Lecture 11 - The microtubule cytoskeleton

The cytoskeleton Gives the cell its shape Allows the cell to organize its components Produces large-scale movements (I.e. muscle contraction, cell crawling, propulsion via cilia and flagella)

The cytoskeleton is composed of networks of 3 different filaments

Cytoskeletal filaments exhibit different physical properties

The cytoskeleton is dynamic

Microtubules are organized to perform specific functions

What do microtubules do? Establish an internal polarity to movements and structures in the interphase cell Participate in chromosome segregation during cell division Establish cell polarity during cellular movement Produce extracellular movement via beating of cilia and flagella

Microtubule structure

Microtubules exhibit a behavior termed dynamic instability Total mass of polymerized tubulin remains constant, but individual microtubules are dynamic Growth: assembly of microtubule Shrinkage: disassembly of microtubule Catastrophe: switching from growth to shrinking Rescue: switching from shrinking to growth

Tubulin subunit addition takes place predominantly at the plus end

Growing microtubules have a “cap” of GTP at the plus end

Microtubule-associated proteins MAPs can function as cross-bridges connecting microtubules. They can affect microtubule rigidity and assembly rate.

The centrosome is the primary microtubule nucleation site in most cells

Centrosomes act to polarize the microtubule network Plus end - fast growing, usually in the cytoplasm Minus end - slow growing, anchored at the centrosome in most cells

Centrosome duplication occurs once per cell cycle

Centrosomes are often abnormal in cancer cells

Why are microtubules dynamic? Microtubule dynamics allow the cell to quickly reorganize the network when building a mitotic spindle Dynamics also allow microtubules to probe the cytoplasm for specific objects and sites on the plasma membrane - search and capture

Search and capture model Search & capture during cell polarization Search & capture during mitosis

Motor proteins Enzymes that convert ATP hydrolysis directly into movement along cytoskeletal filaments Some motors move towards the plus end, others move to the minus end Carry cargo (organelles, protein complexes, RNA) and mediate microtubule/microtubule sliding

First evidence of microtubule motors came from study of axonal transport Extruded axoplasm assays - Cytosol is squeezed from the axon with a roller onto a glass coverslip. Addition of ATP shows movement by videomicroscopy Vesicle movement in this system is about 1-2um/s similar to fast axonal transport.

Motor proteins

There are two families of microtubule motors Kinesins –Move cargo to the plus end –In mitosis, participate in mitotic spindle dynamics –Usually dimers of 2 heavy chains and 2 light chains Dyneins –Move cargo to the minus end –In mitosis, participate in mitotic spindle dynamics –Power beating of cilia and flagella –Large protein complex with many subunits

Structure of kinesin 2 heavy chains + 2 light chains Microtubule and ATP binding sites in the head Cargo-binding site in the tail and light chains

Kinesin “walks” along microtubules

Dynein is a large complex of many proteins

There are two classes of dyneins Cytoplasmic dynein –Carries cargo in the cytoplasm –Involved in mitotic spindle dynamics Axonemal dyneins –Localized exclusively in cilia and flagella –The motors that power cilliary and flagellar beating

General model for kinesin- and dynein-mediated transport

Flagella and cilia are specialized microtubule-based cellular structures

Cilia and flagella Cilia line the epithelial tissue of the respiratory tract to sweep particulate matter out of the airways Cilia line the oviduct to push the egg Non-motile cilia detect signals Flagella allow sperm to swim Flagella are essential for left-right asymmetry during development (Kartagener syndrome: situs inversus, sinusitis, brochiectasis)

Cilia in the respiratory tract

Structure of a motile axoneme

Dynein movement causes flagella to bend

Mutations that disrupt cilia cause multiple diseases Fertility (sperm motility, ectopic pregnancy) Polycystic kidney disease Respiratory infection Retinal degeneration Hearing/balance loss (Usher syndrome)

Sinus invertus: left-right body asymmetry Defects affecting placement of lungs, heart, liver stomach and spleen Morphogens secreted on the right side of the embryo aretransported to the left side by ciliary beating Immotile cilia fail to establish proper morphogen gradients