Module 7: Microtubule Dynamics

Microtubule Dynamics and Regulation

Dynamic Instability

• Microtubules exhibit dynamic instability, where growing ends stochastically transition from growing to shrinking.

• This differs from actin filaments, which treadmill at steady state, maintaining a constant length.

• In a microtubule population, growing and shrinking microtubules can coexist.

In Vitro vs. In Vivo

• Early studies demonstrated dynamic instability in vitro.

• Skepticism existed regarding the physiological relevance of this behavior.

• Researchers injected purified, fluorescently labeled tubulin into living cells.

• Observed dynamic instability in living cells, validating in vitro findings.

Visualizing Microtubules in Living Cells

• Microtubules in mitotic cells are highly dynamic.

• Interphase microtubules are less dynamic.

• Catastrophe promoting factors are more active in mitosis.

• Tenfold increase in catastrophe frequency occurs between mitotic and interphase cells.

Parameters of Dynamic Instability

• Four parameters can be measured:

  • Growth rate: microns \/ minute

  • Catastrophe frequency: transitions from growing to shrinking

  • Shrinkage rate: microns \/ minute

  • Rescue frequency: transitions from shrinking to growing

Regulation by Cellular Proteins

• Microtubules have intrinsic properties that drive transitions.

• Cellular proteins plug in to regulate these parameters.

• This regulation controls when and where filament systems are set up in cells.

GTP Hydrolysis and the GTP Cap

• Tubulin binds and hydrolyzes GTP.

• If the addition rate is fast enough during polymerization, a GTP cap forms.

• The depth of the GTP cap may be as little as one subunit.

• A GTP cap promotes polymerization.

• Loss of the GTP cap triggers a transition to the shrinking state.

Protofilament Structure and Energy Storage

• Alpha and beta tubulin heterodimers have some flex like a joint.

• Proteins sense this flex and the confirmation of the protofilament.

• GTP-bound heterodimers are straighter with less flex.

• GDP-bound heterodimers can bend or flex more.

• Microtubules with a GTP cap contain stored energy.

• Protofilaments want to curl out but are held straight by the GTP cap.

• Losing the GTP cap causes protofilaments to peel away and results in an "explosion".

• This is analogous to a banana peeling.

Mechanical Work

• Microtubules can perform mechanical work.

• Holding onto a depolymerizing microtubule can pull things.

• Polymerizing microtubules can push against barriers or chromosomes, generating force.

• Motor proteins bind and hydrolyze ATP, which leads to conformational changes and movement along the filament.

• Catastrophe factors can be motor proteins that don't walk.

• These factors bind to protofilaments, hydrolyze ATP, and curl the protofilaments.

• This curling triggers catastrophes, even in the presence of a GTP cap.

GTP Hydrolysis Rate

• The basal rate of GTP hydrolysis by tubulin is on the order of growing and shrinking rates.

• Nonhydrolyzable analogs of GTP can be added to create microtubules that don't hydrolyze, remaining in a stable, GTP-bound state.

Microtubule Polarity

• Microtubules have polarity: a plus end and a minus end.

• Plus ends are more dynamic: they grow and shrink more frequently.

• Minus ends are less dynamic.

• Plus ends grow faster and shrink faster compared to minus ends.

• Polarity is NOT related to charge.

• The actual charge of the tube is negative.

Disordered Tails and Post-translational Modifications

• Microtubules have disordered tails rich in glutamic acids.

• These tails are located at the C terminus of both alpha and beta subunits.

• These tails are analogous to histone tails.

• Histone tails are positively charged.

• Microtubule tails are negatively charged.

• These charged tails undergo post-translational modifications (PTMs).

• This is referred to as the tubulin code.

• PTMs change the properties of how things interact with microtubules.

• Detyrosination: An enzyme removes tyrosine residues from the alpha tubulin end, which changes interaction properties.

Charge-Based Interactions

• Charge-based interactions are important for microtubule function.

• The NDC80 complex (at the kinetochore) has a positively charged disordered tail.

• This tail interacts with the negatively charged surface of the microtubule.

• This electrostatic interface is vital for kinetochore-microtubule interactions.

• Kinases can phosphorylate residues in the tail to reduce affinity.

  • In vitro studies have shown that phosphorylation reduces affinity.

Overall Charge

• The microtubule is essentially a negatively charged tube.

• Actin is also negatively charged but lacks disordered tails.

Cellular Organization

• Microtubules are organized in cells.

• Textbook view: radial array of microtubules coming out of the centrosome with plus ends oriented towards the plasma membrane.

• Centrosomes are positioned next to the nucleus.

• Microtubules organize into a spindle during cell division.

• They form specialized structures, e.g., the nine plus two arrangement in flagella and cilia.

Nine Plus Two Microtubule Arrangement

• Found in flagella and cilia.

• Consists of nine doublet microtubules surrounding two single microtubules.

• Doublet microtubules: one complete tube and one incomplete tube attached to the side.

