Microtubules (Week 5-1: Cytoskeletal System I)

Cytoskeletal systems

organized network of interconnected filaments and tubules that extends throughout the cytosol of a cell

• maintain the shape and organization of the cell (highly structured)

• involved in cell motility → allows for movement via structures like cilia, flagella, and lamellipodia

• plays a role in cell division → microtubules form the mitotic spindle, which is crucial for chromosome separation

• constantly being reorganized in response to cellular needs, enabling rapid adaptation to environmental changes

cytoskeletal elements

1. microtubules

2. microfilaments

3. intermediate filaments

microtubules

• composed of tubulin subunits

• 25 nm in diameter

• provide structural support

• acts as tracks for motor proteins (kinesin and dynein)

• plays a role in cell division, intracellular transport, and cell motility

microfilaments

• composed of actin subunits

• 7 nm wide

• involved in cell shape, motility, exocytosis, endocytosis and cytokinesis

• largest structural elements of the cytoskeleton

• hollow, cylindrical structure with a wall consisting of 13 protofilaments

• composed of alpha and beta tubulin dimers

• consist of nucleotide substrate, GTP

intermediate filaments

• variable composition (keratin, vimentin, neurofilaments, lamins)

• 8-12 nm wide

• provide mechanical strength, structural support and anchor organelles

other polymer networks in cells

septins: network composed of proteins that are involved in cytokinesis, vesicle trafficking and cell compartmentalization

prokaryotic cytoskeletal systems

1. MreB

2. FtsZ

3. Crescentin

MreB

• involved in DNA segregation and maintaining cell shape

• similar to microfilaments

FtsZ

• regulates cell division by forming a z-ring at the site of cytokinesis in bacteria

• similar to microtubules

crescentin

• regulator of cell shape

• similar to intermediate filaments

cytoplasmic microtubules

Penetrate through the cytosol and are responsible for a variety of functions:

• Maintaining axons

• Formation of mitotic and meiotic spindles

• Maintaining or altering cell shape

• Placement and movement of vesicles

axonemal microtubules

• Include the organized and stable microtubules found in structures such as:

• Cilia → short, hair-like projections that facilitate movement or fluid flow

• Flagella → long, whip-like structures enabling motility in cells like sperm

• Basal bodies → which anchor cilia and flagella to the cell

cilia

• short, hair like projections that facilitate movement or fluid flow

flagella

• long, whip-like structure enabling motility in cells like sperm

basal bodies

• which anchor cilia and flagella to the cell

tubulin heterdimer

• protein building blocks of microtubules

• each protofilament in microtubules consist of repeating subunits of tubulin heterdimer

• composed of alpha and beta tubulin

• both subunits bind noncovalently to form a stable ab-heterodimer (which does not normally dissociate)

2 GTP-binding sites of tubulin dimers

• alpha tubulin → always bound to GTP, not hydrolyzed

• beta tubulin → binds to GTP, which can be hydrolyzed to GDP

structural domains of tubulin

1. N-terminal GTP binding domain

2. central domain

3. c-terminal

N-terminal GTP-binding domain

• both alpha and beta tubulin have an N-terminal that binds GTP

• however only the GTP bound to beta tubulin is hydrolzed, while the GTP in alpha tubulin gives structural stability

central domain

this domain binds colchicine (drug that disrupts microtubule polymerization by preventing tubulin assembly)

c-terminal

this domain is responsible for interactions with MAPS (microtubule-associated proteins)

heterodimer polarity

• all alphabeta-heterodimers are oriented in the same direction, creating a polarized structure

• the minus end is exposed to alpha-tubulin, which anchors to MTOC (microtubule-organizing center)

• the plus end is exposed to beta-tubulin, where polymerization occurs, allowing microtubule growth

microtubule polarity

• polarized structures that have inherent polarity due to orientation of tubulin dimers

