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Microtubule Structure

• Comprised of 13 protofilaments

• Arrayed circularly to form a tube wall

• They’re staggered to resemble a spiral 

What are the basic subunits of each protofilament (microtubule structure)

Dimers of alpha and beta tubulin proteins

What are the GTP-binding properties of α- and β-tubulin subunits?

• Both α- and β-tubulin bind GTP.

• α-tubulin: GTP is tightly bound

  • never hydrolyzed

  • does not exchange with free nucleotides.

• β-tubulin: GTP is loosely bound

  • hydrolyzed to GDP

  • exchanged for GTP in the cytosol.

How are tubulin subunits added and removed during microtubule assembly?

• α- and β-tubulin subunits are added/removed as dimers.

• αβ–GTP dimers have a higher affinity for the growing microtubule (more stable).

• αβ–GDP dimers have a lower affinity and tend to dissociate from the filament.

Microtubule Polarity

• They’re polar so the two ends have different characteristics and dynamics

  • (+) end = fast growing 

  • (-) = slow growing 

• Within the dimers

  • the beta-subunit is closer to (+)

  • the alpha-subunit is closer to (-)

Microtubule Dynamics

• Dimers with αβ–GTP are added to (+) end

  • Rescue phase 

• Dimers with αβ–GDP are released from shrinking filament

  • Catastrophe

• GTP hydrolysis occurs within polymerized microtubule

  • Most of it consists of dimers containing αβ–GDP

• (+) has GTP cap (unhydrolyzed) which favours growth 

  • αβ–GTP dimers have a 4x slower disassociation rate in comparison to αβ–GdP

  • They thus have higher affinity for their neighbours and stay together 

• (+) end has dynamic instability

  • Oscillates between growth or shortening

  • High [GTP-tubulin] = polymerization

  • Low [GTP-tubulin] = depolymerization

EB1 Protein (Microtubule)

• Plus-end binding protein

• Prevents premature catastrophes

• Acts as positive regulator of microtubule growth 

MAPs - Microtubule Associated Proteins

Proteins controlling the assembly and disassembly of microtubules 

MAPs - Microtubule Associated Proteins (Function)

• Interconnect microtubules to form bundles 

• Inc stability 

• alter rigidity 

• influence assembly rate 

MAPs - Microtubule Associated Proteins (Two Groups) 

1. Those that stabilize microtubules (Ex. Tau and EB1)

2. Those that destabilize microtubules (Ex. catastrophin)

Microtubule Nucleation 

• Starting off growth 

• Involves γ‐tubulin which is present in smaller amounts in the cell compared to alpha/beta tubulin

• Helps form γ‐tubulin ring complex (γ-TuRC)

  • Nucleates at (-) end of a new microtubule 

  • Forms a template for the growing (+) end

• γ-TuRC acts as a cap of the (-) end while microtubule growth occurs at (+) end

MTOC (Microtubule Organizing Center)

• A specific location inside the cell where microtubule nucleation occurs 

• In animal cells, the MTOC is centrosome (red dot)

  • Located near nucleus

MTOC: Centrosome and yTuRC

• Consists of 2 cylindrical structures called centrioles (inside centrosome which is in green) 

• Also has pericentriolar material (PCM) containing many γ‐TuRC complexes (red rings on green ball)

• (-) end of microtubules are nucleated at the γ‐TuRC

• (+) end are directed towards the cell periphery (shown as +)

MTOC role in Mitosis

• The MTOC (centrosome) organizes microtubules that form the mitotic spindle.

• The spindle’s microtubules attach to chromosomes to separate replicated sister chromatids.

• Centrosomes are duplicated before mitosis, creating two MTOCs that move apart to opposite poles.

• Microtubules nucleate from the γ-TuRC complexes at each MTOC, with plus ends growing outward.

Microtubule Toxins: Cholchicine 

• Useful in lab to arrest the cell cycle 

• Ex. cholchicine 

  • Derived from meadow saffron 

  • Inhibits polymerization 

  • Binds and stabilizes αβ‐tubulin dimers

  • Prevents addition/loss of tubulin dimers

  • Arrests cells in metaphase without chromatid seperation

Microtubule Toxins: Taxol

• Useful in lab to arrest the cell cycle 

• Taxol Function

  • Binds to β‐tubulin to increase affinity for (+) end

  • Prevents depolymerization 

  • Prevents assembly of mitotic spindle to inhibit mitosis

• Used in cancer treatment

• Hard to synthesize in lab so it’s derived from pacific yew tree

Kinesin Motor Protein

• (+) directed transport on microtubules, so towards cell periphery away from MTOC

