Module 2: Cytoskeletal System I

Cytoskeletal Systems

• network of interconnected filaments and tubes that extends throughout the cytosol of a cell.

it helps:

• maintain shape and organization of the cell

• involved in cell motility

• transport materials inside the cell

• plays a role in cell division

its not rigid - it can rapidly change and reorganize based on what the cell needs

3 elements of the cytoskeletal system

1. Microtubules

2. Microfilaments

3. Intermediate Filaments

Microtubules

• largest cytoskeletal element (25 nm wide)

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

• contains alpha and beta tubulin monomers

• Consists of GTP (nucleotide substrate)

Microfilaments

• smallest cytoskeletal element (7 nm wide)

• made of actin proteins

• help maintain and change cell shape

• involved in cell movement (like crawling or contracting)

• play roles in endocytosis (take in materials) and exocytosis (release materials)

• form the contractile ring that helps split cells during division (cytokinesis)

Intermediate Filaments

• 8-12 nm wide (medium size)

• made of various proteins such as keratin, vimentin, neurofilaments, and lamin)

• provide mechanical strength

• give structural support to cells

• help anchor organelles in place

Other polymer network in cells

Septins

• proteins that form another network in cells

• involved in cytokinesis (cell division)

• vesicle trafficking (moving materials in vesicles)

• cell compartmentalization (organizing different parts of the cell)

Prokaryotic Cytoskeletal Systems (3 elements)

bacteria and archaea have cytoskeletal-like proteins that function similarly to eukaryotic cytoskeletal elements:

1. MreB

   • similar to actin

   • involved in DNA segregation and maintains cell shape

2. FtsZ

   • similar to tubulin

   • forms Z-ring to regulate cell division

3. Crescentin

   • similar to intermediate filaments

   • regulates cell shape

Disassembly and assembly in cytoskeleton

• highly dynamic structure

• continuous builds up and breaks down

• microtubules and microfilaments change rapidly to allow movement and reshaping

• drugs and proteins can stabilize or break these structures

Functions of microtubules

Cytosolic MTs

1. organization and maintenance of animal cell shape and polarity

2. chromosome movement

   • form mitotic spindle during cytokinesis

3. intracellular transport

   • act as tracks for motor proteins to move materials

Axonemal MTs

• cell motility (via cilia and flagella)

2 types of microtubules

1. Cytoplasmic MTs

   • penetrates throughout the cytosol (fluid part of the cell)

   • more dynamic (constantly growing/shrinking)

   • contains only A tubules → exist as singlets

   • maintains axons

   • forms the mitotic spindle

   • support and shape the cell

   • act as tracks for vesicle movement

2. Axonemal MTs

   • more stable and highly organized

   • contains both B & C tubules → exist as doublets/triplets

   • found in specialized structures that help the cell move

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

     • Flagella - long, whip-like structure that enables motility in cells

     • Basal Bodies - anchors cilia and flagella to the cell

Tubulin Heterodimers

• basic building block of microtubules

• each microtubule is a hollow tube made up of 13 protofilaments

• each protofilament is made of repeating tubulin heterodimers

Composed of:

1. Alpha-Tubulin

2. Beta-Tubulin

   • these stick together noncovalently (using weak forces like hydrophobic interactions)

   • once they form a pair, they do not fall apart

2 GTP-binding sites in tubulins

Each tubulin dimer has 2 GTP-binding sites:

1. Alpha-Tubulin

   • always bound to GTP

   • the GTP is used for stability, not for energy

2. Beta-Tubulin

   • can bind and hydrolyze GTP to GDP

   • this switch controls whether the microtubule grows or shrinks

while tubulin dimers can be added or removed from microtubules, the alpha/beta pair remains intact

Structure of tubulin subunit

• alpha and beta tubulins form a heterodimer, which is the basic unit of microtubules

• they look similar in 3D shape, but are only 40% identical in amino acid sequence

3 structural domains of tubulin

1. N-terminal GTP-Binding Domain

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

   But:

   • alpha tubulin always holds GTP permanently (does not get used or exchanged), its for stability

   • beta tubulin binds GTP that can be hydrolyzed (changed to GDP, which controls microtubule growth and shrinkage

2. Central Domain (Colchicine-Binding Site)

   • binds the drug colchicine

   • colchicine blocks tubulin from assembling into microtubules

   • this domain helps control microtubule dynamics

3. C-terminal Domain (MAP-Interacting Region)

   • this is where MAPs (microtubule-associated proteins) bind

   • MAPs regulate microtubule stability, dynamics and intracellular transport

Microtubule Polarity

• all tubulin dimers are arranged in the same direction, creating microtubule’s inherent polarity (defined plus and minus end)

