MICROTUBULE ORANIZING CENTER (MTOCs)

Microtubule Function
- Depends on location and orientation
- In vitro-studies, assembly of microtubules from αβ-tubulin dimers occur in 2 phases
o Nucleation:
 slow phase
 Small portion of the microtubule is initially formed
o Elongation:
 Rapid phase
- In vivo, nucleation is rapid in cells
o Occurs in association with a variety of specialized structures
o Microtubule-organizing centers (MTOCs)
o Best studied MTOC is the centrosome
Centrosome
- Microtubules of cytoskeleton are nucleated by the centrosome
- Complex structure:
o Two barrel shaped centrioles
o Electron dense pericentriolar matrix (PCM)
o Typically cylindrical (0.2 um diameter, twice as long)
Centrioles:
- Contain nine evenly spaced fibrils
o Each fibril has a band of 3 microtubule (A, B, and C)
o Only A microtubule is complete (with 13 protofilaments)
o B microtubule is incomplete (10 protofilaments), C microtubule is incomplete (11
protofilaments)
o Connected to center by radial spoke
o Pinwheel appearance
- Come in pairs at right angles to each other
Role of centrosome in the initiation and organization of microtubular cytoskeleton:
- Experiment done in a cultured animal cell:
1. Microtubules were depolymerized by incubation in a colcemid (drug that binds to tubulin
subunits and blocks their use by the cell)
2. Microtubule reassembly monitored by removing the chemical, fixing cells at various
intervals, and treating the fixed cells with fluorescent antibodies
3. After removing the inhibitor (colcemid), one or two bright fluorescent spots seen in the
cytoplasm of the cell
4. Within 15-30 minutes, filament number radiating from loci increases dramatically
5. Cells are sectioned and examined via microscope showing newly formed microtubules
radiating outward from a centrosome
* Microtubules do not penetrate into the centrosome, terminates in PCM (which
initiates microtubule formation)
* Centrioles are not directly involved in microtubule nucleation, they play a role in
recruiting the surrounding PCM
Centrosomes as sites of microtubule nucleation
- Microtubule polarity is always the same
o Minus end: associated with the centrosome
o Plus end: the growing end, at the opposite tip
- Elongated at the opposite end of the polymer
 - Growing end may contain specific proteins that help attach the microtubule to a particular
target
Microtubules associated with centrosome
- Highly variable from one cell type to another
- Non-polarized cell (fibroblast): centrosome is typically located near the center of the cell,
remains associated with the minus ends of a large number of microtubules
- Polarized cells (Epithelial cells): microtubules are anchored by the minus ends at dispersed sites
near the apical end of the cell, plus ends extend toward the basal ends of the cell
- Nerve cells: microtubules with no association to centrosome (located in the neuron’s cell body)
o Microtubules formed in conjunction with centrosome
o Severed from MTOC
o Transported into axon by motor proteins
- Certain animal cells, such as mouse oocytes, are acentrosomal
Basal Bodies and Other MTOCs
- Outer microtubules of cilium or flagellum are generated as outgrowths from microtubules in a
basal body which is at the base of the cilium or flagellum
- Basal bodies are identical in structure to centrosomes and can give rise to one another
- Plant cells lack both centrosomes and centrioles or any other type of MTOC
o Microtubules in a plant cell are nucleated throughout the surface of the nucleus and
throughout the cortex
Microtubule Nucleation
- All MTOC play similar roles in cells:
o Control the number of microtubules
o Control microtubule polarity
o Number of protofilaments that make up the walls
o Time and location of their assembly
- All MTOC share a common protein component: γ-tubulin
γ-Tubulin
- Constitutes 0.005 percent of the cell’s total protein (* α- and β- tubulin make up 2.5 percent of
non-neural cells’ protein)
- Insoluble fibers of PCM serve as attachment sites for ring-shaped structures that have the same
