BIOLOGY 2B03 Chapter Notes - Chapter 7: Microtubule Nucleation, Polar Filament, Pericentriolar Material

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BIOLOGY 2B03 - Module 7 Lecture II

Microtubules are Built from Tubulin Dimers

● Comprised of 13 protofilaments arrayed in a circular pattern to form a very strong wall

○ Subunits of the filaments: dimers of alpha and beta tubulin proteins

● Protofilaments: staggered; a string of tubulin monomer appear to spiral up through the

microtubule like a spring

○ The ring formed by the protofilaments is visible under the electron microscope

image

Heterodimers of a and b Tubulin Proteins

● Both alpha and beta monomer subunits are

bound to GTP

○ Alpha- is bound tightly to GTP whereas

Beta- is less tightly bound

○ The alpha-bound GTP is never

hydrolyzed and does not exchange with

nucleotides in solution

○ The beta-bound GTP is cyclically

hydrolyzed to GDP and GDP is

exchanged for GTP

● The alpha- and beta- subunits can be added and

removed as dimers to the filaments

● alpha-beta-GTP has a much higher affinity for

the microtubule filament than alpha-beta-GDP

Microtubules are Dynamic

● Microtubules are polar filament

○ The two ends have different characteristics and dynamics

● The plus-end: fast growing, beta subunit is closer than alpha-subunit

○ The alpha-beta-GTP are added to the plus end growing the filaments → rescue

● The minus-end: slow growing

○ The alpha-beta-GDP are released → catastrophe

● GTP hydrolysis occurs within the polymerized microtubule

○ most of the microtubule consists of dimers containing alpha-beta-GDP

● GTP cap (alpha-beta-TGP): ath the plus end; favours growth rather than shrinkage

○ alpha-beta-GTP has ¼ dissociation rate compared to alpha-beta-GDP dimers

■ Because of the higher affinity of alpha-beta-GTP for its neighbours

EB1 Protein Reveals Microtubule Growth

Growth of the plus-end can be visualized

● ex) living mouse embryonic fibroblasts expressing RFP-tubulin and EB1-GFP

● EB1-GEF: plus-end binding protein

○ Prevents premature catastrophes

○ Acts as a positive regulator of microtubule growth

Dynamic Instability

● The plus-end of the microtubule exhibits dynamic instability

○ An oscillating behaviour between growth and shortening or rescue and catastrophe

● The concentration of free alpha-beta-GTP dimers in maintained at a level that allows

polymerization

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Microtubule Associated Proteins (MAPs)

Microtubule-Associated Proteins (MAPs): proteins that control assembly and disassembly of

microtubules

● Many interconnection to help form bundles (cross-bridges), increase stability, alter rigidity,

and influence assembly rate of the microtubule

● Classified into two groups:

○ Those that stabilize the filaments → ex) Tau and EB1

○ Those that destabilize the filaments → ex) catastrophin

Microtubule Nucleation

● Alpha- and beta- tubulins are the building blocks of microtubules

● Gamma-tubulin: involved in nucleation of microtubules

○ Present much smaller amounts in cells

○ Gamma-tubulin and other associated protein → formation of gamma-tubulin ring

complex (gamma-TuRC)

● gamma-TuRC: the ring nucleates at the minus-end of a new microtubule by forming a

template for the growing plus-end

○ Acts as a cap of the minus end while microtubule growth and dynamic occur at the

plus end

Microtubule Nucleation Occurs at the MTOC

Microtubule Organizing Centre (MTOC): specific location inside the cell where microtubule

nucleation occurs

● Centrosome: MTOC in animal cells; located near the nucleus

○ Consists of two cylindrical structure (centrioles) and a cloud of pericentriolar

material (PCM)

○ PCM contains multiple gamma-TuRC

● The minus-ends of the microtubules are nucleated at the gamma-TuRC

● The pus-ends are directed towards the periphery of the cell

MTOCs in Mitosis

● During the mitosis, the microtubules of the mitotic spindle attach to the chromosomes →

orchestrate replicated sister chromatid separation

○ Mitotic Spindles: dynamic structures; are assembled and disassembled during the

cell cycle

○ The cycle depends on the dynamic instability of the microtubules

● Chromosomes are duplicated in mitosis → creates two MTOC’s →separation of replicated