• Example: sperm with flagella in the fallopian tubes.

• Fallopian tubes have multiciliated cells that move sperm.

• Motor activity slides microtubules within the arrangement, mediating beating motion.

Centrosomes

• Spindle poles contain centrosomes.

• Centrosomes nucleate microtubules.

• They have an interesting internal arrangement.

• Centrioles: specialized structures within centrosomes with a pinwheel arrangement.

• Centrioles consist of triplet microtubules.

• Mother and procentrioles are arranged at 90 degrees to each other.

• Pericentriolar material (PCM): highly disordered proteins that localize to the centrosome.

• These ordered microtubule structures (triplets and nine plus two) are more stable than spindle microtubules.

• The centrosome is a key site of microtubule nucleation.

Gamma Tubulin Ring Complex

• Centrioles recruit the gamma tubulin ring complex.

• Gamma tubulin is enriched in the centrosome.

• The complex templates the microtubule.

• Microtubules have 13 protofilaments.

• The gamma tubulin ring complex has 13 subunits with gamma tubulin exposed at the end.

• Similar to the Arp2/3 complex: provides template subunits for nucleation.

• The gamma tubulin ring complex structure was solved in 2020 by cryo-EM.

• It has 13 exposed gamma tubulin subunits arranged in a cone shape.

• An actin subunit is present as a "plug", with unknown function.

• A conformational change within the complex promotes nucleation.

Gamma Tubulin's Role

• The gamma tubulin ring complex sits at the minus end and caps it.

• Microtubules nucleated from the centrosome have suppressed minus-end dynamics.

• Severing enzymes can cut the lattice, detaching it from gamma turk and allowing dynamics.

• When attached, gamma turk caps the minus end, similar to Arp2/3.

Search and Capture

• The spindle is formed through a search and capture mechanism.

• Searchers: microtubules nucleated by centrosomes.

• Capturers: kinetochores.

• DNA is replicated in S phase and held together by cohesins.

• Condensed chromosomes have sister centromeres.

• Kinetochores: large proteinaceous structures built at centromeres that bind microtubules.

• NDC80: A complex with a positively charged tail is enriched in the outer kinetochore and attaches to microtubules.

• Centrosomes duplicate in S phase.

• In early mitosis, centrosomes migrate to opposite sides of the nucleus.

• The nuclear envelope dissolves, and microtubules search for targets.

• This leads to spindle assembly and chromosome segregation.

Regulation of Centriole Duplication

• Centriole duplication is highly regulated to maintain two per cell.

• Process begins at the end of mitosis.

• Kinases such as polo-like kinase are involved.

• Overexpression of these kinases can result in too many centrosomes.

• Spindles can form without centrosomes.

• Too many centrosomes can lead to chromosome missegregation.

• Multipole spindles can form, especially in cancer cells.

• Fail-safe mechanisms, such as clustering, can occur: multiple centrosomes cluster together to form a bipolar structure.

• Blocking clustering mechanisms can drive cancer cells to undergo massive chromosome missegregation and die.

Computational Model of Search and Capture

• A model was created to test how long search and capture takes.

• Simulation with centrosomes nucleating microtubules took 511 minutes to achieve alignment.

• Mitosis typically takes 20 minutes to an hour.

• Increasing microtubule numbers searching only reduced time to 200 minutes.

• Biased search and capture: When centrosomes orient microtubules towards chromosomes, the model works more efficiently.

Microtubule Branching

• Analogous to actin branching via Arp2/3.

• Augment complex: binds to preexisting mother microtubules and recruits gamma turk.

• Gamma turk nucleates a daughter microtubule off of the mother microtubule.

• Gamma tubulin is observed sitting on the mother microtubule.

• Daughter microtubules are nucleated from the gamma tubulin site.

• The branch hits the membrane and then catastrophes and goes back.

Augment Complex

• The augment complex colocalizes with gamma tubulin and nucleates the daughter microtubule.

• Steps: Augment complex binds to a microtubule.

• Fifteen seconds later, gamma turk is recruited.

• Fifteen seconds later, the daughter microtubule is nucleated.

• The process takes 30 seconds from beginning to end.

Microtubule Lifetimes

• Astro microtubules are very dynamic and have short lifetimes (30-60 seconds).

• Kinetochore microtubules (attached to kinetochores) are stabilized and can last for 15 minutes.

Branching and Kinetochore Fibers

• Consider two populations: microtubules attached to kinetochores, and those that are not.

• Nonkinetochore microtubule catastrophes before daughter microtubules can be made.

• Kinetochore microtubules live long enough to become mothers and initiate branching.

• Augment complex is important for building kinetochore fibers.

• Kinetochores attach to 10-20 microtubules; 46 pairs of kinetochores exist.

• The augment complex is essential for building k fibers.

Biased Search and Capture

• Kinetochore microtubules branch.

• The bias towards microtubules already associated with kinetochores occurs because they live long enough.

• Nucleation is closer to the target kinetochore.

