Lecture 14

Nucleosomes are a basic unit of eukaryotic chromosome structure

• Histones are responsible for the first and most basic level of chromosomes packing, the nucleosome.

  • Each individual nucleosome core particle consists of a complex of 8 histone proteins.

  • 2 molecules of each histone H2A, H2B, H3, and H4, and double stranded DNA that is 147 nucleotides long.

  • The histone octamer forms a protein core around which the double stranded DNA is wound.

• The region of linker DNA that separates each nucleosome core particle from the next can vary in length from a few nucleotide pairs up to about 80.

• Nucleosomes pack on top of one another, generating arrays in which the DNA is even more highly condensed.

  • Much of the chromatin is seen to be in the form of a fiber with a diameter of 30 nm.

The genome is organized through a hierarchy of long-range interactions: topologically associating domains (TADs)

• When far apart DNA sequences come together, it can be beneficial.

• A lot of times, enhancers are far away from gene sequences.

  • Because of TADs, they can be brought very close to the start site.

  • If one side has an enhancer, and the other is the gene sequence, a transcription factor can bind to the enhancer and activate genes.

  • Cohesin holds the loops together.

• Condensin plays a role in stabilizing the loops

Mechanical events of mitosis

• Microtubules emanate from the centrosome

  • Many animal cells have a single, well defined MTOC (microtubule organizing center) called the centrosome.

  • This is where microtubules are nucleated at their minus ends, so the plus ends point outward and continuously grow and shrink.

• Embedded in the centrosome are the centrioles, a pair of cylindrical structures arranged at right angles to each other in an L-shaped configuration.

  • A centriole consists of a cylindrical array of nine triplet microtubules arranged into a barrel shape.

  • The mother centriole is more complex with distal appendages

• During DNA replication, the centrosome duplicated and splits into 2 parts, each containing a centriole pair.

  • The two centrosomes move to opposite sides of the nucleus when mitosis begins, and they form the two poles of the mitotic spindle.

The eukaryotic cell cycle usually consists of four phases

• Most cells require much more time to grow and double their mass of proteins and organelles than the require to duplicate their chromosomes and divide.

• To allow for this growth, most cell cycles have gap phases.

  • G1 phase between M and S phase

  • G2 phase between S phase and mitosis.

• Thys, the eukaryotic cell cycle is traditionally divided into four sequential phases: G1, S, G2, and M.

  • G1, S, and G2 together are called interphase.

• The gap phases provide time for the cell to monitor the internal and external environment to ensure conditions are suitable and preparations are complete before the cell commits itself to the major upheavals of S phase and mitosis.

Prophase: mitosis begins and the mitotic spindles form

• At prophase, the replicated chromosomes, each consisting of two closely associated sister chromatids, condense.

• Outside the nucleus, the mitotic spindle assembles between the two centrosomes, which have moved apart.

Metaphase: mitotic spindle assembly

• At metaphase, the chromosomes are aligned at the equator of the spindle, midway between the spindle poles.

The mitotic spindle is a microtubule based machine

• The spindle is a bipolar array of microtubules, which pulls sister chromatids apart in anaphase.

• The plus ends of interpolar microtubules overlap with the plus ends of microtubules from the other poe.

• The plus ends of kinetochore microtubules are attached to sister chromatid pairs at large protein structures called kinetochores, which are located at the centromere of each sister chromatid.

• Many spindles also contain astral microtubules that radiate outwards from the poles, and aren’t attached to anything.

  • They undergo dynamic instability because of the free floating plus ends.

Motor proteins govern the assembly and function of the mitotic spindle

• The function of the mitotic spindle depends on numerous microtubule dependent motor proteins.

• Kinesin related proteins usually move towards the plus end of the microtubules.

  • In the mitotic spindle, these motor proteins generally operate at or near the ends of the microtubules.

  • Kinesin-5 proteins contain 2 motor domains that interact with the plus ends of antiparallel microtubules in the spindle midzone. Because the two motor domains move towards the plus ends, they slide the two antiparallel microtubules past each other towards the spindle poles, pushing the poles apart.

  • Kinesin-14 are minus end directed motors with a single motor domain and other domains that can interact with a neighboring microtubule. They can cross-link antiparallel microtubules in the spindle midzone and tend to pull the poles together.

  • Kinesin-4, 10 are plus end directed motors that associate with chromosome arms and push the attached chromosome away from the pole.

• Dyneins are minus end directed proteins that organize microtubules at various locations in the cell.

  • They link the plus ends of astral microtubules to components of the actin cytoskeleton, and can pull the spindle poles towards the cell cortex and away from each other.

