02.11.11Lecture 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

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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 - Cytosolis squeezed from the axon with aroller onto a glass coverslip.

Addition of ATP shows movementby videomicroscopy

Vesicle movement in this systemis about 1-2um/s similar to fast axonaltransport.

Motor proteins

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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

Kinesin “walks” along microtubules

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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)

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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)

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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