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Questions and Answers
According to Mitchison and Kirschner’s experiments, what is surprising about microtubule behavior after dilution below the critical concentration (Cc)?
According to Mitchison and Kirschner’s experiments, what is surprising about microtubule behavior after dilution below the critical concentration (Cc)?
- The critical concentration (Cc) has no effect on microtubule dynamics.
- Microtubules exhibit a brief period of accelerated growth before shrinking.
- Some microtubules continue to grow despite the expectation that they should shrink. (correct)
- All microtubules immediately depolymerize, ceasing all growth.
In Mitchison and Kirschner's experiment, with tubulin at 50 µM, what were the approximate free tubulin concentrations in Condition 1 and Condition 2 after dilution?
In Mitchison and Kirschner's experiment, with tubulin at 50 µM, what were the approximate free tubulin concentrations in Condition 1 and Condition 2 after dilution?
- Condition 1: ~15 µM, Condition 2: ~30 µM
- Condition 1: ~15 µM, Condition 2: ~7.5 µM (correct)
- Condition 1: ~30 µM, Condition 2: ~15 µM
- Condition 1: ~7.5 µM, Condition 2: ~3.75 µM
Mitchison and Kirschner observed that the length information of microtubules did not align with previously understood biochemical theories. Considering the experimental setup, which factor MOST likely contributed to this discrepancy, challenging assumptions about microtubule dynamics?
Mitchison and Kirschner observed that the length information of microtubules did not align with previously understood biochemical theories. Considering the experimental setup, which factor MOST likely contributed to this discrepancy, challenging assumptions about microtubule dynamics?
- The inherent instability of tubulin dimers at high concentrations.
- Limitations in the resolution of electron microscopy techniques used for measurement.
- Experimental errors in maintaining precise temperature control during elongation.
- Dynamic instability, where individual microtubules can switch between growing and shrinking phases independently of the overall tubulin concentration. (correct)
What is the primary effect of end-binding proteins on filament dynamics?
What is the primary effect of end-binding proteins on filament dynamics?
If a solution of polymerizing actin filaments is treated with a capping protein, what would be the expected effect on the actin filaments' plus-ends?
If a solution of polymerizing actin filaments is treated with a capping protein, what would be the expected effect on the actin filaments' plus-ends?
What is the most accurate definition of 'stabilization' in the context of filament dynamics?
What is the most accurate definition of 'stabilization' in the context of filament dynamics?
How does the addition of a capping protein influence the actin elongation rate at the plus-end?
How does the addition of a capping protein influence the actin elongation rate at the plus-end?
A researcher observes that a particular microtubule binding protein dramatically reduces the catastrophe frequency of microtubules in vitro. However, when the same protein is added to cells, the microtubules become surprisingly unstable, exhibiting frequent catastrophes and rapid turnover. Which of the following could best explain this result?
A researcher observes that a particular microtubule binding protein dramatically reduces the catastrophe frequency of microtubules in vitro. However, when the same protein is added to cells, the microtubules become surprisingly unstable, exhibiting frequent catastrophes and rapid turnover. Which of the following could best explain this result?
What does 'n' represent in the equation $\frac{dn}{dt} = k_{on} subunit - k_{off}$ regarding filament dynamics?
What does 'n' represent in the equation $\frac{dn}{dt} = k_{on} subunit - k_{off}$ regarding filament dynamics?
Which of the following best describes the primary function of accessory proteins in the context of cytoskeletal dynamics?
Which of the following best describes the primary function of accessory proteins in the context of cytoskeletal dynamics?
A researcher observes that the addition of a specific drug leads to a significant decrease in the average length of microtubules within a cell. Which of the following mechanisms could explain this observation?
A researcher observes that the addition of a specific drug leads to a significant decrease in the average length of microtubules within a cell. Which of the following mechanisms could explain this observation?
In vitro, both actin filaments and microtubules demonstrate dynamic behavior. Which statement accurately describes how their dynamics differ?
In vitro, both actin filaments and microtubules demonstrate dynamic behavior. Which statement accurately describes how their dynamics differ?
