Protein targeting (protein sorting)CHAPTER 18 Cell Organization and Movement II:
Microtubules and Intermediate Filaments
Microtubules and Intermediate Filaments
© 2008 W. H. Freeman and Company
Lung cell in mitosis
Chromosomes (blue) Kertain intermediated filament (red) Centrosomes (magenta) Microtubule (green)
Microtubule are found in many different locations and all have similar structure
Cells contain stable and unstable microtubules (MTs).
SEM of the surface of ciliated epithelium of rabbit oviduct Microtubule base
motor protein
Tubulin subunit are formed by α
and β One subunit bind to two GTP, has GTPase
activity. 55kDa monomers in all eukaryotes γ-tubulin are formed by α
and β.
GTP reversibly
add
In mammals at least 6 alpha and 6 beta isoforms have been identified
The proteins are highly conserved (75% homology between yeast and human)
Most variability is found in the C- terminal region of the molecules and is likely to affect interactions with accessory proteins
A tubulin homologue, FtsZ, is expressed in prokaryotes
_
Arrangement of protofilaments in singlet, doublet, and triplet MTs.
The tubule is a complete microtubule cylinder, made of 13 protofilaments
Microtubules or actions of microtubule motor protein →
polymerization, depolymerization → movement
motility; Located near the nucleus, assembly an orientation of
microtubules ,the direction of vesicle trafficking, and orientation
of organelles.
MTOC becomes responsible for establishing the polarity of cell
an direction of cytoplasmic processes in both interphase and
mitotic cells.
Microtubules are assembled from microtubule organization centers (MTOC)
In non-mitotic cell, the MTOC=centrosome near nucleus (+) end of microtubule toward to periphery
During mitosis, MTOC=spindle pole

Centrosome contains a pair of orthogonal () centrioles in most animal cells
Centrioles (C) a pair, C and C’ Pericentriolar (PC) matrix: γ-tubulin & pericentrin; surrounding the centrioles
MTOC = microtubule organizing center Microtubules assemble from the MTOC. In mammals, the centrosome () is
- -
+
+
pericentriolar matrix
MTOC
The center of the center of the cell… Centrioles in a centrosome connect to centromeres
MTOC=microtubule organizing center
γ-TURC
Microtubules nucleated by the γ-tubulin ring complex appear capped at one end, assumed from other data to be the minus end.
γ-Tubulin, which is homologous to α
& β
tubulins, nucleates microtubule assembly within the centrosome.
Several (12-14) copies of γ-tubulin associate in a complex with other proteins called “grips” (gamma ring proteins).
This γ-tubulin ring complex is seen by EM to have an open ring-like structure resembling a lock washer, capped on one side.
γ-tubulin ring complex
lockwasher shape cap
assembly
MTOC organization: pericentriolar material (including protein and γ
tubulin) →nucleating ( ) microtubule
assembly → anchoring γ-TuRC the has 8
polypeptides and 25 nm diameter
Under Cc, γ-TuRC directly nucleate microtubule assembly
γ-TuRC Formed only one end (-).
Stain with an anti-gamma tubulin antibody. Gamma-tubulin at initiates synthesis at one end (-) (green).
Intracellular tubulin concentration: 10-20 μM
Cc: 0.03 μM
More easy polymerization
Microtubule are dynamic structures due to kinetic differences at their ends
Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end)
Similar to actin
[tubulin] < Cc depolymerization [tubulin] ~ Cc dynamic
-- microtubule assembly/disassembly occurs preferentially at the (+) end
Temperature affects whether MTs assemble or disassemble.
High Temp: assembly (need GTP) Low Temp: disassembly
Cc: critical concentration Up: dimers polymerize into microtubules; below: depolymerize MAP : microtubule associated protein regulated polymerzation of microtubule
Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place
preferentially at the + end)
tubulin dimers →short protofilaments → unstable and quickly associate more
stable curved sheets → wraps around microtubule with 13 protofilaments →composing the microtuble wall → incorporate αβ
(β
hydrolysis GTP) → bind + end
Slowed 2 fold than +
Rate of MT growth in vitro is much slower than shrinkage ().

Disassembly quick (7μm/min) Assembly slowly (1μm/min)
Fluroresence microscopy reveals in vivo growth and shrinkage of individual MTs.
