Lecture 3 - the eukaryotic cell cycle

Question 1

What are the phases of mitosis?

Answer 1

Interphase microtubules are long and diffuse, emanating from a centrosome

Prophase chromosomes condense

Prometaphase the nuclear envelope breaks down

Metaphase chromosomes align at the spindle equator

Anaphase sister chromatids separate along the spindle

Telophase cleavage furrow constricts around the central spindle and two nuclei re-assemble.

Question 2

How is chromosome movement achieved?

Answer 2

Chromosomes move by remaining attached to shortening + end of kinetochore MTs
Kinetochore is the part of the chromosome which attaches to microtubules
Astral microtubules attach centrioles to periphery of the cell - anchoring, prevent centrioles from moving inwards
Kinetochore microtubules attached to chromosomes themselves, interpolar microtubules connect the two centrioles. Movement driven by kinetochore microtubules. One end (+) shortens and chromosome remains attached to these shortening ends, as it shortens it gets pulled to the poles.

Question 3

What evidence is there for how chromosomes move?

Answer 3

Microtubules in images are fluorescently tagged - bleached with a laser. Bleached zone followed as cell passed through mitosis

Bleached zone remains unchanged

Chromosomes move relative to bleached zone

Chromosomes remain attached to shortening + end of kinetochore MTs

Question 4

How can cell synchrony be tested for experimentally?

Answer 4

Flow cytometry
Fluorescence of an intercalating dye
Fluorescent molecules intercalate between DNA bases - fluorescence - if they do not intercalate there is not fluorescence so the intensity of fluorescence corresponds to the amount of DNA in the cell
Chemical inhibitors are typically used to block the cell cycle just before mitosis or in early S phase
(Nocodazole, cytochalasin B, thymidine, aphidicolin)