Plus End (+)

• exposed beta-tubulin

• more dynamic → rapid polymerization and depolymerization

• kinesin (motor protein) moves toward this end

• where most of the microtubule growth occurs

Minus End (-)

• exposes alpha-tubulin

• less dynamic → grows slowly or stays stable

• often anchored at the MTOC (microtubule-organizing center)

• dynein (motor protein) moves toward this end

3 different structures that microtubules can form

1. Singlet Microtubules

   • a simple hollow tube made of 13 protofilaments

   • found in the cytoplasm of most eukaryotic cells

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

2. Doublet Microtubules

   made of:

   • one complete A tubule (13 protofilaments)

   • one incomplete B ring (10-11 protofilaments) attached

     • found in cilia and flagella

     • forms the axoneme (structural core that allows movement)

     • involved in cell motility and fluid flow across surfaces

3. Triplet Microtubules

   made of:

   • one complete A tubule

   • two incomplete B & C rings (10-11 protofilaments)

     • found in centrioles and basal bodies

     • serves as MTOCs (microtubule-organizing centers)

     • helps organize spindle fibers during mitosis

     • act as anchor for building cilia and flagella

Tubulin Polymerization

• microtubules are built by linking together alpha/beta tubulin heterodimers

• process is dynamic (microtubules are constantly growing/shrinking)

• requires GTP and Mg²+ for proper assembly

How do microtubules assemble?

1. Nucleation (Lag Phase)

   • tubulin dimers group together to form small oligomers (seeds for microtubule growth)

   • slow step because it takes time to form these stable seeds

   • called the lag phase because polymerization starts slowly and the initial structures are unstable

2. Elongation (Rapid Growth Phase)

   • once seeds are formed, tubulin dimers are quickly added to both ends of the microtubule

   • the plus end (+) grows faster

   • the minus end (-) is anchored at the MTOC, growing slowly or remains stable

3. Dynamic Instability (Plateau Phase)

   • microtubules undergo continuous assembly and disassembly

   • GTP-bound tubulin at the plus end helps stabilize and promote growth

   • GDP-bound tubulin causes the microtubule to shrink (depolymerize)

   • eventually, the system reaches a steady state, where the rate of polymerization = the rate of depolymerization

Critical Concentration (Cc)

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

1. Cytoplasmic MTs have GTP-tubulin, while axonemal MTs have GDP-tubulin

2. Cytoplasmic MTs can have A, B, or C tubules, while axonemal MTs only have A tubules

3. Cytoplasmic MTs are found in cilia, while axonemal MTs are found in flagella

4. Cytoplasmic MTs exist as singlets, while axonemals MTs exist as doublets or triplets 

5. Cytoplasmic MTs are comprised entirely of alpha-tubulin, while axonemal MTs are comprised entirely of beta-tubulin

Cytoplasmic MTs exist as singlets, while axonemals MTs exist as doublets or triplets 

Treadmilling

• occurs in microtubules

when the free tubulin concentration is:

• above Cc at the plus end → tubulin can be added

• below Cc at the minus end → tubulin is removed

so,

• one end is growing

• the other end is shrinking

• the microtubule stays the same length, but subunit flow through

Why does treadmilling occur?

• the plus end has a lower Cc → needs less free tubulin to grow

• the minus end has a higher Cc → needs more free tubulin to grow

What happens if Cc changes?

• if Cc decreases → minus end can now grow

  • both ends grow and treadmilling stops

• if Cc increases → plus end cannot grow

  • both ends shrink and treadmilling stops

3 scenarios for microtubule disassembly

if free tubulin concentration is

1. below Cc of both plus and minus ends

   • the microtubule shrinks

2. above Cc for plus end, but below Cc for minus end

   • treadmilling occurs (length stays the same)

3. above Cc for both plus and minus ends

   • the microtubule grows

What affects microtubule stability?

• GTP hydrolysis of beta tubulin

Each tubulin dimer binds 2 GTPs:

• alpha-tubulin: binds GTP permanently

• beta-tubulin: hydrolyzes GTP into GDP after added to microtubule

Why this matters:

• GTP-bound tubulin is stable and promotes growth

• GDP-bound tubulin is unstable and leads to shrinkage

• the switch from GTP to GDP causes dynamic instability

Dynamic Instability Model

• describes how microtubules switch between growth & shrinkage

Growing Microtubules

• when GTP-bound tubulin adds to plus end, it forms a GTP cap

• this cap stabilizes the microtubule and prevents shrinking

Shrinking Microtubules (Catastrophe)

• if the GTP cap is lost (because GTP is hydrolyzed to GDP), the end becomes unstable

• the GDP-bound tubulin falls apart easily

• this leads to rapid shrinkage, called a catastrophe

Rescue (Regrowth)

• if new GTP-tubulin is added before complete depolymerization, the microtubule can regrow

Tubulin concentration is above the critical concentration of the (+) end, but below that of the (-) end. What will happen to microtubule assembly?