• plus end is exposed to beta tubulin, more dynamic (rapid polymetiztion and depolymerizarion), kinesis motor proteins move toward this end

• minus end is exposed to alpha tubulin, less dynamic as it is anchored to MTOC, dynein motor proteins move toward this end

microtubule structural variation

MTs can assemble into different structural forms depending on their function within the cell

1. singlet microtubules

2. doublet microtubule

3. triplet microtubule

singlet microtubule (variation of structures)

• simple, hollow tube made up of 13 protofilaments

• found in the cytoplasm of most eukaryotic cells

• plays a role in intracellular transport, mitotic spindle formation and cell shape maintenance

doublet microtubule (structural variation)

• contains one complete 13-protofilament A tubule

• has additional incomplete ring (B-tubule) with 10-11 protofilaments

• found in cilia and flagella, forming the axoneme (structural core of these motile organisms)

• essential for cell movement and fluid flow across surfaces

triplet microtubule (structural variation)

• contains one complete 13-protofilament A tubule

• has two additional incomplete (B and C tubules) with 10-11 protofilaments each

• found in centriole and basal bodies, which serve as MTOCs

• plays a critical role in spindle formation during mitosis and serve as the base for cilia and flagella assembly

microtubule formation

• microtubules form through the reversible polymerization of alphabeta- tubulin heterodimer at their ends

• process is highly dynamic and requires GTP and mg2+ for proper assembly

steps of microtubule assembly

1. Nucleation (lag phase)

2. elongation (rapid growth phase)

3. dynamic instability (plateau phase)

nucleation (lag phase)

• tubulin dimers accumulate into small oligomers, which act as seeds for microtubule growth

• this is a slow step because it requires formation of small tubulin oligomers (rate-limiting step)

• considered lag phase because polymerization occurs slowly as tubulin dimers assemble into short, unstable structures

elongation (rapid growth phase)

• once a nucleation seed is formed, tubulin dimers are added to either ends, extending the microtubule (faster phase)

• the two ends of an MT differ chemically, plus end grows faster, while minus end is anchored at MTOC

dynamic instability (plateau phase)

• microtubules undergo constant assembly and disassembly

• some MT continue to grow, while others shrink (dynamic instability)

• the GTP bound tubulin at the plus end stabilizes growth, while GDP-bound tubulin leads to depolymerization

• MTs reach a steady length where the rate of polymerization = rate of depolymerization

• this occurs when the mass of MTs reach a point where the amount of free tubulin is diminished

• at this point, the assembly is balanced by disassembly

critical concentration

• the tubulin dimer concentration at which microtubule assembly and disassembly are exactly balanced

• if free tubulin concentration is above Cc → microtubules grow

• if free tubulin concentration is below Cc → microtubules shrink

what distinguishes cytoplasmic and axonemal microtubules?

cytoplasmic MTs exist as singlets, while axonemal MTs exist as doublets or triplets

treadmilling

• when free tubulin concentration is above Cc at the plus end but below Cc for the minus end

• tubulin dimers are continuously added at the plus end

• tubulin dimers are simutaneously removed from minus end

• the microtubule remains at relative constant length, but tubulin subunits cycle through the structure

what happens if Cc changes?

• If Cc₋ (minus-end) decreases, treadmilling may stop, because both ends may start growing

• If Cc₊ (plus-end) increases, treadmilling may stop, because both ends may start shrinking

3 scenarios for microtubule disassembly

if free tubulin concentration is

1. below Cc of both plus and minus ends: subunit removal at both ends

2. above Cc of plus end but below Cc of minus end: treadmilling

3. above cc of both plus and minus end: subunit addition at both ends

what affects microtubule stability

GTP hydrolysis contributes to the dynamic instability of microtubules

• each tubulin heterodimer binds 2 GTP molecules

• alpha tubulin binds GTP permanently

• beta tubulin binds GTP temporarily, which can be hydrolyzed to GDP after it is added to the microtubule