• The nucleation is directionally biased: microtubules are born in the same direction as the mother.

• Molecular explanation for how you can bias a search and capture model.

Spindle Formation Without Centrosomes

• Bipolar spindles can form around DNA beads in frog egg extracts.

• These eggs lack centrosomes, centromeres, and kinetochores.

• The only requirement is that the DNA beads be 5kb.

RAN GTP Gradient

• A gradient of RAN GTP is generated around mitotic chromatin.

• This is due to the GEF (RCC1) being stuck on mitotic chromosomes.

• Microtubules that grow toward chromosomes are stabilized and live longer.

Nuclear Import and RAN

• RAN drives the directionality of nuclear import and export.

• GAP (Rnt1) accelerates hydrolysis to GDP.

• GEF (RCC1) opens up RAN; because there is 1000x GTP versus GDP, GTP rushes in to recharge RAN GTP.

• RCC1 is on the chromatin in the nucleus.

• GAP is on the cytoplasmic face of the nuclear core complex.

• RAM is GDP in the cytoplasm and RAM GTP in the nucleus.

• RAN GTP binds to import receptors (importins).

• This causes a conformational change that releases cargo into the nucleus.

• With exportins, RAN GTP promotes association with cargo, then it goes out; hydrolysis releases the cargo.

• RAN GTP is key to driving the directionality of nuclear import and nuclear export.

Serendipity in Science

• New faculty studied nuclear import.

• It turns out the signal is this.

• When the nuclear envelope breaks down, there isn't a the nuclear envelope anymore; but there is still RCC1 on the mitotic chromosomes, creating a locally high concentration of brand GTP; those creating a RAN GTP zone.

• Import receptors release regulators (spindle assembly factors), which bind, stabilize, and nucleate microtubules.

• When spindle assembly factors are bound to the import receptor, they are inactive; it diffuses over near the chromosomes, encounters RAM GTP, then releases the spindle assembly factor; and now it can start to make microtubules.

• Motor proteins organize and sort them to make the structure of the cell.

Nuclear Localization Signal

• Nucelar localization signals are charged.

• Some of these were being regulated because they hide this charged NLS, and preventing it from interacting with the microtubules; because what's the charge of the Micro tube will? Negative.

• And there's a number of different factors that look that are regulating in this fashion in mitosis.

Molecular Motors and Organelle Organization

• ER tubules are extended along microtubules by motor proteins (kinesins).

• Motor proteins can do "tip tracking" along the growing end of microtubules, generating force to extend the tubule.

• The ER is a vast network of tubules distributed throughout the cell.

• The Golgi is localized near the centrosome because it is positioned by dynein.

• Dynein is a motor protein that moves towards the minus ends of microtubules.

• Disrupting dynein function causes Golgi disbursement.

• Vesicles moving from the ER to the Golgi are moved by dynein; vesicles moving from the Golgi to the plasma membrane are moved by kinesins.

• Motors position organelles (ER, Golgi) and other cellular structures.

Squid Axons

• Woods Hole's MBL (Marine Biological Laboratory) is important for squid studies because of the large population.

• Squid giant axons: used to study nerve signaling.

• Axoplasm: the cytoplasm squeezed out of the axon.

• Removed cytoplasm, and then an empty tube of just membrane; and they could change the solution on the inside, and the outside; and look at what was basically required for, like, the action potential.

• Showed that you needed sodium on the outside, and when you triggered the simulus, then it would cause, ah, the sodium to flow in.

• By the way, was all figured out.

ER Lumen and Cytosol

• Proteins popped out of the ER lumen pass through the membrane.

• Signal sequences target and feed proteins through the membrane.

Membrane Orientation

• First stretch in membrane.

• Second loop in the cytosol.

• Signal peptidase clips the protein, which becomes packaged into a vesicle and sent to the Golgi apparatus, then to the plasma membrane.

Plasma Membrane Configuration

• Cytosolic face remains facing the cytosol.

• Extracellular world faces the sinusoidal space.

• Modifications like glycosylation occur in the ER lumen or Golgi, so sugars are on the cell's outside surface.

• Lipid bilayer leaflets maintain their orientation: cytosolic leaflet remains cytosolic.

• Flipases can flip leaflets, but generally, the cytosolic leaflet remains cytosolic when it becomes part of the plasma membrane.

• Soluble proteins deposited in the lumen are secreted.

• Proteins not engaging with the signal recognition particle remain cytosolic.

Protein Trafficking

• Membrane proteins journey from the endoplasmic reticulum to the plasma membrane.

• Proteins disperse throughout the ER membrane network and move to exit sites.

• Transport vesicles cluster and move towards the Golgi apparatus near the cell's center.

• Movement is mediated by dynein on microtubules.

• Vesicles then move from the Golgi, potentially containing transmembrane proteins that diffuse laterally within the plasma membrane upon fusion, or secreted proteins transiently visible.

• Depolarizing microtubules halts directed movement, causing diffusion.

• Vesicle coating aids membrane pinching and removal.