• Tetrameric complex made of 2 heavy chains and 2 light chains 

• The globular heads (motor domains) cyclically bind to microtubules 

  • Generates movement through ATP hydrolysis 

• The tails determine specificity of cargo binding

  • The tails are highly variable

Kinesin Mechanochemical Cycle

• The lagging head is bound to ATP

• The leading head is bound to ADP

• ATP kinesin has a higher affinity for the microtubule than ADP bound kinesin

• The ATPase motor lagging head hydrolyzes ATP to ADP + Pi

  • Reduces affinity of lagging head for microtubule

• ADP is exchanged for ATP in leading head

  • Increases affinity of leading head

• The binding of ATP induces conformational change causing lagging head to swing in front to another microtubule binding site

• This resets cycle to the top

How does kinesin move along microtubules?

• Kinesin moves in a “hand-over-hand” fashion.

• It has two motor heads (domains), and one is always attached to the microtubule.

• The two heads work in a coordinated cycle, each in a complementary stage of ATP binding or hydrolysis.

In-vitro assays for kinesin movement

• Nomarski Microscope

  • Following plastic beads tethered to kinesin

  • The track is anchored to the dick made from purified tubulin 

• Gliding mobility assay

  • kinesin are tethered to a glass slide at their cargo (Tail) ends

  • They can then move fluorescently labeled microtubules added to solution above slide

Dynein

• (-) directed, moving towards MTOC 

• 2 main forms: Cytoplasmic and Axonemal 

• Has 2 heavy chains and a variety of intermediate and light chains

Two forms of dynein

• Cytoplasmic

  • Associated with microtubules 

  • Direct movement of organelles and vesicles in cytoplasm

• Axonemal 

  • Found in structures powering movement of whole cells

  • Ex. cilia or flagella 

How does dynein move cargo along microtubules?

• Movement is powered by a power stroke in the linker arm (near the cargo attachment site).

• In a dynein dimer, the two motor units alternate power strokes, producing continuous movement.

Describe the steps of dynein’s ATP-driven power stroke

• ATP binding releases motor head group from microtubule

• ATP hydrolysis creates dynein-ADP+Pi that can now attach to the microtubule

• The release of Pi powers the power-stroke of the liner

  • Pulls the cargo 

• Each power stroke, the cargo moves towards the (-) end by 8mm

Bidirectional Vesicle Movement: Neural Cells 

• Microtubules span the axons of neural cells 

• The (-) ends are anchored to MTOC 

• The (+) ends extend along the axons towards synapse cell membrane 

• Vesicles with NTs are carried from cell body to synapse along microtubules 

Microtubule Tug of War

• Model describing the movement of proteins if they’re bidirectionally transported

• The final direction of movement is the winner of this ‘battle’ 

• There are regulatory proteins controlling direction in response to cell signals 

Change of Direction (Microtubule Transport) Application: Melanosomes in Fish

• Melanosomes: Pigment-filled organelles 

• Movement of it changes skin cells in response to behavioural signalling 

• This movement is done by molecular motors carrying it to the cell periphery or center 

  • Dynein: Move towards (-) end MTOC 

  • Kinesin: Move towards cell periphery (+) end

• Dispersion to periphery = cell appears darker 

• Concentrated in middle = Cell appears lighter 

• This is controlled by signals using cAMP as a secondary messenger 

What are the 3 types of filaments making up the cytoskeleton

1. actin

2. microtubules

3. intermediate filaments

Function of Cytoskeleton

• Provides shape and structure

• Responsible for the specialized structures in cells

  • Microtubules in cilia

  • Actin filaments in imcrovilli 

• The shape depends on functions

What is the dynamic nature of cytoskeleton important for

• Cells that move 

• Cells that undergo migration or cell division 

What are the three types of fibres in eukaryotic cells defined by (cytoskeleton)

• Diameter

• Type of subunit used to build the filament

Actin Filament Labelling (2)

• Labeled using fluorescently-tagged phalloidin

  • Toxin derived from death cap mushroom 

  • Binds to actin monomers with high affinity and specificity 

  • Stabilizes the filmament when bound

• Labeled with antibody 

• Labeled with protein fusion (Actin:GFP)

Intermediate filament labeling

• Labeled using an antibody specific to a monomeric subunit

• Labeled using GFP-fusion 

Microtubule labeling

• Labeled using antibodies specific to one of the tubulin subunits 

• Labeled using protein fusion (Tubulin:GFP)