• GTP bound tubulin dimers have a high affinity for other dimers and promote polymerization

• GTP is needed to promote heterodimer interactions and additions to MTs, but its hydrolysis is not required for MT assembly

dynamic instability model

• describes how microtubules switch from growth and shrinkage

• growing microtubules contain GTP tubulin at the plus end

• these form a stable GTP cap that prevent depolymerization

• when GTP at the plus end is hydrolyzed to GDP, the microtubule becomes unstable

• GDP-bound tubulin has a lower affinity for other dimers, causing rapid depolymerization

• this rapid disassembly is called catastrophe

• if new GTP-tubulin dimers are added before complete depolymerization, the microtubule can resume growth

• the GTP cap at the plus end prevents subunit removal and stabilizes the growing microtubule

• if the GTP cap is lost, the microtubule undergoes catastrophe

catastrophe

rapid depolymerization when the GTP cap is lost

GTP-tubulin and dynamic instability

• high GTP tubulin concentration→ fast addition of tubulin → large GTP cap forms → microtubule remains stable and grows

• low GTP tubulin concentration → tubulin addition slows down → GTP cap shrinks —> microtubule becomes unstable

• if the concentration falls, the rate of tubulin addition decreases

• if GTP concentration falls too low, the rate of GTP hydrolysis exceeds the rate of tubulin addition, leading to cap shrinkage

microtubule catastrophe

• the switch from growth to shrinkage

• if the GTP cap disappears altogether, the microtubule becomes unstable

• GDP bound tubulin is more likely to dissociate, leading to rapid depolymerization (shrinkage)

microtubule rescue

• the sudden return to polymerization

• if free GTP tubulin becomes available again, it can rebind to the plus end

• this restores the GTP cap, allowing the microtubule to switch back to growth

what factors facilitate microtubule assembly?

• centrosome

• y-tubulin

centrosome

• primary MTOC in animal cells

• located near the nucleus

• associated with 2 centrioles surrounded by pericentriolar material

• centriole walls are composed of 9 triplet microtubules

• they are oriented at right angles (perpendicular to each other)

• cells without centrioles → poorly organized spindle fibers

y-tubulin

• specialized type of tubulin that is found only in centrosomes

• essential for microtubule nucleaton

• y-tubulin acts as a template for MT polymetization, whereas alpha and beta tubulin form microtubules

y-tubulin ring complexes

• nucleate the assembly of new microtubules away from the centrosome

• they serve as a template for microtubule polymerization

• ensuring the minus end remains anchored, while plus end extends outward for growth

• if y-TuRCs are lost, microtubule nucleation cannot occur

what could you add to a reaction tube to stop microtubules from tread milling?

tubulin GTP dimers

MTOCs (microtubule organizing centers) organization and polarity

• MTOCs initiate microtubule polymerization, using y-tubulin ring complexes

• the minus end is anchored at the MTOC, while the plus end extends outwards

• this fixed polarity is maintained because tubulin dimers are only added to plus end, preventing depolymerization, while the minus end remains anchored

microtubule binding proteins regulate microtubule stability

1. MAPs (microtubule associated proteins)

2. +TIP proteins

3. microtubule-destabilizing/severing proteins

MAPs (microtubule binding proteins)

• bind along the microtubule wall

• help stabilize microtubules and organize them into bundles, preventing depolymerization

• some MAPs cross-link microtubules with other filaments and cellular structures for structural support

+TIP protein (+ end tubulin interacting proteins)

• regulate plus end dynamic

• stabilize plus end by binding and protecting the growing microtubule tip

• prevents catastrophe and help capture microtubule at target sites

microtubule de-stabilizing/severing proteins

• promote microtubule depolymerization or fragmentation

• catastrophin is a type of protein that induces rapid shrinkage by destabilizing the plus end

• severing proteins cut microtubules into smaller fragments, which then depolymerize