lecture 14 cytoskeleton: components. cytoskeleton proteins revealed by commassie staining...

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Lecture 14 Cytoskeleton: components

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Lecture 14

Cytoskeleton: components

Cytoskeleton proteins revealed by Commassie staining

Cytoskeleton: filament system

Internal orderShape and remodel surfaceMove organellesMovementCell division

Three types of filamentsand accessory proteins(assembly of cytoskeleton, motorproteins that move organellesor filaments)

Intermediate filaments:mechanical strength and resistance to shear stress

Microtubules: positions of membrane-enclosed organelles, intracellular transport

Actin filaments:shape of the cell’s surfaceand whole cell locomotion

Dynamic and adaptable

5-9 nm diameter

25 nm diameter

10 nm diameter

Cytoskeletal filaments are all constructed from smaller protein subunits

Intermediate filaments: smallerelongated and fibrous subunits

Actin and microtubule filaments:compact and globular subunits

All form as helical assembliesof subunits

Noncovalent interactions:rapid assembly and disassembly

Multiple protofilaments give strength and adaptability

Ends are dynamic

Intermediate filaments are resistent to bending or stretching forces

The time course of actin polymerization in a test tube

GTP

GTP!

The structure of a microtubule and its subunits

13 parallel protofilaments

hollow and cylindrica and polar

heterodimer

monomer

ATP

polar

two parallel protofilamentsthat twist around each otherin a right-handed helix

The structure of an actin monomer and actin filament

Flexible but cross-linked andbundled together by accessoryproteins in a living cell

The preferential growth of microtubules at the plus end

Plus end: polymerize and depolymerize faster thanminus end

Actin filamentsPlus end- barbed endMinus end- pointed end

Microtubules:Plus end- subunitMinus end- subunit

The treadmilling of an actin filament

D form polymer leans towards disassembly

Structural difference between the two ends

Treadmilling behavior of a microtubuleas in a living cells

The extent of treadmilling inside the cellIs uncertain.

Actin treadmilling is observed in vitro.

A treadmilling-like phenomenon is seen inliving cells for microtubules

Tubulin conjugated with fluorescent dye1/20 subunit is fluorescent

“Microtubule lattice”

Dynamic instability:predominant in microtubules

GTP hydrolysis “catch up”

Treadmilling: predominant in actin filaments

GTP hydrolysis causes filament to curve

Lateral bonds force GDP-containingprotofilaments into a linear conformation

Direct observation of the dynamic instability of microtubules in a living cell

Dynamic instability of individual actin filaments cannot be observed readily-difference between two ends are not so extreme

However the actin filament turn over is rapid: individual filamentpersists for a only few minutes

The dynamic behavior of filaments allows cells to change structures rapidly and

Giant multinuclearcell of a fly earlyembryo

Actin filaments:redMircotubules:green

One divisionper 10 minutes

Actin and tubulin are highly conserved: they have to bind tomany proteins directly and indirectly

Accessory proteins and intermediate filament proteinsare not as conserved

Intermediate filamentsare only found in some metazoans:vertebrates,nematodes,molluscs

Not required inevery cell type

Ancesters: nuclear lamins

Parallel

Antiparrel

“subunit”No polarity!

8 parallel protofilaments

Easily bentHard to break

A model of intermediate filament construction

Mechanical properties of actin, tubulin and Intermediate filament polymers

viscometer

Microtubules: easilydeformed and then rupture

Actin filaments are morerigid and also ruptureeasily

Intermediate filaments:easily deformed and don’trupture--maintain cell integrety

Keratin filaments in epithelial cells

“desmosomes”

The most diverse family20 in human epithelial cells10 more in hair and nails

Intermediate filaments impart mechanical stability to animal cells

Diagnosis of epithelialcancers (carcinomas)

Blistering of the skin caused by mutant keratin genes

Epidermolysis bullosa simplex:the skin blisters in response tovery slight mechanical stress

Other blistering diseases:mouth, esophageal lining and cornea of the eye--mutations of different keratins

Truncated keratin (missing both the N- C- domains) Tg mice

Two types of intermdiate filaments in cells of the nervous system

Neurofilaments:axonsNF-L, NF-M, NF-H proteins coassemble

NF-M and NF-H have long C-terminal tailsThat bind to neighboring filaments:uniform spacing

When axons grow, subunits are added at the filament endsand along the filament length; axon diameter increase 5 fold

In ALS (Lou Gehrig’s Disease), there is an accumulation and abnormal assembly ofNeurofilaments in motor neuron cell bodies and axon--interfere with normal axon transport

Regular spacing

axon glia

Actin filaments and microtubules are targets ofmany plant toxins

Amanita Phalloids (death cap)(Eat raw meat)

Effect of the drug taxol on microtubule organization

treatment of cancers

Summary

1. Three types of cytoskeletal filaments, protofilaments;2. Subunits, polymerization, treadmilling, dynamic

instability;3. Intermediate filaments, cell integrity, diseases caused

by mutations in the intermediate filament genes4. Natural toxins and cytoskeleton