Fluorescently-labeled tubulin microinjection into fibroblasts. Microtubule is dynamic instability.
Two factor influence the stability of MT: 1. Cc (critical concentration): > or = Cc → growth; < Cc →
shrink (). 2.
subunit bind to GTP or GDP.
Dynamic instability depends on the presence or absence of a GTP-β-tubulin cap

Microtubule growth from MTOC
(1) colchicines/colcemid: mitotic inhibitors --its effect is reversible --binds to tubulin dimer
blocks the addition or removal of other tubulin subunits to the ends of microtubule
disruption of microtubule dynamics
--cells are blocked at “metaphase” after colchicines treatment cytogenetic studies cell synchronization ()
(2) taxol, vinblastin: --bind to microtubules and stablize microtubule by inhibiting the lengthening
and shortening of microtubules --used for cancer treatment
Colchicine and other drugs disrupt MT dynamics.
Blocked at metaphase
Player : MAPs (microtubule associate proteins)
Microtubule are stabilized by side and end binding protein
Similar to actin Tau family (MAP2, Tau) → spacing
MAP regulating kinase (MARK): A key enzyme for phosphorylating MAPs → phosphorylated MAPs → unable to bind to microtubules
Cyclin-dependent kinase (CDK)→ phosphorylation of MAP4 → controlling the activity of various proteins in the course of the cell
cycle.
Spacing of MTs depends on length of projection domain in bound MAPs.
MAP2 and Tau can regulate microtubule spacing
When MT bundles are induced in cells
overexpressing MAP2 (left) and tau (right), the bundles formed by MAP2 have wider spacing between MT than those formed by tau.
MAP2 COOH end binds along the MT lattice while NH2 terminal end projects out from the microtubule
Tau binds similarly but its projection arm is much shorter than arm of MAP2
MAP2 Tau
Cross-link MT
Some MAP bind to (+) end of microtubule, such as TIPs
Tubulin: green + TIP protein EB1
EB1 specific bind to (+)
Kinesin-13 enhanced the disassembly
ATPase
polymerization)
• Severing proteins Gelsolin (severs and caps (+) ends)) ADF (actin depolymerizing factor); cofilin
microtubules regulatory proteins
• Stabilizing proteins MAPs (phosphorykated MAPs do not bind to microbutules) Tip proteins
• Destabilizing proteins kinesin-13 (+) stathmin (+) katanin (-)
Myosins: Motor proteins that ‘walk’ along actin filaments
Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change
Kinesin and dynein: Motor proteins that ‘walk’ along microtubules
MT associated motor proteins:
kinesins: towards + end (anterograde transport) Golgi to ER traffic
dyneins: towards - end (retrograde transport) ER to Golgi traffic wave-like motion of flagella and cillia
The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis.
Anterograde transport goes towards the axon terminal (cell body → synaptic terminals), such as vesicles.
Retrograde transport goes towards the axon hillock (synaptic terminal → cell body), such as old membrane
Fast: membrane-limited vesicles, ~250 mm/day. Slow: tubulin subunits, neurofilaments. Intermediate: mitochondria.
Progression of organelles along axons requires microtubules and the motor proteins: kinesin (toward +) and dynein (toward -).
Also dependent on motor proteins: Transport of vesicles for exocytosis/endocytosis or between the endoplasmic
reticulum and Golgi Extension of the endoplasmic reticulum Integrity and reassembly of the Golgi apparatus
Organelles in axons are transported along microtubule in both direction
DIC microscopy demonstrates MT-based vesicle transport in vitro.
Has anterograde and retrograde
Squid axon
Kinesin-1 powers anterograde transport of vesicles down axons toward (+) end of microtubule
cargo
-
+
Kinesin I powers anterograde transport of vesicles in axons.
most common structure comprised of two heavy chains and two light chains; and processive + end directed motor protein (most)
MT bind to the helix region in the head; binding is regulated by ATP hydrolysis Plastic bead coated with kinesin will slide along a microtubule towards an end 10 families identified - mainly 2 types, cytosolic and mitotic kinesins; Some structural similarity to myosin
Structure of kinesin: two heavy chain and two light chain
Model of kinesin-catalyzed vesicle transport.