diameter as microtubules (25nm) and contain γ-tubulin
- Minus ends of microtubules are embedded within the PCM of the centrosome
- structure of microtubules:
o γ-tubulin in a helical array form an open, ring shaped template
o αβ-tubulin dimers begin to assemble on the ring template
o only α-tubulin of the heterodimer can bind to the ring of γ-subunits
- the γ-tubulin ring complex (γ-TuRC) determines the polarity of the entire microtubule and forms
a cap at the minus end, preventing the gain or loss of microtubulin subunits
The Dynamic Properties of Microtubules
- all microtubules are similar morphologically, but they are different in their stability
- Microtubules are stabilized by MAPs and other factors such as posttranslational modifications to
tubulin subunits
- Stability of microtubules:
o Mitotic Spindle of Cytoskeleton: extremely labile, sensitive to disassembly
o Mature neurons: less labile
o Centrioles, cilia, flagella: highly stable
- Disassembly can be induced by:
 o cold temperature
o hydrostatic pressure
o elevated Ca2+ concentration
o variety of chemicals such as colchicine (binds with tubulin), vinblastine (binds with
tubulin), vincristine (binds with tubulin), nocodazole , podophyllotoxin
o Taxol (binds with polymer) inhibits disassembly and prevents assembly of new
microtubular structures
- Microtubules are polymers formed by the noncovalent association of protein building blocks
- They are normally subject to depolymerization and repolymerzation as cell requirements change
from time to time
- Dynamic character is well illustrated by plant cells:
1. Interphase –
o microtubules are widely distributed throughout the cortex
o nucleation factor localized along lengths of cortical microtubules
o new microtubules are formed directly on the surface of existing microtubules
o daughter microtubules are severed from parent microtubule and incorporated into
the parallel bundles that encircle the cell
2. Approaching mitosis –
o microtubules disappear from most of the cortex
o a single transverse band (preprophase band) is left of the microtubules and marks
the site of the future division plane
3. During Mitosis –
o preprophase band is lost
o microtubules reappear as mitotic spindle
4. After chromosomes split –
o mitotic spindle disappears
o a bundle of microtubules (phragmoplast) replaces the mitotic spindle
o phragmoplast plays a role in the formation of the cell wall that separates the two
daughter cells
- Changes in organization is accomplished by the combination of two mechanisms
1. Rearrangement of existing microtubules
2. Disassembly of existing microtubules, reassembly of new ones in different regions of the cell
(microtubules that make up the preprophase band are formed from the same subunits as
the cortical array or the phragmoplast)
The Underlying Basis of Microtubule Dynamics
In vitro microtubule assembly experiment
- Done by Richard Weisenberg
- Cell homogenates should possess all the macromolecules required for the assembly process
- obtained tubulin polymerization in brain homogenates (37 deg) by adding Mg2+, GTP, and EGTA
(which binds Ca2+, an inhibitor of polymerization)
- Microtubules could be disassembled and reassembled by lowering and raising the temperature
- in vitro assembly is greatly aided by the addition of MAPs or by pieces of microtubules or
structures that contain microtubules that act as templates
GTP’s importance in microtubule assembly
- Assembly of tubulin dimers require a GTP molecule be bound to the β-tubulin molecule
- β-tubulin is not only a structural protein, it is also an enzyme (GTPase)
- GTP is hydrolyzed to GDP after the dimer is incorporated into a microtubule, the resulting GDP
remains bound to the assembled polymer
 - During disassembly, a dimer is released from a microtubule and enters the soluble pool, GDP is
then replaced by a new GTP
- This nucleotide exchange recharges the dimer and allows it to serve as a building block for
polymerization again
Why did the costly polymerization pathway evolve?
