Microtubules and Intermediate Filaments Chapter 18

Bolded terms from slides defined

Tubulin

A family of globular cytoskeletal proteins that polymerize to form the cylindrical wall of microtubules.

Microtubules

Cytoskeletal fiber (about 25 nm in diameter) that is formed by polymerization of ⍺,β-tubulin monomers and exhibits structural and functional polarity. Microtubules are important components of cilia, flagella, and mitotic spindle, and other cellular structures.

Each contains 13 protofilaments aligned longitudinally along its length.

Each protofilament is assembled by polymerization of heterodimer building blocks: ⍺ and β-tubulin subunits.

The dimers are held together by non-covalent electrostatic interactions.

Dyneins

A class of motor proteins that use the energy released by ATP hydrolysis to move toward the (-) end of microtubules. Dyneins can transport vesicles and organelles, are responsible for the movement of cilia and flagella, and play a role in chromosome movement during mitosis.

Retrograde transport back toward the cell body is driven by dynein.

Kinesins

A class of motor proteins that use energy released by ATP hydrolysis to move toward the (+) end of a microtubule. Kinesins can transport vesicles and organelles and play a role in chromosome movement during mitosis.

Anterograde transport of vesicles is driven by kinesin (+) end directed motors

Structure of Microtubules

• Both ⍺ and β subunits bond one GTP molecules

• The β subunit hydrolyzes GTP to GDP and P_i after polymerization. P_i is released.

• Polymerization occurs at only the (+) end which terminates with an exposed β subunit.

• The (-) end terminates with an ⍺ subunit- UNLIKE actin polymers, the (-) end of microtubules does not polymerize.

GTP is required for microtubule assembly- both ⍺ and β monomers bind GTP

Single microtubule

13 protofilaments- most cytoplasmic microtubules are singlets

Doublet microtubule

an additional wall of 10 protofilaments from a second tubule (the B microtubule); found in the axonemes of cilia and flagella.

Triplet microtubule

two 10-protofilament walls on a 13-protofilament microtubule; found in the centriole and basal bodies.

Microtubule Organizing Center (MTOC)

General term for any structure (e.g., centrosome, spindle pole, basal body) that organizes microtubules in cells.

where:

• catalyze nucleation

• anchor (-) ends

Centrosome (cell center)

Structure located near the nucleus of animal cells that is the primary microtubule-organizing (MTOC); it contains a pair of centrioles embedded in a protein matrix and duplicates before mitosis, with each centrosome becoming a spindle pole.

Centrosome (spindle pole body)

An organelle that serves as the main microtubule organizing center for animal cells. Centrosomes are made from arrangements of two barrel-shaped clusters of microtubules, called centrioles, and a complex of proteins that help additional microtubules to form.

Spindle pole bodies

Functionally analogous structure of centrosomes in yeast.

Basal Body

Structure at the base of cilium or flagellum from which microtubules forming the axoneme assemble; structurally similar to a centriole.

Centriole

Either of two cylindrical structures within the centrosome of animal cells and containing nine sets of triplet microtubules; structurally similar to a basal body.

Pericentriolar Material

Amorphous material seen by thin-section electron microscopy surrounding the centrioles of animal cells. Pericentriolar material contains many components, including the gamma-tubulin ring complex (gamma-TuRC), which promotes nucleation of microtubule assembly.

ᵞ-tubulin

a third tubulin is found in the PCM and is critical for microtubule nucleation.

associates with other PCM proteins forming cone shaped structures called γ-tubulin ring complexes (γ-TURCs)- each the same diameter as a microtubule.

γ-tubulin associates only with ⍺-tubulin and therefore establishes microtubule polarity: (-) ends in the centrosome and (+) ends polymerizing out into the cell.

γ-tubulin ring complexes (γ-TURCs)

Serve as:

1. The seed site for polymerization of the ⍺/β-tubulin dimers

2. A protective cap to prevent the (-) end from depolymerizing

Augmin Complex

A protein complex required for building robust mitotic spindles in Metazoa cells.

During M phase augmin increases microtubule desity within the mitotic spindle. It binds the sides of existing microtubules, recruits γ-tubulin ring complexes (γ-TURCs) and nucleates the assembly of new microtubules in a branching pattern.

Catastrophe

If the rate of GTP hydrolysis outpaces dimer polymerization at the (+) end, the stabilizing cap will degrade, exposing the weakly bound GDP-β dimers

If this happens a microtubule will spontaneously and rapidly disassemble- this is called catastrophe.

Dynamic Instability

A phenomenon: microtubules that are not stabilizes by (+) end associated proteins are in a constant state of polymerization followed by catastrophe and rescue

Search Capture Model

Microtubules constantly reach out into the cell by polymerization in search of a cap or complex to bind to for stability.

Clochicine

Extracted from meadow saffron. It binds tubulin dimers and blocks polymerization. It’s used experimentally to block proliferating cells at the G2-M phase transition (synchronizing cells in culture).

Also been used for years to treat gout- bon joint inflammation- by preventing white blood cells from migrating into, and enhancing, sites of inflammation.

Taxol

An alkaloid from Yew trees, binds microtubules and prevents depolymerization. This stops cells from dividing and promotes cells death. Since 1992, Taxol has been a major chemotherapy drug used to treat breast, lung, and ovarian cancer.

Microtubule-Associated Protein (MAP)

Any protein that binds to microtubules and regulates their stability.

• most enhances stability by inhibiting depolymerization

• many promote interactions between microtubules, vesicles, and intermediate filaments

• some determine spacing between microtubules

• MAP activity is regulated by phosphorylation state, and there are several regulatory kinases and phosphates.