Actin composition

• Thinnest filament 

  • 5-9nm

• Made of 2 strands of helical polymers that spiral around eachother

• Each strand is built from single actin monomers

  • G-actin

Microtubule Composition

• Thickest fibres 

• Made of dimeric subunits of alpha and beta-tubulin

Intermediate filament (IF) composition

• There are many types 

• Each is assembled from a different protein or set of proteins

Epithelial Cell 3 cytoskeletal fibre Distribution

1. Actin (red) forms the shape of the microvilli at apical side of cell surface 

2. IF (blue) span to provide structural support 

3. Microtubules (green) form networks for transport 

Filament-Specific Motor Proteins

• Move along the actin and microtubules

• No motor proteins found for IF

Which motor protein moves along actin filament

Myosin

Which motor proteins move along microtubules

• Kinesin 

• Dynein 

General Structure of Motor Proteins

• They step along their respective fibres using cycling chemical reactions

• The head domains bind to a cytoskeletal fibre

• Tail domain attaches to cargo 

• ATP hydrolysis provides energy for this movement

Actin-Based Structures

• Highest density of actin is at cell periphery to determine shape and movement of cell surface 

  • Establishment of microvilli 

  • Formation of contractile bundles forming sarcomeres

  • Contractile ring directing cytokinesis 

Actin filament organization variance within a single cell

1. Contractile Stress fibres (seen throughout)

2. Gel-like network (seen at cell cortex)

3. tight parallel bundles (seen in filopodia)

Actin Filament Polarity 

• No visible without the myosin proteins

• They bind to actin in one orientation, pointing away

• This defines (+) / (-) end of the filament based on rate of actin polymerization

• (+) grows more quick and has barbed appearance

• (-) end grows slower, or may shrink and has pointed appearance

G-actin structure

• Has 4 structural domains

• Large cleft between domains 2/4

• The cleft forms ATP-nucleotide binding site

• This binding site is pointed towards the minus-end

  • Makes them hidden as the monomers bind

  • Only 2 monomers at the end have exposed sites 

• Each actin monomer is polar so the microfilament is polar 

Actin Dynamic Polymerization

• Depolymerization and polymerization can occur at both the plus and minus ends 

• More growth tends to occur at (+) while there’s shrinkage at (-)

• This is bc of ATP

  • When monomers are bound to ATP, they can join

  • Intrinsic ATPase activity hydrolyes ATP to ADP

  • ADP never gets released as the binding site in covered 

• (+): Actin-ATP monomers are added

• (-): Actin-ADP comes off

• Cytosol: Free actin-ADP exchanges ADP for ATP

Critical Concentration

• Concentration where the rate of actin monomer addition is equal to the rate of removal 

• No net growth at that end

• If [monomer] exceeds this, polymerization exceeds rate of depolymerization (filament grows) 

• If [monomer] is lower, depolymerization exceeds (filament shrinks)

• The critical and working concentrations are different at each end 

Proteins involved in actin polymerization/depolymerization

• Profilin binds to actin-ATP 

  • Activates monomer 

  • Promotes ATP binding

• Profilin-actin dimers accumulate at plus end 

  • Increases [monomer] at that end 

• Thymosin binds to actin monomers

  • inhibits polymerization

• Thymosin-actin dimers accumulate at plus end

  • Creates a buffer of stored actin monomers

Treadmilling

• When there is no net increase in actin filament length

• Happens when rate of polymerization at (+) = depolymerization at (-)

• The relative position of the filament changes to move forward

• Helpful for cell movement/migration

Actin Filaments and Cell Migration

• Powers cell movements through organization of actin filaments to push out cell membrane

• Observed through formation of filopodia and lamellipodia in a migrating cell

  • Forms leading edge of cell

  • Forms fan-like expansions of cell membrane (lamellipodia)

  • Forms finger-like filopodia extensions of cell membrane

  • Initiates movement to desired direction

Myosin Motor Protein Types 

• Power intracellular cargo trafficking 

• Myosin I / II / V are in all euk cells

• Have motor domain (head) at N-terminus 

  • Binds actin filaments

  • Hydrolyzes ATP to drive motor

• Have different tail domains

  • Carries cargo at different rates

• They usually move toward the (+) end of actin

Myosin II Structure 

• Has 2 heavy chains forming a coiled-coil motif (green)

• Has four light chains (blue)