MTOC’s → microtubule nucleation at the gamma-TuRC and plus-end grows outwards

○ Some plus ends emanate towards the cell periphery and anchor the spindle

○ Others grow towards each other → creates the spindle and attach to the

condensed replicated chromosomes

Microtubules Reorganize During Mitosis

● Tubulin is generally labeled using an antibody to either of the tubulin subunits or using GFP

fusion constructs

● In interphase: the network of microtubules fill the cell and the nucleus is stained using

fluorescent DNA dye (DAPI)

● In metaphase: chromosomes are compacted and associated with bipolar mitotic spindle

○ Chromosomes aligned at the equator by the attachment between the microtubules

stretching from the poles of the spindle to attachment sites at the centromeres of the

chromosome

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Document Summary

Comprised of 13 protofilaments arrayed in a circular pattern to form a very strong wall. Subunits of the filaments: dimers of alpha and beta tubulin proteins. Protofilaments: staggered; a string of tubulin monomer appear to spiral up through the microtubule like a spring. The ring formed by the protofilaments is visible under the electron microscope image. Both alpha and beta monomer subunits are bound to gtp. Alpha- is bound tightly to gtp whereas. The alpha-bound gtp is never hydrolyzed and does not exchange with nucleotides in solution. The beta-bound gtp is cyclically hydrolyzed to gdp and gdp is exchanged for gtp. The alpha- and beta- subunits can be added and removed as dimers to the filaments. Alpha-beta-gtp has a much higher affinity for the microtubule filament than alpha-beta-gdp. The two ends have different characteristics and dynamics. The plus-end: fast growing, beta subunit is closer than alpha-subunit. The alpha-beta-gtp are added to the plus end growing the filaments rescue.

Related Questions

Activity: Dynamic Instability of Microtubules

Part A

What causes catastrophe of the microtubule in vitro?

the lack of tubulin heterodimers

non-motor Microtubule Associated Proteins (MAPs)

GTP hydrolysis

mutation of the Î²-tubulin

Part B

What is the role of GTP in microtubule polymerization?

GTP binds the alpha and beta tubulin subunits together to form the tubulin monomer.

GTP hydrolysis provides the energy for the polymerization of the microtubule.

GTP is a second messenger that signals the need for polymerization/de-polymerization.

GTP stabilizes the tip of the microtubule, allowing more monomers to be added.

Part C

What would happen in the treadmilling experiment if a non-hydrolyzable analogue of GTP were used?

The monomers would be unable to add to the plus end, and the microtubules would shrink until they disappeared.

The microtubule would treadmill until the new tubulin, with non-hydrolyzable GTP, reached the minus end, and then it would only extend at the plus end.

The microtubule would add monomers at both the plus and minus ends, growing in both directions.

The non-hydrolyzable GTP would stabilize both ends, causing treadmilling to stop.

Part D

What is the difference between the plus and minus ends of the microtubule in in vitro experiments?

The beta subunit of the tubulin is exposed on the minus end.

Polymerization occurs at the plus end.

The plus end has a lower critical concentration for tubulin heterodimers.

Catastrophe occurs at the minus end.

Part E

How would the drug taxol affect the in vitro dynamic instability and treadmilling experiments?

Taxol would block catastrophe at the plus end in the dynamic instability experiment but not depolymerization at the minus end in the treadmilling experiment.

Taxol would block the addition of tubulin in both experiments, leading to a destruction of the microtubules.

Taxol would stabilize the microtubules in both experiments, leading to polymerization without catastrophe.

Taxol would block depolymerization at the minus end in the treadmilling experiment but not catastrophe at the plus end in the dynamic instability experiment.

7 questions could use some help on for Molecular Biology. Pleasehelp. Just answers will do (I'll figure out), but wouldn't mindexplanations. If answered correctly 5 star guarantee & goodattempt definite 'best answer'. Thanks!

Question1

Which of the following cytoskeletal components is thelargest?

Microtubules
Microfilaments (actin)
Intermediate filaments

Question2

During mitosis, all microtubules emanate from the centrosome.Therefore, the centrosome is an example of:

a microtubule organizing center (MTOC)
A +TIP protein
A catastrophin
A monomer sequestering protein

Question3

Tubulin is a G-protein. It can stabilize microtubules when boundto which of the following?

