Cytoskeleton Assembly & Intermediate Filaments Study Notes

BIOL 2120- Cell Structure and Function

Cytoskeleton Assembly & Intermediate Filaments

  • This lecture will cover the assembly of the cytoskeleton, with a specific focus on intermediate filaments and their biological significance.

Introduction to the Cytoskeleton

  • The cytoskeleton is an essential structure within eukaryotic cells, composed of various types of protein filaments that provide shape, support, and movement.

Dynamics of Filament Polymerization and Disassembly

GFP-tubulin

  • Green Fluorescent Protein (GFP) Tubulin: A tool used to visualize tubulin within the cytoskeleton to study polymerization dynamics.

What Drives Dynamic Cycles of Filament Polymerization and Disassembly?

  • The edges of cells undergo significant filament dynamics, influenced by several factors:

    • Polymer formation involves a balance of subunit addition and subtraction at filament ends.

    • Each filament end possesses a Critical Concentration (Cc).

Polymer Formation

Characteristics of Critical Concentration (Cc)

  • Each end of a filament will have a specific Critical Concentration (Cc).

  • Balance of Subunits:

    • When the subunit concentration in the cell equals the Cc, the rate of subunit addition is balanced with the rate of subtraction.

    • If subunit concentration exceeds Cc, filament growth occurs.

    • If subunit concentration drops below Cc, filament disassembly occurs.

Rates of Subunit Addition and Subtraction

  • The polymerization dynamics of filaments are characterized by two key rates:

    • kON: Rate at which subunits are added to the filament.

    • kOFF: Rate at which subunits are removed from the filament.

    • When subunit concentration is at Cc, the relationship between these rates can be expressed as:

    • kOFF/kON=1kOFF / kON = 1

    • Different filament ends may exhibit different Critical Concentrations, kOFF, and kON values.

Factors Controlling Critical Concentration and End Behaviors

  • ATP/GTP Hydrolysis Influence:

    • The hydrolysis of ATP/GTP is crucial in regulating Cc and thus filament stability and formation.

    • Individual monomers slowly hydrolyze ATP/GTP, whereas subunits within filaments hydrolyze ATP/GTP rapidly.

    • The longer a subunit remains in a filament, the more likely it is to be in an ADP/GDP state, further impacting polymerization dynamics.

  • Function as GEF/GAP:

    • The filament can be conceptually viewed as functioning like a Guanine Exchange Factor (GEF) or a GTPase-activating protein (GAP) for the subunits.

Consequences of ATP/GTP Hydrolysis

Effects on Filament Dynamics

  • Hydrolysis results in the following:

    • ADP/GDP is trapped within the filament.

    • The energy released during hydrolysis causes conformational changes which increase kOFF in GDP-bound subunits.

    • This energy diminishes filament affinity, altering polymerization dynamics.

Comparison of Critical Concentrations

  • The Critical Concentration for ADP/GDP subunits will differ from that of ATP/GTP-bound subunits. Under typical conditions:

    • Cc for ATP/GTP-bound subunits promotes addition.

    • Cc for ADP/GDP-bound subunits fosters disassembly.

    • There exists a competitive dynamic between hydrolysis and the addition of new subunits.

Treadmilling in Filaments

Concept of Treadmilling

  • Treadmilling occurs when one end of the filament is predominantly composed of ADP/GDP-bound subunits (minus end disassembling) while the other end is ATP/GTP-bound (plus end assembling).

  • This dynamic facilitates processes such as cell migration, as illustrated by Actin-GFP experiments that show filament length remains relatively constant.

Microtubule Dynamics

Role of the GTP Cap

  • In microtubule assembly, a terminal GTP cap at the plus end serves as:

    • A stabilizing feature made of GTP-bound heterodimers that allows continued assembly.

    • The cap prevents catastrophic depolymerization and constrains the curvature of the microtubule. Unlike GDP-bound subunits, which induce mild curvature.

Occurrence of Catastrophe

  • Catastrophe refers to the rapid disassembly that occurs when the GTP cap is lost, leading to significant polymerization instability and switching behaviors in microtubules.

  • GDP-bound tubulin contributes to this instability due to its inherent curvature.

Intermediate Filaments

General Characteristics

  • Intermediate filaments are distinct from actin and microtubules, primarily found in cells exposed to mechanical stress, such as epithelial cells.

  • Notably, insects lack cytoplasmic intermediate filaments.

  • Two main categories of intermediate filament proteins include:

    • Nuclear Lamins: Provide structure to the nuclear envelope and organize nuclear pores.

    • Cytoplasmic Keratins: Can form diverse structures, including within keratin treatments.

Formation of Intermediate Filaments

  • Intermediate filament assembly involves:

    • Nuclear lamins which are localized to the nucleus, contributing to the nuclear envelope's integrity.

    • Cytoplasmic intermediate filaments consist of elongated subunits with a coiled-coil domain, forming dimers and tetramers through complex interactions:

      • Dimer formation occurs in parallel, followed by antiparallel arrangement to create tetramers (protofilaments).

      • Eight tetramers coalesce to form the final filament structure.

## Structural Diagram of Intermediate Filaments

Coiled-Coil Domains:

  • Diagram illustrating subunit arrangement:

  • Monomers consist of an a-helical region that forms dimers through coiled-coil interactions.

  • Further arrangements result in staggered tetramers stacking together, ultimately creating a ropelike filament approximately 10 nm in diameter.

Cell-Cell and ECM Anchoring

  • Intermediate filaments are crucial for structural integrity, anchoring to cell-cell contact points via desmosomes and to the extracellular matrix (ECM) through hemidesmosomes.

  • Conditions such as Epidermolysis bullosa simplex underscore the importance of these structures in maintaining tissue integrity.

Conclusion

  • Next Steps: Discussion will shift to cytoskeletal regulation, potentially exploring topics such as the interactions between fungi (e.g., mushrooms) and cytoskeletal components, particularly focusing on how certain toxins can disrupt filament dynamics and lead to cell dysfunction.