- Due to GTP hydrolysis, microtubule is not an inexpensive cellular activity
- when a microtubule is growing, the plus end is seen as an open sheet to where GTP-dimers are
added
- during times of rapid microtubule growth, tubulin dimers are added more rapidly than GTP can
be hydrolyzed
- Presence of a cap to tubulin-GTP dimers at the plus ends of protofilaments favor the addition of
more subunits and the growth of the microtubule
- microtubules with open ends undergo a spontaneous reaction that leads to closure of the tube
which is accompanied by the hydrolysis of the bound GTP (generating GDP-bound tubulin
subunits)
- GDP-bound tubulin subunits are less suited to fit into a straight protofilament than GTP-bound
tubulin which causes a strain that destabilizes the microtubule
- Strain energy is released as the protofilaments curl outward from the plus end of tubule and
undergo catastrophic depolymerization
- GTP hydrolysis is a fundamental component of the dynamic quality of microtubules, the strain
energy stored in a microtubule as a result of GTP hydrolysis makes the microtubule unstable and
capable of disassembling soon after its formation
- Microtubules shrink very fast and allows a cell to disassemble its microtubular cytoskeleton very
rapidly
Dynamic Character of Microtubules
- because switch from growth to shrinkage (catastrophe) occurs with high frequency in vivo, most
microtubules disappear from the cell in a matter of minutes and are replaced by new
microtubules that grow out of centrosome
Dynamic Instability
- Timothy Mitchison and Marc Kirschner
- Reported properties of individual microtubules and proposed that microtubule behavior in vivo
could be explained by dynamic instability
- explains:
1. Grown and shrinking microtubules can coexist in the same region of a cell
2. a given microtubule can switch back and forth unpredictable (stochastically) between
growing and shortening phases
- dynamic instability is an inherent property of the microtubule and the plus end where subunits
are added during growth and lost during shrinkage
- Dynamic instability can be influenced by external factors
o cells contain a diverse array of proteins (+TIPs) that bind to the dynamic edge of the
microtubule
o +TIPs regulate the rate of microtubule’s growth or shrinkage or the frequency of
interconversion between the two phases
o other +TIPs mediate the attachment of the plus end to a specific cellular structure
(kinetochore or actin cytoskeleton)
o once attached to a structure the dynamic properties can exert force on the structure
o Polymerization of a microtubule can push on an attached object; depolymerization can
pull on an attached object
 - provides a mechanism by which the plus ends can rapidly explore the cytoplasm for appropriate
sites of attachment which temporarily stabilizes microtubules and allows the cell to build
complex cytoskeletal networks
- allows cells to rapidly respond to changing conditions that require remodeling of the
microtubular cytoskeleton
- unlike microtubules of mitotic spindle/ cytoskeleton, microtubules of organelles lack dynamic
activity and are highly stable
CILIA AND FLAGELLA: STRUCTURE AND FUNCTION
- Cilia and flagella are hairlike, motile organelles that project from the surface of a variety of
eukaryotic cells, and are essentially the same structure
- Bacteria possess flagella but are simple filaments that are not evolutionarily related to
eukaryotic flagella
Cilia
- likened to an oar
- moves the cell in a direction perpendicular to the cilium
- Power stroke – cilium in rigid state, pushes around surrounding medium
- Recovery stroke – cilium in flexible state, little resistance to medium
- occurs in large numbers with coordinated beating
- in multicellular organisms, moves fluid and particulate material through various tracts
Flagella
- occur singly or in pairs
- exhibit a variety of beating patterns (waveforms) depending on the cell type
Structure of cilia and flagella
- entire projection is covered by a membrane continuous with the plasma membrane of the cell
- Core of cilium (axoneme) contains an array of microtubules that run longitudinally through the
organelle
- Axoneme consists of nine peripheral doublet microtubules surrounding a central pair of single
microtubules (9+2 array)
- All microtubules have the same polarity, with the plus ends at the tip and the minus ends at the
base
- Peripheral doublets consist of one complete microtubule (A tubule with 13 profilaments) and
one incomplete microtubule (B tubules with either 10 or 11 protofilaments)
- Central tubule enclosed by the central sheath connected to the A tubules by a set of radial
spokes
- Doublets connected to each other by an interdoublet bridge composed of nexin, an elastic
protein
- a pair of dyenein arms (2 headed inner and 3 headed outer) project from the A tubule
- longitudinal section shows the continuous nature of microtubules and discontinuous nature of
the other elements
- Cilium/ flagellum emerge from a basal body, the A and B tubules of the basal body elongate to
form the cilia/ flagella doublets
- if cilia/ flagella is sheared from cell surface, a new organelle will be regenerated from the basal
body
- the growth of the axoneme occurs at the plus (outer) end of the microtubules