A novel drug, 'Cytohalt,' is being tested on mouse fibroblast cells. It is observed that cells treated with Cytohalt exhibit a complete cessation of lamellipodia formation and a significant increase in cellular stiffness. Furthermore, fluorescence recovery after photobleaching (FRAP) experiments reveal a near-zero recovery rate for actin filaments in the treated cells. Which of the following mechanisms of action for Cytohalt is most consistent with these observations?
A novel drug, 'Cytohalt,' is being tested on mouse fibroblast cells. It is observed that cells treated with Cytohalt exhibit a complete cessation of lamellipodia formation and a significant increase in cellular stiffness. Furthermore, fluorescence recovery after photobleaching (FRAP) experiments reveal a near-zero recovery rate for actin filaments in the treated cells. Which of the following mechanisms of action for Cytohalt is most consistent with these observations?
In the context of actin dynamics, what is the likely effect of adding capping proteins?
In the context of actin dynamics, what is the likely effect of adding capping proteins?
Formins are known to regulate actin dynamics. Which of the following activities is directly influenced by formins?
Formins are known to regulate actin dynamics. Which of the following activities is directly influenced by formins?
In a kymograph of actin dynamics, how does the addition of formin typically affect the polymerization rate, as indicated by the slope of the line?
In a kymograph of actin dynamics, how does the addition of formin typically affect the polymerization rate, as indicated by the slope of the line?
Consider a scenario where the concentrations of both G-actin and capping proteins are significantly elevated. What combined effect would this have on critical concentrations (Cc+ and Cc-) and actin filament dynamics?
Consider a scenario where the concentrations of both G-actin and capping proteins are significantly elevated. What combined effect would this have on critical concentrations (Cc+ and Cc-) and actin filament dynamics?
A researcher is studying a novel protein that interacts with both formin and capping proteins at the plus-end of actin filaments. If this protein enhances the binding affinity of formin while simultaneously reducing the dissociation rate of capping proteins, predict the long-term effect on actin filament length and stability within the cell.
A researcher is studying a novel protein that interacts with both formin and capping proteins at the plus-end of actin filaments. If this protein enhances the binding affinity of formin while simultaneously reducing the dissociation rate of capping proteins, predict the long-term effect on actin filament length and stability within the cell.
Flashcards
Cytoskeleton
Cytoskeleton
A network of protein filaments that provide structural support and facilitate movement within cells.
Actin Treadmilling
Actin Treadmilling
The process where actin filaments add subunits at the plus end while losing them at the minus end, resulting in a movement of the filament.
Dynamic Instability
Dynamic Instability
The rapid switching between growth and shrinkage observed in microtubules.
Accessory Proteins
Accessory Proteins
Proteins that bind to and regulate the dynamics and organization of cytoskeletal filaments.
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Net Assembly (n)
Net Assembly (n)
The net assembly represents the overall increase in the amount of filaments. It's calculated by subtracting the dissociation rate from the association rate.
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Critical Concentration (Cc)
Critical Concentration (Cc)
Critical concentration (Cc) is the tubulin concentration at which microtubule assembly is in equilibrium with disassembly. Above Cc, microtubules grow; below Cc, they shrink.
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Dilution Assay
Dilution Assay
The process of diluting a sample to reduce the concentration of a substance, often used to study microtubule dynamics by altering free tubulin levels.
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MT Growth Below Cc
MT Growth Below Cc
Microtubules can continue growing even when tubulin concentration is below the critical concentration (Cc) after dilution, challenging simple biochemical expectations.
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Tubulin Concentration Effect
Tubulin Concentration Effect
Experimentally changing tubulin concentration affects microtubule behavior, such as growth and shrinkage.
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Microtubule Density Measurement
Microtubule Density Measurement
Quantitative analysis of length and number of microtubules under different conditions.
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Monomer-binding protein
Monomer-binding protein
Proteins that bind to individual building blocks (monomers) of filaments, regulating their assembly and disassembly.
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Plus/Minus-end binding proteins
Plus/Minus-end binding proteins
Proteins that preferentially attach to either the plus-end or minus-end of a filament, influencing its behavior.
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Stabilization (Filaments)
Stabilization (Filaments)
The process of making a filament less dynamic, reducing both polymerization and depolymerization rates.
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Capping proteins
Capping proteins
Proteins that bind to the plus-end of actin filaments, preventing further addition of monomers and stabilizing the filament.