Medical Relevance of Microtubule-associated proteins

excessive phosphorylation of one neuron-specific MAP, called tau, leads to its aggregation as tangles. This is implicated in several neurological disorders, including Alzheimer’s Disease.

+TIPS proteins

(+ end tracking proteins) stabilize the (+) ends and inhibit catastrophe, either directly or by linking microtubules to various cellular structures.

only bind the growing end of microtubules, not a (+) end undergoing catastrophe

Kinesin-13

A member of the kinesin motor protein family promotes depolymerization at (+) (-) ends

“Motor” doamin in the middle of the heavy chain but has lost motor activity

• destabilizes microtubule (+) and (-) ends driving depolymerization

Stathmin

Binds selectively to a pair of dimers in collapsing, curved protofilaments, enhancing depolymerization.

• Stathmin is activated by phosphorylation. At a migrating cell’s leading edge this phosphorylation is blocked. As a result, microtubules grow at the leading edge without catastrophe, and help actin push the lamellapdia forward.

Katanin

Named after the Japanese katana sword, severs microtubules resulting in unstable (+) ends that rapidly collapse.

Kinesin-1

powers anterograde vesicle movement toward microtubule (+) ends.

homodimer of two identical heavy chains two light chains associated with the tail of each heavy chain bind receptors on vesicles.

• Heavy chain head motor domain binds microtubules

• Coiled stalk domains dimerize

• Flexible linker domain- connects head to stalk

• Head domain binding ATP causes linker to rotate, driving processive movement of kinesin along microtuble.

Kinesin-5

Four identical heavy chains organized in a bipolar structure- slide antiparallel microtubules past each other- critical during telophase and cytokinesis.

Cytoplasmic Dynein

A large multi-subunit complex functioning as a (-)end-directed microtubule motor. It contains two heavy chains that possess motor activity and function coordinately like a pair of legs to processively walk along a cytoplasmic microtubule

Various smaller subunits bind the stems and promote dimerization of the two heavy chains and interactions with regulatory proteins: they are called

• light

• light-intermediate

• intermediate chains

Heavy Chain

A huge multi-domain protein with:

• Globular, force-generating head domain

• Attached stalk domains

• Associated linker and stem domains

• Stem domains of cytoplasmic dynein dimerize generating a structure with two heads and stalks

Dynactin

Protein complex links the heavy chain stems to cargo via carious adapter protein. It contains multiple regulatory subunits that can control dynein activity.

AAA ATPase Family

A group of proteins that couple hydrolysis of ATP with large molecular movements usually associated with unfolding of protein substrates or the disassembly of multisubunit protein complexes.

AAA+ ATPase Domain

Each head domain is a structure conserved in several proteins with a spinning or rotating function (like DNA helicase)

made up of six similar tandem linked regions called AAA+ ATPase modules arranged in a wheel conformation

Power Stroke

The force of the linker snapping back straight across the AAA+ ATPase domain rotates the head and moves the cargo 8nm toward (-) end

Mitotic Spindle

A specialized temporary structure, present in eukaryotic cells during mitosis, that captures the chromosomes and then pushes and pulls them to opposite sides of the diving cell; also called mitotic apparatus.

Cilium (pl. cilia)

Short, membrane-enclosed structure extending from the surface of eukaryotic cells and containing a core bundle of microtubules. Cilia usually occur in groups and beat rhythmically to move a cell (e.g., single-celled organism) or to move small particles or fluid along a surface (e.g., trachea cells).

Flagellum (pl. flagella)

Long locomotory structure (usually one per cell) extending from the surface of some eukaryotic cells (e.g., sperm), whose whiplike bending propels the cell through a fluid medium. Bacterial flagella are smaller and much simpler structures.

Primary Cilia

Single cilia found on epithelial cells that function as antenna to receive specific signaling molecules

Axonome

A bundle of microtubules and associated proteins present in cilia and flagella and responsible for their structure and movement.

consisting of microtubules in a 9+2 arrangement

nine doublets of microtubules (one complete and one incomplete wall) surround a central microtubule pair.

A protein rich central sheath connects the inner to outer tubules by radial spoke protein complexes.

the 9 doublets are connected to one another by an interdoublet bridge composed of the protein nexin

Protofilaments

Tetramers linked end-to-end

Protofibrils

four protofilaments associate laterally

mature intermediate filament: contains four coiled protofibrils with the globular domains forming beaded clusters on the surface

Keratin

A group of intermediate filament proteins found in epithelial cells that assemble into heteropolymeric filaments

provide tensile strength and integrity to epithelial cells and are the most diverse IF family (54 of all 70 IF types are keratins)

Hard Keratins

Found in epidermal appendages and are further classified into ⍺-keratins (⍺-helix based; found in mammalian hair, wool, quills, horns, and nails) and β-keratins (β-sheet based; found in avian feathers and reptilian tissues).

Cytokeratins

Soft keratins

Primarily found in epithelial cells where they form intracellular intermediate filament networks that provide a scaffold for epithelial cells and tissues to withstand mechanical stress and maintain structural integrity.

Keratinocytes

Basal layer of skin is composed of these constantly proliferating cells

after they leave the basal layer, these cells express abundant cytokeratins which associate with protein junctions between cells and provide tension and abrasion resistance.

Desmin

A subunit protein of the intermediate filaments found in a smooth and skeletal muscle.

The principle IF of muscle cells, both smooth and skeletal.

Lamins

A group of intermediate filament proteins that form a fibrous network, the nuclear lamina, on the inner surface of the nuclear envelope.

Nucelar Lamina

Fibrous network on the inner surface of the nuclear envelope composed of lamin intermediate filaments.

Plakins

A family of proteins that help attach intermediate filaments to other structures