cargo
binds only one monomer at a time in a processive manner
ATP hydrolysis coupled to movement EM data suggests binding primarily to β-
tubulin
Dimer of a heavy chain complexed to a light chain Mr= 380kD Three domains: Large globular head Binds microtubules and ATP 2) Stalk 3) Small globular head Binds to vesicles
Step size –
Kinesins form a large protein family with diverse functions
14 classes Not all function has
known Cargo: organelle,
motor, for depolymerization
ATP
Not all kinesins have the same subunit structure but all have the globular head domain
Kinesin-1 uses ATP to “walk” down a microtubule
Motor protein
Convergent structural evolution of the ATP-binding core of myosin head an kinesin
How does kinesin
ATP binding to the leading head initiates neck linker docking
Neck linker docking is completed by the leading head, which throws the partner head forward by 160
Å
The new leading head docks tightly onto the binding site
The trailing head hydrolyzes ATP to ADP-Pi
The trailing head, which has released its Pi and detached its neck linker (red) from the core, is in the process of being thrown forward.
Adapted from: Figure 1 in Vale & Milligan (2000) Science, Vol
288, Issue 5463, 88-95
The Way Things Move: Looking Under the Hood of Molecular Motor Proteins
Ronald D. Vale and Ronald A. Milligan
Science, 2000 288:80-95
Dynein motors transport organelles toward the (-) end of microtubules (retrograde)
Cytoplasmic dynein retrograde toward (-)
Need dynactin
Not directly attached to cargo
Dynactin is complex: Besides dynamitin, dynactin contains – a filament made of Arp1 that is actin like and binds with spectrin – Spectrin binds ankyrin which associates with the vesicle/ organelle – p150 Glued binds microtubules and vesicles – ankyrin, spectrin, and Arp1 are thought to form a planar cytoskeletal array.
Dynactin complex
light chain
Cytosolic dyneins are (-) end-directed motor proteins that bind cargo through dynactin.
Dynein also has heavy chains like kinesin but it mediates transport towards the (-) end of the microtubule;
Its light chain associates dynamtin, that is part of a large protein complex called dynactin, which is responsible for interacting with the organelles, vesicles, or chromosomes that are being transported.
Transport require dynactin, to links vesicles and chromosomes to dynein light chain Arp1 actin related protein, interact with spectrin Dynactin interact with light chains of dynein
Very large multimeric
The power stroke of dynein
Kinesin and dyneins cooperate in the transport of organelles throughout the cell
ERGIC: ER to Golgi intermediate compartment
Cooperation of myosin and kinesin at the cell cortex.
actin
Myosins: Motor proteins that ‘walk’ along actin filaments
Motor proteins are enzymes that couple the hydrolysis of ATP to a conformational change
Cytoplasm
Dynein Kinesin
Movement of pigment granules in frog melanophores
Microtubule: green Nucleus: blue Pigment: red
Cilia an flagella: microtubule based surface structure
Eukaryotic cilia and flagella contain a core of doublet MTs
studded with axonemal dyneins.
Structural organization of cilia and flagella
Video microscopy shows flagellar movements that propel sperm and chlamydomonas forward
nexin
Ciliary and flagellar beating are produced by controlled sliding of outer double MTs.
In vitro dynein-mediated sliding of doublet MTs requires ATP.
Intrafllagellar transport (IFT) moves material up and down cilia and flagella
Intraflagellar transport
Retrograde anterograde
For growth
Defects in IFT cause disease by affecting sensory primary cilia
Autosomal recessive polycystic kidney disease (ADPKD)
Bardet-biedl sndrome
Retial degeneration, can not smell
Defective primary cilium in mouse mutants lacking components of the IFT particle
SEM of epithelial cell
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Centrosome contains a pair of orthogonal () centrioles in most animal cells
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Addition of MT fragments demonstrates polarity of tubulin polymerization. (MT assembly and disassembly take place preferentially at the + end)
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Fluroresence microscopy reveals in vivo growth and shrinkage of individual MTs.
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Spacing of MTs depends on length of projection domain in bound MAPs.
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The rate of axonal transport in vivo can be determined by radiolabeling and gel electrophoresis.
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Model of kinesin-catalyzed vesicle transport.
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Cytosolic dyneins are (-) end-directed motor proteins that bind cargo through dynactin.
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Organelle transport uses motor proteins
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Eukaryotic cilia and flagella contain a core of doublet MTs studded with axonemal dyneins.
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