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Catastrophe frequency
Catastrophe frequency
The number of catastrophe events within a filament per unit of time. A higher frequency means more instances of rapid depolymerization.
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Effect of capping proteins on actin elongation
Effect of capping proteins on actin elongation
The rate of actin subunit addition at the plus end decreases significantly, represented graphically by a flattening of the dn+/dt line.
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Multifunctional accessory proteins
Multifunctional accessory proteins
Accessory proteins can regulate multiple functions within the cell.
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Formin
Formin
A protein that binds to the plus-end of actin filaments and enhances polymerization.
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Formin's effect on nucleation
Formin's effect on nucleation
Formin increases the nucleation rate, as shown by more filaments appearing over time in the presence of formin.
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Formin's effect on polymerization rate
Formin's effect on polymerization rate
Formin increases the rate of polymerization.
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- BIOL 362 Cellular Dynamics, Module 1-3: Cytoskeletal Dynamics III
- Date: January 16, 2025
- Focus: Cytoskeletal dynamics, accessory proteins, drugs controlling cytoskeletons
Lecture Outline
- What is the cytoskeleton?
- Common properties exist for F-actin and microtubule dynamics
Actin Filament Dynamics
- Actin filament dynamics in vitro include treadmilling
Microtubule Dynamics
- Microtubule dynamics in vitro include dynamic instability
Regulation
- Accessory proteins exist to regulate filament dynamics in vivo
- Drugs that modulate cytoskeletal dynamics are key
Additional Resources
- Relevant chapter in MBoC (7th edition): Chapter 16
Learning Goals
- Understand accessory proteins role in regulationg the cytoskeleton
- Understand how drugs control cytoskeletons
Net Assembly
- Represented as (n) in equations
- Net assembly (n) = Number x Length
Test Tube
- Clicker Question: Test tube 2 will contain the correct answer
- Tube 1 calculation = (100 * 10) + (8 * 20) = 1160
- Tube 2 calculation = (50 * 10) + (8 * 20) + (5 * 1000) = 5660
MT Dilution Assay
- Experiments were done in 1984
- Tubulin started at 50 μM
- Elongation step occurs
- A reaction mix was diluted
- Condition 1: uses [free tubulin] ~ 15 μM
- Condition 2: uses [free tubulin] ~ 7.5 μM
- Cc ~ 14 μM
Additional Info for MT Dilution Assay
- Surprisingly, some microtubules kept growing after the subunit dilution below C
- Electron microscopy was performed
- Length information did not follow biochemical theories learned before
Condition Outcomes
- Condition 1: Net increase in mass
- Condition 2: Net decrease in mass
- This is consistent with biochemical theories noted
Dynamic Instability
- Rapid growth happens with GTP-capped end
- Random loss of GTP cap leads to a catastrophe
- Rapid Shrinkage happens
- Regain of GTP cap leads to rescue of the dynamics
Info Handling In Course
- Actin filament: Biochemical data is key (length x number)
- Microtubules: Microscopy data is key (mostly focusing on length)
- Nucleation steps: Nucleation steps will be considered qualitatively rather than quantitatively in both cases
MT Length Data
- MT length information is best captured using microscopy
- Movies are not a convenient form of data to present in scientific literature
Kymograph
- Timelapse data are often presented in a form of data called a kymograph
Kymograph Info
- A kymograph in cell biology is made from a stack of time series images
- In image form it captures a movie of a single MT and stacks them vertically
Clicker Question
- The expected kymograph will be "A"
- A kymograph shows temporal changs of MT length over time
Accessory Proteins
- They modify filament dynamics in vivo
Types of Accessory Proteins
- End-binding protein
- Non-end binding protein
- Monomer-binding protein
Binding Types
- Many proteins demonstrate have plus-end or minus-end preference for binding site
- Tubulin/end binding protein (EB3)
End-Binding Proteins
- They modify filament dynamics at both minus and plus ends
Minus-End Effects
- Minus-end binding proteins: affect Nucleation and stabilization
Plus-End Effects
- Plus-end binding proteins: affect Polymerization, Nucleation & stabliziation Also affects catastrophe & rescue