GDP
GTP
Either GTP or GDP
Neither GTP nor GDP

Question4

In the formation of microtubules, dimers are built when analpha-tubulin protein binds to a beta-tubulin protein. This is anexample of what level of protein structure?

Primary structure
Secondary structure
Tertiary structure
Quartenary structure

Question5

Different tissues in the body express slightly differentversions of alpha tubulin. Which of the following could lead toslightly different alpha-tubulin molecules with a modified proteinsequence?

One tissue has more DNA methylation upstream of thealpha-tubulin gene.
One tissue has more miRNAs produced than the other.
One tissue uses different splice sites than the other.
One tissue has different trans-elements than the other.

Question6

How is confocal microscopy different from traditionalfluorescent microscopy?
The molecules are labelled red instead of green.
The molecules are observed over time.
The molecules are observed in many thin planes, which are thenstacked together to create a crisper image.
The confocal microscope has higher objective power than regularfluorescent microscopes.

Question7

Taxol is a chemotherapy drug that permanently stabilizesmicrotubules and prevents their degradation. Describe howmicrotubules are typically destroyed and why permanentstabilization would kill dividing cells. Note: you may need toreview the basics of cell mitosis from a previous course.

ultramarinepig913

1. Kinesin-1 motors can move long distances on microtubule tracks without dissociating. By contrast, myosin II motors in skeletal muscle take only one or a few steps before letting go. What is most likely the difference between these two motors that allow two different types of movement?

A. Differences in length of the stalk of each respective motor allow for differences in step size

B. Differences in ATP versus GTP hydrolysis alter the motor head movement along filaments

C. Actin filaments are smaller in diameter and have less space for movement

D. Binding affinity of the Kinesin-1 motor head to microtubules is high, whereas binding affinity of the myosin II motor head to actin filaments is low

E. Microtubules are larger in diameter and have more space for movement

2. Dynamic instability causes microtubules either to grow or to shrink rapidly. What is the most likely reason for a microtubule to undergo rapid growth?

A. GTP hydrolysis exceeds the rate of heterodimer addition

B. GTP hydrolysis of alpha tubulin causes an outward bend in the heterodimer conformation

C. A low concentration of tubulin heterodimers are in solution

D. Heterodimer addition exceeds the rate of GTP hydrolysis

E. Increased trafficking of microtubule motor proteins favors stability

3. There are no known motor proteins that move on intermediate filaments (IFs). What property of intermediate filaments would most likely explain the lack of motor proteins?

A. Tissue specific expression of IFs prevent a single type of motor protein to evolve over time

B. Lateral bundling of IFs prevent motor movement

C. IFs are less dynamic than microtubules and actin filaments

D. IFs are unpolarized

E. IFs do not bind a nucleotide

4. You discover a mutation that results in less rapid hydrolysis of GTP by the beta-tubulin subunit. What would most likely occur?

A. There would be no change in filament assembly

B. There would be enhanced instability of the microtubule

C. There would be enhanced stability of the microtubule

D. Microtubules would not form

E. There would be enhanced treadmilling

5. Which of the following statements describes common features of cadherins and integrins?

A. They can link to microtubules using specific adapter proteins

B. They mediate heterophillic interactions

C. They achieve high binding affinity through clustering

D. They are responsible for interactions of a cell with the extracellular matrix

E. They are found in the apical plasma membrane of an epithelial cell

6. Intermediate filaments are uniquely non-polar and built by lateral bundling in five steps: 1) synthesis of an alpha-helical monomer, 2) formation of a coiled-coil dimer, 3) formation of a staggered tetramer of two coiled-coil dimers, 4) lateral association of 8 tetramers, and 5) addition of 8 tetramers to a growing filament. At what step is polarity lost?

A. Step 1

B. Step 2

C. Step 3

D. Step 4

E. Step 5

7. Collagenase is an enzyme that breaks peptide bonds in collagen to help destroy extracellular structures. What would be the most likely reason to use collagenase in a cell biology laboratory?

A. Disrupt cadherin interaction with the extracellular matrix

B. Disrupt cell-cell contacts to passage cells in cell culture

C. Dissociate epithelial cells from the underlying basement membrane to separate tissues

D. All of the answers are likely uses for collagenase

E. Lyse cells to expose intracellular proteins and organelles

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BIOLOGY 2B03 Chapter Notes - Chapter 7: Microtubule Nucleation, Polar Filament, Pericentriolar Material

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