Kinetochores attach sister chromatids to the spindle

• Spindle microtubules become attached to each chromatid at its kinetochore, a giant, multilayered protein structure that is built at the centromeric region of the chromatid.

  • In metaphase, the plus ends of kinetochore microtubules are embedded head-on in specialized microtubule attachment sites within the outer region of the kinetochore.

  • The kinetochore of an animal cell can bind to 10-40 microtubules.

• Attachment of each microtubule depends on multiple copies of a rod-shaped protein complex called the Ndc80 complex

  • The Ndc80 complex is anchored in the kinetochore at one ends and interacts with the sides of the microtubules at the other, thereby linking the microtubule and kinetochore while still allowing the addition or removal or tubulin subunits at this end.

Bi-orientation is achieved by trial and error

• The success of mitosis demands that sister chromatids in a pair attach to opposite poles of the mitotic spindle, so that they move to opposite ends of the cell when they separate in anaphase.

  • This is called bi-orientation

• Incorrect attachments are corrected by a system of trial and error that is based on a simple principle: incorrect attachments are highly unstable and do not last, whereas correct attachments become locked in place.

• Tension regulates the proper bi-orientation of the spindle pole

  • When a sister-chromatid pair is properly bi-oriented on the spindle, the two kinetochores are pulled in opposite directions by strong poleward forces.

  • Sister chromatid cohesion resists these poleward forces, creating high levels of tension within the kinetochores.

  • When chromosomes are incorrectly attached, the tension is low and the kinetochore sends an inhibitory signal that loosens the grip of its microtubule attachment site, allowing detachment.

• The tension-sensing mechanism depends on the protein kinase Aurora-B, which is associated with the kinetochore and is thought to generate the inhibitory signal that reduces the strength.

  • When there is no tension, Aurora-B sticks out and phosphorylates several proteins, which decreases the site’s affinity for a microtubule plus end.

  • If there is tension, Aurora-B isn’t long enough so no proteins become phosphorylated. This allows high affinity binding with microtubules.

Multiple forces act on chromosomes in the spindle

• The first major force pulls the kinetochore and its associated chromatid along the kinetochore microtubule toward the spindle pole.

  • It is produced by proteins at the kinetochore itself.

• A second poleward force is provided by microtubule flux (treadmilling) whereby the microtubules themselves are pulled towards the spindle poles and dismantled at their minus ends. The mechanism is not clear.

  • The addition of new tubulin at the plus end compensates for the loss of tubulin at the minus end, so that microtubule length remains constant despite the movement of microtubules towards the spindle pole.

Telophase: Formation of contractile ring

Actin and myosin II in the contractile ring generate the force for cytokinesis

• As sister chromatids separate in anaphase, actin and myosin II begin to accumulate in the rapidly assembling contractile ring.

  • Assembly of the contractile ring results in part from the local formation of new actin filaments, which depends on formin proteins that nucleate the assembly of parallel arrays of linear, unbranched actin filaments.

• After anaphase, the overlapping arrays of actin and myosin II filaments contract to generate the force that divides the cytoplasm in two.

The cell-cycle control system depends on cyclically activated cyclin-dependent protein kinases (Cdks)

• Central components of the cell-cycle control system are members of a family of protein kinases known as cyclin dependent kinases (Cdks).

  • The activity of these kinases rise and fall as the cell progresses through the cycle, leading to cyclical changes in the phosphorylation of intracellular proteins that initiate or regulate the major events of the cell cycle.

• Cyclical changes in Cdk activity are controlled by a complex array of enzymes and other proteins.

  • The most important of these Cdk regulators are proteins known as cyclins.

  • Cdks are dependent on cyclins for their activity: unless they are bound tightly to a cyclin, they have no protein kinase activity

  • Cyclins undergo a cycle of synthesis and degradation in each cell cycle. The levels of the Cdk proteins, by contrast, are constant.

  • Cyclical changes in cyclin protein levels result in the cyclic assembly and activation of cyclin-Cdk complexes at specific stages of the cell cycle.

• There are 4 classes of cyclins, each defined by the stage of the cell cycle at which they bind Cdks and function. All eukaryotic cells require three of these classes:

  1. G1/S-cyclins: activate Cdks in late G1 and thereby help trigger progression through start, resulting in a commitment to cell-cycle entry. Their levels fall in S phase.

  2. S-cyclins: Bind Cdks soon after progression through start and help stimulate chromosome duplication. S-cyclin levels remain elevated until mitosis, and these cyclins also contribute to the control of some early mitotic events.

  3. M-cyclins: activate Cdks that stimulate entry into mitosis at the G2/M transition. M-cyclin levels fall in mid-mitosis

• In the absence of cyclin, the active structure in the Cdk protein is partly obscured by a protein loop.

