Module 6: Cytoskeleton (Actin) Notes

Introduction to the Cytoskeleton (Actin)

Learning Goals

• Describe how biochemistry can be used to answer cell biological questions.

• Explain how the cytoskeleton can be dynamic without changing protein concentration.

• Describe how to determine a sample size in cell biology (time depending).

Actin Polymerization

• Actin polymerizes, transitioning between:

  • G-actin (globular monomeric actin)

  • F-actin (filamentous actin polymer)

• Actin filaments are thin and flexible.

  • Monomers polymerize into a helical chain.

  • Diameter: 7 nm.

  • Plus end: fast-growing.

  • Minus end: slow-growing.

Functions of Actin Filaments

• Actin filaments drive cell movement and have other functions:

  • Villi.

  • Contractile bundles.

  • Sheet-like and finger-like protrusions (lamellipodia and filopodia).

  • Contractile ring.

• Actin architecture and function are governed by actin-binding proteins.These proteins modulate actin polymerization, stability, and interactions with other cellular components, playing crucial roles in processes such as cell motility, shape, and division.

Biochemical Purification

• Take advantage of different material properties to separate components.

Isolating Actin

• How to isolate actin from all the other cellular components:

  • Changing the buffer changes the state of actin:

    • G-buffer: favors G-actin (monomers).

    • F-buffer: favors F-actin (filaments).

Steps for Actin Isolation

1. Homogenize in G-Buffer:

   • Determine if actin is in monomeric or filamentous form and how hard it is to pellet.

   • Start with cell suspension or tissue, disrupt cells to obtain a cell homogenate containing organelles.

2. Centrifuge in G-Buffer:

   • Low-speed centrifugation to pellet whole cells, nuclei, and cytoskeleton.

   • Medium-speed centrifugation to pellet mitochondria.

   • High-speed centrifugation to pellet microsomes and small vesicles.

3. Switch to F-Buffer:

   • Promotes actin polymerization.

4. High-Speed Centrifuge:

   • Pellet the F-actin filaments.

• Result: Purified actin.

Biochemical Purification to Find Actin Regulators

• Requires an assay.

• Pyrene-actin fluorescence:

  • Fluorescence increases only with polymer formation.

  • Monitor fluorescence over time to assess polymerization.

  • Add an activator of actin polymerization to observe changes.

Actin Polymerization Reconstitution Assay

• Pyrenyl-actin assay:

  • Measure relative pyrene fluorescence.

  • Compare actin control to actin with accessory proteins.

  • Observe increased F-actin formation with accessory proteins.

Actin Networks in Cells

• Many cells have three distinct networks that merge in and out of each other:

  • Filopodia: Finger-like protrusions.

  • Lamellipodia: Sheet-like protrusions.

  • Stress fibers: Contractile bundles.

  • These networks can exist simultaneously.

Regulation of Actin Structures

• Different structures (filopodia, lamellipodia, stress fibers) are made of the same polymer (actin).

Tom Pollard's Message

• If you understand how the polymer assembles and you look at the cellular behavior, you can understand how it is regulated in the cell.

Comparing In Vitro and In Vivo Behavior

• Compare:

  • Behavior of part(s) in a tube (reconstitution).

  • Behavior in the cell.

• Aim for a complete understanding of how the polymer assembles alone and a good description of the polymer in vivo.

• Examples: Tubulin and Actin.

• Find what's missing.

Actin Polymerization Dynamics

• Mass of filaments over time:

  • Nucleation: Slow, no nuclei.

  • Elongation: Fast, lots of free monomer.

  • Steady State: Equilibrium, not much free monomer.

Comparing In Vitro and In Vivo Actin Behavior

1. In Vitro:

   • Nuclei are slow to form (polymerization slow).

   • Once nuclei form, the more monomer, the faster the growth (because you only have a few nuclei).

   • If nuclei form slowly, you get long filaments.

   • At steady state, the filaments are stable - just grow and shrink - there is no excess monomer.

2. In Vivo:

   • Polymerization happens fast.

   • Filaments are short and branched.

   • Filaments are not stable; they turn over.

Controlling Actin Polymerization

• To make polymerization happen fast where you want it:

  • Have a controlled seed (nucleus).

• Key point of control: Nucleus and free ends (they are the same thing!).

• K_d represents the dissociation constant.

Controlling Filament Length

• If you want filaments to be short:

  • Cap the filaments to stop elongation.

  • Fractionate cells looking for factors that make short filaments.

  • Assay: Need to stop elongation by capping.

Controlling Actin Filaments

• Need a way to pull apart the old filaments we don’t want!

• Control through:

  • Nucleators.

  • Cappers.

  • Severing proteins.

Visualizing Actin Polymers

• Using TIRF (Total Internal Reflection Fluorescence) microscopy to visualize individual actin polymers.

Missing Components for Complete Control

• Still missing things to get the structures in the cell!

  • Control of length and rate.

Arp2/3 Complex: A crucial regulator of actin filament nucleation and branching, facilitating rapid actin polymerization and enabling cellular processes such as motility and shape change.