1. The microtubule will get longer

2. The microtubule will get shorter

3. The microtubule will lose subunits off both the (+) / (-) end

4. The microtubule (-) end will get longer, but the (+) end will get shorter

5. Treadmilling will occur 

Treadmilling will occur 

GTP Cap

• forms when GTP-bound tubulin is added to the plus end

• the cap stabilizes the microtubule

• prevents shrinking

• if cap is lost → the end becomes unstable and shrinks

• if cap is present → microtubule is stable and grows

How does GTP-tubulin control microtubule stability?

• microtubules grow and shrink based on the balance between adding GTP-tubulin and hydrolyzing it to GDP-tubulin

High GTP-Tubulin Concentration

• tubulin is added quickly to the plus end

• large GTP cap forms

• microtubule remains stable and grows

Low GTP-Tubulin Concentration

• tubulin addition slows down

• GTP cap shrinks

• if GTP is hydrolyzed faster than new tubulin is added, the cap is lost

• microtubule becomes unstable and shrinks (catastrophe)

Microtubule Catastrophe

• happens when the GTP cap is lost at the plus end

• without the GTP cap, the microtubule becomes unstable

• the GDP-bound tubulin falls apart easily

• result: the microtubule shrinks rapidly

Microtubule Rescue

• if free GTP-tubulin becomes available again, it can bind to plus end

• it must be added before complete depolymerization

• this rebuilds the GTP cap, making the microtubule stable again

• result: the growth resumes

4 factors that facilitate microtubule assembly

• microtubules do not grow randomly - they begin at special sites in the cell called the MTOCs

  1. Centrosome

     • main MTOC in animal cells

     • found near the nucleus

     • helps organize microtubules and control where they grow

     • contains 2 centrioles (cylindrical structures)

  2. Centrioles

     • each centriole is made of 9 triplet microtubules

     • surrounded by PCM - area that helps start microtubule growth

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

     • without centrioles, poorly-organized spindle fibers and inefficient chromosome segregation

  3. Y-Tubulin

     • specialized form of tubulin

     • found only in the centrosome

     • helps start the process of microtubule growth (nucleation)

     • differ from alpha/beta tubulin that builds microtubules

  4. Y-Tubulin Ring Complex (y-TURCs)

     • large ring-shaped protein structures made of y-tubulin

     • serves as a template for microtubule polymerization

     • anchors the minus end and allows plus end to grow outwards

     • if y-TURCs are lost → microtubule nucleation cannot occur

       • cells fail to organize microtubules for intracellular transport, spindle formation and cell division

What could you add to a reaction tube to stop microtubules from treadmilling?

1. Tubulin-GTP dimers

2. Tubulin-GDP dimers

3. GTP

4. GDP

5. Alpha-tubulin

Tubulin-GTP dimers

MTOCs (Microtubule-Organizing Centers)

• structures inside the cell that initate microtubule growth

• uses y-tubulin ring complexes to help build and organize microtubules

Polarity:

• minus end is anchored at MTOC

• plus end grows outwards toward the edges of the cell

Growth Site:

• plus end is where tubulin is added/removed

  • main site for dynamic instability

• minus end stays anchored to prevent shrinkage

Dynamic instability at the Plus End:

• if GTP-tubulin is added faster than its hydrolyzed to GDP, the microtubule grows

• if GTP is hydrolyzed faster than new tubulin is added, the microtubule shrinks

• allows for rapid reorganization of microtubule network in response to cellular needs

3 proteins that regulate microtubule stability

• microtubules constantly grow and shrink → dynamic

• regulated by microtubule-binding proteins

  1. Microtubule-Associated Proteins (MAPs)

     • bind along the sides of microtubules

     • stabilizes microtubules by organizing them into bundles

     • some MAPs link microtubules to other cell structures, giving extra support

  2. +TIP Proteins

     • regulates plus-end dynamics

     • helps stabilize growing tip

     • prevents catastrophe (sudden shrinkage)

     • guides microtubules to target sites

  3. Microtubule-Destabilizing / Severing Proteins

     • breaks down microtubules

       Catastrophin:

       • causes rapid shrinkage by destabilizing the plus end

       Severing Proteins:

       • cut microtubules into smaller fragments to easily depolymerize