts, endothelial cells, white blood cells, embryonic cells
3) Desmin: muscle cells
4) Neurofilaments: strength to axons and determining axon diameter
5) Nuclear Lamins: nuclear structure/organization
Intermediate Filament Relationship to Extracellular Structures
IFs run between desmosomes (cell-cell interactions), hemi-desmosomes (cell-extracellular matrix), and nuclei (for strength and rigidity)
intermediate Filament Final Structure?
Network or Bundle, typically cross-linked with other structures using Intermediate Filament Binding Proteins
Septin Structure Description
Binds and hydrolyzes GTP
Known to colocalize with F-actin in stress fibers and contractile ring
Forms nonpolar heterohexamers or heterooctamcers that can become filaments, rings, or cages
Septin Functions (4)
Cytokinesis, Scaffolding, Diffusion Barrier (Compartmentalization), Organellle/Vesicle Trafficking and Dynamics