  • Cyclin binding causes the loop to move away from the active site, resulting in partial activation of the Cdk enzyme.

• Full activation of the cyclin-Cdk complex then occurs when a separate kinase, the Cdk-activating kinase (CAK), phosphorylates an amino acid near the entrance of the Cdk active site.

  • This causes a small conformational change that further increases the activity of the Cdk, allowing the kinase to phosphorylate its target proteins effectively and thereby induce specific cell-cycle events.

Cdk activity can be suppressed by inhibitory phosphorylation and Cdk inhibitor proteins (CKIs)

• The rise and fall of cyclin levels is the primary determinant of Cdk activity during the cell cycle. However, several additional mechanisms help control Cdk activity at specific stages of the cycle.

  • Phosphorylation at a pair of amino acids in the roof of the kinase active site inhibits the activity of a cyclin-Cdk complex.

  • Phosphorylation of these sites by a protein kinase known as Wee1 inhibits Cdk activity.

  • Dephosphorylation of these sites by a phosphatase known as Cdc25 increases Cdk activity.

• Binding of Cdk inhibitor proteins (CKIs) inactivates cycliln-Cdk complexes.

  • CKI binding stimulates a large rearrangement in the structure of the Cdk active site, rendering it inactive.

  • CKI p27 (a tumor suppressor) binds to both the cyclin and the Cdk in the complex, distorting the active site of the Cdk. It also inserts into the ATP-binding site, further inhibiting the enzyme activity.

M-Cdk drives entry into mitosis

• M-Cdk brings about all of the diverse and complex cell rearrangements that occur in the early stages of mitosis.

  • M-Cdk does not act alone to phosphorylate the key protein involved in early mitosis. Two additional families of protein kinases, the Polo-like kinases, and the Aurora kinases, also make important contributions to the control of early mitotic events.

• M-Cdk directly phosphorylates key regulatory proteins to:

  • Assemble the mitotic spindle and appropriate sister chromatid attachment

  • Chromosome condensation

  • Nuclear envelope breakdown

  • Actin cytoskeleton rearrangement

  • Reorganization of the Golgi apparatus

Dephosphorylation activates M-Cdk at the onset of mitosis

• M-Cdk activation begins with the accumulation of M-cyclin. This leads to a corresponding accumulation of M-Cdk as the cell approaches mitosis.

• Although the Cdk in these complexes is phosphorylated at an activating site by the Cdk-activating kinase (CAK), the protein kinase Wee1 holds it in an inactive state by inhibitory phosphorylation at the two neighboring sites.

  • Thus, by the time the cell reaches the end of G2, it contains an abundant stockpile of M-Cdk that is primed and ready to act but is suppressed by the phosphates that block the active site of the kinase.

• The crucial event is the activation of the protein Cdc25, which removes the inhibitory phosphates that restrain M-Cdk.

• At the same time, Wee1 is being suppressed, further ensuring that M-Cdk activity increases.

• Cdc25 can also be activated by its target, M-Cdk. M-Cdk may also inhibit the inhibitory Wee1.

  • The ability of M-Cdk to activate its own activator (Cdc25) and inhibit its own inhibitor (Wee1) suggests that M-Cdk activation in mitosis involves positive feedback looks.

  • It turns out that Cdc25 is activated by Polo kinase.

Regulated proteolysis triggers the metaphase to anaphase transition

• Progression through the metaphase to anaphase transition is triggered not by protein phosphorylation, but by protein degradation, leading to the final stages of cell division.

• The key regulator of the metaphase to anaphase transition if the anaphase-promoting complex (APC).

  • It is a member of the ubiquitin ligase family of enzymes, specifically it’s an E3 ligase.

• The APC catalyzes the ubiquitylation and destruction by proteasome of two major types of proteins:

  • The first is securin, which protects the protein linkages that hold sister chromatid pairs together in early mitosis.

  • The second major target is M-cyclin, which, when destroyed, inactivates most Cdks in the cell.

• The APC is activated by association with Cdc20, which recognizes specific amino acid sequences in M-cyclin and other target proteins.

  • With the help of E1 and E2, the APC assembles the polyubiquitin chains on the target protein.

  • IT is then recognized and degraded in the proteasome.

• Before anaphase, securin binds to an inhibits the activity of a protease called separase.

  • The destruction of securin at the end of metaphase releases separase, which is then free to cleave one of the subunits of cohesin.

  • The cohesins fall away, and the sister chromatids separate.

• Activation of APC is unknown, although it is known that APC activation requires Cdc20.