• Arp2/3 complex:

  • Makes branches when combined with actin.

  • ONLY nucleates filaments off old filaments, creating branches.

Controlling Arp2/3 Activity

• Something was needed to turn Arp2/3 on and localize nucleation!

• Listeria utilizes this by building a comet tail of actin on one side and rocketing around the cytoplasm.

• ActA is a nucleator found on Listeria that activates Arp2/3.

  • If you take this protein, put it on beads, and drop them into cell extract, it will rocket around.

Mechanism of Nucleation

• How Nucleators + ARP 2/3 work:

  • Later, the human nucleator was found based on similarity to ActA (called WASP).

  • 2 subunits in ARP2/3 look like actin.

  • The nucleator brings in the third actin monomer.

  • 3 actins = nucleus!

• Localizing WASP to a membrane creates a proximity sensor.

  • Requires: WASP is active and a preexisting filament!

• Cytoskeleton Overview
  A system that enables cellular organization and movement, providing structural support to the cell. It influences behavior and morphology across evolutionary time, playing a crucial role in numerous cellular processes such as division, intracellular transport, and shape maintenance. Understanding the cytoskeleton, particularly the actin cytoskeleton, is critical in cellular biology due to its involvement in diverse functions ranging from motility to signal transduction.

• Agenda for the Session

  • Discuss how the actin cytoskeleton works in various cell types

  • Introduce the changes in the cytoskeleton across different eukaryotic images, highlighting evolutionary adaptations

  • Practical discussion on designing cell biology experiments tailored to study cytoskeletal dynamics

  • Overview of learning goals, establishing a framework for understanding actin's role in cellular functions

• Learning Goals

  1. Explain the role of biochemistry in addressing unfamiliar questions, particularly in cytoskeletal research.

  2. Describe the dynamic nature of the cytoskeleton without altering protein concentrations, enhancing understanding of cellular adaptability.

  3. Determine sample sizes in cell biology experiments to ensure statistically valid results, focusing on actin dynamics.

• Actin Cytoskeleton

  • Migration observed in white blood cells chasing bacteria, illustrating the actin's role in immune responses.

  • Actin polymerization plays a crucial role in various cellular activities:

  • G-actin (globular actin) vs. F-actin (filamentous actin): G-actin polymerizes to form F-actin, supporting cell structure and function.

  • Actin grows at a plus (barbed) end and a minus (pointed) end, exhibiting dynamic properties characterized by cycles of polymerization and depolymerization.

  • Actin is used collectively for:

    • Maintaining cell shape, mediating migration, and increasing surface area (e.g., microvilli)

    • Muscle contraction through interaction with myosin filaments, crucial for muscle function.

    • Cell division by forming the contractile ring during cytokinesis.

• Understanding Actin Functionality

  • Actin dynamics are examined using knockout studies, fluorescence, and comparative analysis to discern actin’s regulatory mechanisms.

  • Various proteins influence actin behavior:

  • Nucleators (e.g., Arp2/3 complex) initiate filament formation, critical for branched actin networks.

  • Motor proteins (e.g., myosins) collaborate with actin for contraction, facilitating cellular movements.

  • Side-binding proteins (e.g., tropomyosin) alter actin polymer longevity and properties, influencing stability and interaction with other proteins.

• Actin Purification Methods

  • Utilizing different buffers (G-buffer for monomers, F-buffer for polymers) to facilitate actin studies.

  • Homogenization followed by gradient centrifugation separates components based on size and density, essential for purification.

  • Cycling through buffers allows for purifying actin through its polymerization states, enabling functional assays to assess activity.

• Assays to Study Actin Behavior

  • Pyrene-actin assay detects polymerization through fluorescence during reconstitution experiments, allowing measurement of kinetic parameters.

  • Comparison of actin behavior in test tubes versus living cells exposes differences, such as the effects of cellular context on actin dynamics.

• Comparative Studies in Polymer Dynamics

  • Test tube (in vitro) studies show long, stable filaments as opposed to the dynamic, branched structures seen in cells, emphasizing environmental influences.

  • Experiments indicate mechanisms that speed up nucleation and organize actin for specific cellular functions, essential for processes like motility and tissue development.

• Actin Regulation Mechanisms

  • Nucleators and capping proteins (e.g., CapZ) regulate the length and growth of actin filaments, ensuring proper assembly and disassembly in cells.

  • Severing proteins (e.g., cofilin) allow for filament breakdown, contributing to dynamic organization and rapid reorganization in response to cellular signals.

  • Techniques using TURF microscopy visualize filament dynamics in real time within cells, providing insights into actin behavior under physiological conditions.

• Emergent Properties of Actin Networks

  • Simple actin rules lead to complex behaviors in regulation and cellular functions, showcasing the importance of actin for cellular architecture.

  • Use of different actin regulators can lead to diverse structures such as lamellopodia, filopodia, and stress fibers, allowing cells to adapt and react to environmental changes.

  • The interplay of actin dynamics underscores the adaptability of eukaryotic organisms, reflecting evolutionary pressures on cytoskeletal structures.