Stabilization of Filaments
- Stabilization is a less dynamic filament state
- It affects Catastrophe frequenc,y which is the number of catastrophe events per time
Capping Proteins
- Preferentially bind to the actin plus-end to stabilize them
- Without capping proteins, both actin plus/minus ends are dynamic
- With capping protein, the plus end is stabilized, and the minus end remains dynamic
Clicker Question
- Adding a capping protein will generate statement A: dn+/dt line becomes flat
Accessory Protein Functions
- One protein may regulate more than one protein function
Formins
- Formin is an actin plus-end binding protein
Formin functions
- Speeds up Polymerization rate
- Speeds up Nucleation rate
Formin Dynamics
- Kymograph describes actin dynamics with formin
- The polymerization rate is elongation per total time
- If the kymograph dotted line is:
- More vertical = rate is low
- More horizontal = rate is high
Parameter Modified With Formin
- The affected parameter is kon
Key Term
- dn/dt = kon[subunit] – koff
- It also affects koff
Bulk Assembly
- Bulk actin assembly dynamics are described when combined with formin
- Cc = koff/kon
- Bigger Kon values lead to smaller the Cc when Formin is present
Elongation Considerations
- dn/dt = kon[free subunit]-koff
- The change in elongation can be based on free subunit concentration
MT Stabilization
- The γ-tubulin ring complex stabilizes MT minus-end and promotes nucleation
- Plus-end of MT's have fast dynamics
- Minus-end of MT's have slow dynamics
Growth Consideration
- MT growth and catastrophe from the minus end is limited due to stabilization by the y-tubulin complex
Other Examples
- Plus-end binding XMAP215 and catastrophe factor promote specific actions
- MT polymerization and catastrophe, respectively
XMAP215 Effects
- Decrease in Catastrophe frequency
- Increases Growth rate
- Increases in Nucleation rate
- Time + XMAP215
Catastrophe Factor Effects
- increases frequency of catastrophes
Non-End Binding Proteins
- Some regulate filament stability from the lattice
Function of Non-End Binding Proteins
- Stabilization
- Severing (gelsolin)
- Cofilin binds for ADP-actin filaments, accelerates disassembly
Bundling
- Non-end binding proteins also regulate filament organization
- This creates bundling and crosslinking effects
Monomer Binding
- Affects Monomer Binding to regulate Monomers
The goal is
- Sequestration (proteins unable to polymerize)
- Regulation
Factors that influence the binding mode of cytoskeleton
- Nucleation
- Polymerization
- Catastrophe (MT)
- Stabilization
- Severing
- Disassembly
- Organization
- Sequestration
- ADP-ATP exchange (Actin)
Cytoskeletal Drugs
- Drugs exist to control filament dynamics
Source of Drugs
- Most were mainly isolated from living organisms
Actin Drugs
- Actin-targeting drugs exist
- Cytochalasin B Latrunculin & Phalloidin
- Isolated from Fungi, Sponge, and Amanitia Mushroom
Microtubule Drugs
- Colchicine, Nocodazole, Taxol & Artificial compounds
- Isolated from Crocus, Yew Tree
Long Term use
- Dates back through history in human time
Applications
- Colchicine for ancient Egypt (treatment for gout)
- Colchicine is also useful for Horticulture (creating different flower patterns)
- Taxol is useful for Cancer chemotherapy
Effectiveness
- Taxol is one of the most affordable and best-selling chemotherapy drugs
- A $1 billion USD annual revenue is generated
Chemotherapy
- Used for both early and locally advanced breast cancer
- Different brands, actions and delivery forms
Drug Delivery
- Delivered either by Pill or IV drug (given by vein through an IV)
Drug Actions
- DNA/RNA synthesis inhibition for repair & duplication inhibition
- Microtubule inhibitors
Drug Mechanism
- Work by the mechanism of action of cytoskeletal drugs
Drug Types
- Phalloidin = Stabilization
- Plus-end of filaments is the place where most drugs act
Cytochalasin B
- Inhibits polymerization dynamics
- Nocodazole Colchicine Latrunculin all inhibit subunit incorporation
Overlap
- There exist some similarity between the mechanisms of action of cytoskeletal drugs and accessory proteins
Summary Points
- Kymograph is a useful tool to visualize filament dynamics
- There are four categories of accessory proteins
- Each accessory protein has a specific function- some of them modulate kon, koff, (therefore Cc)
- Cytoskeletal drugs control filament dynamics
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