20250212_Cytoskeleton Lecture 3_v2

Cytoskeleton Overview

• Cytoskeleton is a complex network that provides structural support, enables cellular movement, and facilitates intracellular transport.

• Composed mainly of three types of filaments: actin filaments, microtubules, and intermediate filaments.

Microtubules

• Microtubules are dynamic structures composed of tubulin protein subunits, critical for maintaining cell shape and organizing cellular components.

• Tubulin heterodimers (α/β-tubulin) assemble head to tail to form protofilaments, which align to form microtubules.

Microtubule Organizing Centers (MTOC)

• Most animal cells contain a centrosome as the MTOC, featuring:

  • Pairs of centrioles, modified microtubule arrays surrounded by pericentriolar material.

  • b-TuRCs (tubulin ring complexes) embedded in pericentriolar material ensuring microtubule nucleation.

  • Microtubule plus ends oriented towards cell periphery.

• Other organelles like Golgi and nucleus can also function as MTOCs.

Microtubule Nucleating Proteins

Augmin

• Augmin binds to pre-existing microtubules, facilitating the nucleation of new microtubules at angles, important for generating branched microtubule networks, especially during mitosis.

• It recruits b-TuRC through electrostatic interactions between negatively charged tubulin and positively charged MAPs (Microtubule Associated Proteins).

Tubulin Sequestering Proteins - Stathmin

• Stathmin binds two tubulin heterodimers, preventing their addition to microtubule ends, favoring microtubule shrinkage.

• Phosphorylation reduces stathmin's affinity for tubulin, promoting microtubule growth, particularly during mitosis influenced by cyclin-dependent kinases.

Microtubule Accessory Proteins

• Various accessory proteins modulate microtubule dynamics and stability:

  • Kinesin-13: Induces microtubule disassembly (catastrophe).

  • XMAP215: Stabilizes plus ends and promotes rapid growth.

  • Katanin: Severing microtubules to facilitate remodeling.

Microtubule End-Binding Proteins

• Stabilizing microtubule minus ends and promoting dynamics at plus ends.

• Kinesin-13 binding to microtubule sides, inducing curvature and regulating stability.

• XMAP215 aids in the addition of new GTP-bound dimers to plus ends.

Katanin - Microtubule Severing Protein

• Katanin consists of a small p60 AAA ATPase subunit and a large p80 subunit.

• Its action promotes microtubule growth by creating GTP-capped regions that facilitate repair following severing.

Microtubule Motor Proteins - Kinesin

• Kinesins are a large superfamily of microtubule-associated motor proteins responsible for positive transport of cargo within cells.

• Structure:

  • Composed of monomers, dimers, or tetramers with a motor domain at the N-terminus.

• Function:

  • Bind and move cargo (vesicles, organelles) toward microtubule plus ends.

Kinesin-1 Binding Cycle

1. Lagging head binds ATP, leading head ADP-bound.

2. ATP hydrolysis in the lagging head loosens binding, allowing movement forward.

3. ATP exchange on the leading head induces further conformational changes, driving stepwise movement.

Kinesin Cargo Recognition

• Kinesin tails enable cargo binding directly or through adaptor proteins.

• Example: Kinesin-3 carries vesicles to axon terminals utilizing a PH domain for direct binding to PIPs and adaptor MADD.

Microtubule Motor Proteins - Dynein

• Dyneins move cargo towards microtubule minus ends, with two types: cytoplasmic and axonemal dyneins.

• Structure includes a flexible stalk for binding microtubules and tail regions for dimerization and cargo attachment.

Directional Trafficking by Kinesin & Dynein

• Kinesins generally transport cargo outward towards the cell periphery, while dyneins move cargo inward.

• In neurons, microtubules exhibit complex organization with consistent plus-end orientation in axons and mixed polarity in dendrites.

Cytoskeletal Filaments

• Actin Filaments: Provide cell shape and enable dynamic projections (filopodia) or stable structures (microvilli).

• Microtubules: Form transport networks and contribute to mitotic spindle formation during cell division.

• Intermediate Filaments: Provide structural integrity, lining the nuclear envelope and enhancing mechanical strength in specialized cells.

Intermediate Filaments Structure

• Composed of coiled-coil dimers forming antiparallel tetramers, packed into bundles.

• Examples include nuclear lamins (all cells), keratins (skin, hair), neurofilaments (neuronal axons), and desmins (muscle).