Transcript ch9 FA 11 - Cal State LA

The Cytoskeleton
• Functions
– Structural scaffold creating and supporting cell shape
• Framework positioning organelles within cytoplasm
– Network of molecular “roads” for intracellular transport of materials
– Framework for whole cell movement
– Framework for cell division
The Cytoskeleton
• Three major structural components
– Microtubules
• Major role: support, intracellular transport
– Intermediate filaments
• Major role: mechanical strength to resist physical stresses
– Microfilaments
• Major role: muscle contraction, motility
The Cytoskeleton
• Microtubules (MTs)
– Major role
• Intracellular transport
– Motor proteins drag cargo along them
• Structural support
– Resist compression forces
– Resist shear (bending) forces
– Hollow, rigid
– 25nm diameter, 4nm wall thickness
– Radiate outward toward plasma membrane
from near nucleus (MTOC)
The Cytoskeleton
• Microtubules (MTs)
– Unit = alpha / beta tubulin
heterodimer
• alpha subunit + beta subunit
• Heterodimer is asymmetric
• Beta end is called “plus” end
• Alpha end is called “minus” end
– Not referring to a charge
difference
plus-end
minus-end
The Cytoskeleton
• Microtubules (MTs)
– alpha / beta (a/b)-tubulin heterodimer
– Beta subunit is a GTPase
• Assembly
– Polymer grows by addition of units at
the “plus” end
– GTP-bound tubulin can add
– GTP form hydrolyzes to GDP form
– GDP-bound tubulin cannot add
– GDP-bound tubulin can release only
from “plus” end
– GDP-bound tubulin cannot release
from “minus” end or from central
region
The Cytoskeleton
• Dynamic instability
– MTs can assemble/disassemble at
different rates in different
locations within a single cell
– Various proteins can bind and
stabilize MTs
The Cytoskeleton
• Microtubule-associated proteins (MAPs)
– Form bridges crosslinking adjacent MTs for parallel alignment
– Increase MT stability
– Promote assembly
– Regulated by phosphorylation state
Anti-tubulin antibody stain
The Cytoskeleton
• Microtubule organizing centers (MTOCs)
– GTP-bound a/b-tubulin spontaneously
assembles into MTs very slowly
– GTP-bound a/b-tubulin add to an existing
MT very rapidly
– MTOCs are the nucleation points for MT
assembly
• Centrosome
• Basal body
The Cytoskeleton
• Microtubule organizing centers (MTOCs)
– Centrosome
• 2 centrioles at right angles to each other near nucleus
– Contain gamma-tubulin subunit
– Nucleate “minus” end of a/b-tubulin
» Plus-end is oriented outward toward plasma
membrane
The Cytoskeleton
• Microtubule organizing centers (MTOCs)
– Basal body
• Single centriole at the base of cilia and flagella
• Eukaryotic cilia and flagella
– Hair-like motile organelle projecting from
cell surface
– Covered by plasma membrane
• Eukaryotic cilia and flagella
– Central protein core is called an “axoneme”
• Eukaryotic cilia and flagella
– Central protein core is called an “axoneme”
• Composed of 11 MTs arranged in a “9+2” array
– 9 outer MTs
– 2 central MTs
– Connected by various MAPs
– Locomotion caused by sliding outer tubules past each other
» Action of motor proteins (dynein)
The Cytoskeleton
• Motor proteins that “walk” on MTs
– Kinesin gene family
• Plus-end directed
– Outward or
“anterograde”
transport
– Dynein gene family
• Minus-end directed
– Inward or
“retrograde”
transport
The Cytoskeleton
• Kinesins are composed of 2 heavy and 2 light polypeptides
– Cargo-interaction domain “tail”
• Different kinesins have different specificities
– ATPase “head”
• Binds to MT
• ATP hydrolysis propels heads forward
• Highly processive
The Cytoskeleton
• Kinesins are composed of 2 heavy and 2 light polypeptides
– ATPase “head”
• Binds to MT
• ATP hydrolysis propels heads forward
• Highly processive
The Cytoskeleton
• Motor proteins that “walk” on MTs
– Dynein gene family
• Minus-end directed
– Inward or “retrograde” transport
• Very large (1.5MDa)
– Involved in cilia/flagella movement
The Cytoskeleton
• Three major structural components
– Intermediate filaments (~65 genes)
• Major role: mechanical strength to resist physical stresses
– Hemidesmosomes and desmosomes
• Intermediate filaments (IFs)
– Animal specific
Epidermolysis
– Strong, rope-like
Bullosa Type
Simplex
Junctional
Dystrophic
Genes Mutated
Keratin-5; Keratin-14; plectin
laminin-5; collagen XVII; a6b4 integrin
collagen VII
• Intermediate filaments (IFs)
– Animal specific
– Strong, rope-like
– Bridged together with other
cytoskeletal elements
• (e.g. plectin crosslinks MTs and IFs)
The Cytoskeleton
• Intermediate filaments
– Composition and assembly
• Monomers form dimers
• Dimers form tetramers
lacking polarity
• Tetramers form larger fibers
• Incorporation into existing
filaments not limited to end
regions
The Cytoskeleton
• Three major structural components
– Microfilaments (MFs)
• Major role: muscle contraction,
motility
• Solid, branched
• 8nm diameter
• Molecular unit= actin
The Cytoskeleton
• Microfilaments (MFs)
– Actin molecule is asymmetric
• “plus”-end versus “minus”-end
– Actin is an ATPase
– ATP-bound actin can be
incorporated into growing MFs
– plus-end of MFs grows 10x faster
than minus-end
– Higher dissociation rate from
minus-end leads to treadmilling
The Cytoskeleton
• Microfilaments (MFs)
– Drugs
• Cytochalasin D blocks plus-end addition leading to complete MF depolymerization
• Phalloidin blocks turn-over locking MFs into polymerized state
+ cytochalasin D
• Actin binding proteins
The Cytoskeleton
+ cytochalasin D
The Cytoskeleton
• Motors that walk on Microfilaments (MFs)
– Myosin gene family
• ATPase “head” domain
• Cargo-interacting “tail” domain
The Cytoskeleton
• Motors that walk on Microfilaments (MFs)
– Myosin gene family
• Type V can walk on actin filaments carrying a bound cargo
• Type II forms bipolar filaments via tail - tail interactions
The Cytoskeleton
• Myosin type II in muscle contraction
– Muscle fiber
• Large cell, 100mm long, 10100 microns thick
• Contain >100 nuclei
• Derived from the fusion of
many myoblast cells
– Myofibrils
• thin protein strands composed
of repeating units called
“sarcomeres” that give muscle
its “striated” appearance
– Sarcomere
• Z, I, A, H and M regions
Sliding filament model of muscle contraction
Sliding filament model of muscle contraction