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