• Concluding Thoughts

  • Acknowledgment of the importance of observational studies in understanding actin functionality and its contributions to cell biology.

  • Understanding actin's basic properties allows for the exploration of complex behaviors and properties during cellular functions, paving the way for future discoveries in cell biology.

• Homework and Future Discussions

  • Reading assigned for journal club, focusing on recent advances in cytoskeletal research and reviewing reproducibility in experiments.

  • Future topics will discuss the diversity and evolution of the cytoskeleton across eukaryotes, highlighting its significance in various

Cytoskeleton Overview

A system that enables cellular organization and movement, providing structural support to the cell. It influences behavior and morphology across evolutionary time, playing a crucial role in numerous cellular processes such as division, intracellular transport, and shape maintenance. Understanding the cytoskeleton, particularly the actin cytoskeleton, is critical in cellular biology due to its involvement in diverse functions ranging from motility to signal transduction.

Agenda for the Session

Discuss how the actin cytoskeleton works in various cell types

Introduce the changes in the cytoskeleton across different eukaryotic images, highlighting evolutionary adaptations

Practical discussion on designing cell biology experiments tailored to study cytoskeletal dynamics

Overview of learning goals, establishing a framework for understanding actin's role in cellular functions

Learning Goals

3. Explain the role of biochemistry in addressing unfamiliar questions, particularly in cytoskeletal research.

4. Describe the dynamic nature of the cytoskeleton without altering protein concentrations, enhancing understanding of cellular adaptability.

5. Determine sample sizes in cell biology experiments to ensure statistically valid results, focusing on actin dynamics.

Actin Cytoskeleton

Migration observed in white blood cells chasing bacteria, illustrating the actin's role in immune responses.

Actin polymerization plays a crucial role in various cellular activities:

G-actin (globular actin) vs. F-actin (filamentous actin): G-actin polymerizes to form F-actin, supporting cell structure and function.

Actin grows at a plus (barbed) end and a minus (pointed) end, exhibiting dynamic properties characterized by cycles of polymerization and depolymerization.
- The plus (barbed) end is where actin monomers are preferably added, leading to faster growth. ATP-bound actin monomers bind to the barbed end, hydrolyzing ATP to ADP after incorporation into the filament. This hydrolysis destabilizes the filament and promotes depolymerization at the pointed end.
- The minus (pointed) end experiences slower growth. ADP-actin monomers dissociate from this end.
- Treadmilling occurs when the rate of addition of actin monomers at the barbed end equals the rate of dissociation at the pointed end, maintaining constant filament length.

Actin is used collectively for:

Maintaining cell shape, mediating migration, and increasing surface area (e.g., microvilli)

Muscle contraction through interaction with myosin filaments, crucial for muscle function.

Cell division by forming the contractile ring during cytokinesis.

Understanding Actin Functionality

Actin dynamics are examined using knockout studies, fluorescence, and comparative analysis to discern actin’s regulatory mechanisms.

Various proteins influence actin behavior:

Nucleators (e.g., Arp2/3 complex) initiate filament formation, critical for branched actin networks.

Motor proteins (e.g., myosins) collaborate with actin for contraction, facilitating cellular movements.

Side-binding proteins (e.g., tropomyosin) alter actin polymer longevity and properties, influencing stability and interaction with other proteins.

Actin Purification Methods

Utilizing different buffers (G-buffer for monomers, F-buffer for polymers) to facilitate actin studies.

Homogenization followed by gradient centrifugation separates components based on size and density, essential for purification.

Cycling through buffers allows for purifying actin through its polymerization states, enabling functional assays to assess activity.

Assays to Study Actin Behavior

Pyrene-actin assay detects polymerization through fluorescence during reconstitution experiments, allowing measurement of kinetic parameters.

Comparison of actin behavior in test tubes versus living cells exposes differences, such as the effects of cellular context on actin dynamics.

Comparative Studies in Polymer Dynamics

Test tube (in vitro) studies show long, stable filaments as opposed to the dynamic, branched structures seen in cells, emphasizing environmental influences.

Experiments indicate mechanisms that speed up nucleation and organize actin for specific cellular functions, essential for processes like motility and tissue development.

Actin Regulation Mechanisms

Nucleators and capping proteins (e.g., CapZ) regulate the length and growth of actin filaments, ensuring proper assembly and disassembly in cells.

Severing proteins (e.g., cofilin) allow for filament breakdown, contributing to dynamic organization and rapid reorganization in response to cellular signals.

Techniques using TURF microscopy visualize filament dynamics in real time within cells, providing insights into actin behavior under physiological conditions.

Emergent Properties of Actin Networks

Simple actin rules lead to complex behaviors in regulation and cellular functions, showcasing the importance of actin for cellular architecture.

Use of different actin regulators can lead to diverse structures such as lamellopodia, filopodia, and stress fibers, allowing cells to adapt and react to environmental changes.

The interplay of actin dynamics underscores the adaptability of eukaryotic organisms, reflecting evolutionary pressures on cytoskeletal structures.