Myosin II Mechanism

• MLCK (myosin light chain kinase) phosphorylizes myosin light chains

• Drives polymerization of myosin by

  • initiating extension of their tails

  • activating actin-binding domains on head

• 15-20 myosin II form a bipolar filament 

  • Myosin II thick filament 

• Myosin ll doesn’t carry cargo

  • Generates contractile forces needed for many cellular activities

Myosin II Bipolar Thick Filament

• Has myosin motor heads on both sides of a bare patch (zone of myosin tails) 

• Motor heads are exposed to be associated with actin filaments

Myosin II Function in Skeletal Muscle Fibers

• Sarcomeres: Structure where myosin II thick filaments associated with thin actin filaments

• (+) of actin are fixed to Z-discs within the sarcomere 

• Between parallel actin fibers, myosin thick filaments are present 

• They’re also attached to Z-discs, but with titin 

  • It’s a giant molecular spring 

• During muscle contraction, myosin thick filaments interact with actin to move the Z lines closer together 

Muscle Contraction Mechanism

• Myosin heads associate with actin filaments 

• They get pulled past myosin toward the middle 

• Occurs by the cyclical association of actin filaments with myosin motor heads

  • Myosin head cycles through ATP binding and hydrolysis

  • Allows it to move along actin filaments 

  • Moves towards (+) end 

• Causes sarcomere shortening without changing any filament length 

• The process is calcium-dependent 

Calcium Dependence of Muscle Contraction

• Allows for exposure of myosin binding sites along actin filaments 

• After contraction Ca++ dissociates from actin filaments 

• Myosin heads then release the actin 

• The filaments slide past eachother to allow for muscle relaxation

Muscle Contraction: Chemical and Mechanical Energy

• Muscle contraction involves converting chemical energy into mechanical 

• This is mediated by myosin 

• It undergoes a series of conformation changes (Mechanical) regulated by ATP binding/hydrolysis (chemical)

• The steps of both cycles are interlinked to form the myosin cycle 

Cycle for Single Myosin Motor Head

1. Myosin is attached to actin 

2. ATP binding to myosin releases actin

3. ATP is hydrolyzed into ADP and Pi by myosin head

   1. Changes myosin conformation returning it to relaxed

4. Release of Pi increases affinity of myosin head for actin

   1. Allows binding

5. Release of ADP from myosin head changes conformation

• Since myosin is attached to actin, it pulls the filament

• Puts cycle back in step 1

• ATP binding will then release myosin from actin again 

• This cycle repeats many times during muscle contraction 

• One ATP molecule binding/hydrolysis moves the myosin motor a few nm along the actin track 

Myosin V

Powers intracellular trafficking of cargo along actin 

Myosin V: Melanosomes

• Melanosomes are membrane-enclosed organelles containing melanin in melanocytes (skin cell type) 

• Each melanocyte has several dendrites stretching to connect with many keratinocytes

• Incorporation of melanin into the keratinocytes of skin cells and distribution of this pigment protects cell’s DNA from UV damage

  • Tanning

• Myosin V distributes melanosomes along actin filaments

Loss of myosin V function in animals

• Leads to a phenotype called the dilute phenotype

• Pigments associated with fur colour are not ditributed into the fur

• Resulting colour is diluted

In-vitro study of myosin movement

• Myosin proteins are attached by their tails to a microscope slide 

• Fluorescently-labelled actin filaments can be applied to the slide with addition of ATP

• The chemical cycling of ATP binding, hydrolysis, ADP, and Pi power a mechanical cycle visible under microscope 

• Seen as movement of fluorescent actin filaments 

Rates of Myosin Protein Movement

• Varied with different myosin proteins 

• Can range from 0.2-60 micrometers/second

• Rate depends on cycle of ATP nucleotide binding and hydrolysis 

• This varies with 

  • Rate of ATP hydrolysis by ATPase in myosin head

  • The proportion of time myosin is bound to actin filament due to affinity

• Myosin V spends 90% of cycle bound to actin

• Myosin II spends 5% 

• Myosin V will move more slowly in comparison to Myosin II

Myosin Step Size

• Depends on lever arm length

• This is the distance by which the power stroke propels myosin 

• Myosin V lever is 3x longer than myosin II 

• Step size 

  • Myosin II: 7nm

  • Myosin V: 36nm

• Cargo-carrying proteins (myosin V) move in hand-over-hand fashion 

  • Trailing myosin head detaches from actin 

  • Gets propelled towards the (+) end of actin during power stroke of leading head

  • The trailing head becomes the new leading head