Spindle formation in female meiosis

Question 1

What can be the result of chromosome mis-segregation in female meiosis?

Answer 1

• Infertility
• Miscarriages
• Birth defects

Question 2

What is the function of centrosomes in mitosis?

Answer 2

• Define where the spindle forms (nucleate MTs).
• Define spindle bipolarity.

Question 3

What is unique about division (meiosis) in oocytes?

Answer 3

A bipolar spindle is formed without centrosomes.

Question 4

Why are unfertilised oocytes proposed to not have centrosomes?

Answer 4

So they cannot divide / begin developing (causes problems). The sperm brings the centrosomes which facilitates division, ie division is coupled with fertilisation.

Question 5

How can oocyte division and development be stimulated?

Answer 5

Injection of centrosomes.

Question 6

Are centrosomes required for spindle formation in mitosis?

Answer 6

No - spindles can assemble without them and mitotic division can occur.

Question 6

What is the effect on drosophila with knocked down centrosomes?

Answer 6

Division can occur and they develop almost normally, but they don’t have cilia.

Question 7

What happened when M-phase Xenopus egg extract was combined with sperm?

Answer 7

Spindles (with centrosomes) form.

Question 8

What happened when M-phase Xenopus egg extract was combined with beads coated in phage DNA?

Answer 8

Bipolar spindles (without centrosomes) formed around the beads.

Question 9

What inference can we make from phage DNA promoting spindle assembly in Xenopus eggs?

Answer 9

• Any DNA can recruit cellular factors and assemble MTs
• Centrosomes or KTs are not required for spindle bipolarity.

Question 10

Why are spindles thought to be bipolar?

Answer 10

Because MTs have polarity.

Question 11

What is RCC1?

Answer 11

The Ran GEF (switches from GDP to GTP bound state).

Question 12

What is the Ran GAP called?

Answer 12

Just RanGAP.

Question 13

Where is RCC1 localised?

Answer 13

The nucleus. It is chromatin bound.

Question 14

Where is RanGAP localised?

Answer 14

The cytoplasm.

Question 15

What form is Ran in the cytoplasm?

Answer 15

GDP bound - RCC1 is predominant.

Question 16

What form is Ran in the nucleus?

Answer 16

GTP bound - RanGAP is predominant.

Question 17

How do cytoplasmic nuclear proteins move through the nuclear pore into the nucleus?

Answer 17

They are bound by importin.

Question 18

How are nuclear proteins prevented from exiting the nucleus into the cytoplasm through the nuclear pore?

Answer 18

Importin is removed from them so they can no longer pass through the nuclear pore.

Question 19

Why does importin bind nuclear proteins in the cytoplasm?

Answer 19

Importin has a higher affinity for nuclear proteins than Ran GDP.

Question 20

Why is importin removed from nuclear proteins in the nucleus?

Answer 20

Importin has a higher affinity for RanGTP than the nuclear proteins.

Question 21

How are spindle assembly factors (SAFs) (proteins) hypothesised to work?

Answer 21

When bound by importin (cytoplasm) they are inactive, but when not bound by importin (nucleus) they are active and a spindle is formed.

Question 22

How was FRET used with the YRC protein construct to detect localisation of RanGTP?

Answer 22

• The YRC was made up of a central RanGTP binding protein, YFP on one end and CFP on the other.
• When RanGTP is bound, the fluorescent proteins can’t come into close proximity / interact.
• High RanGTP = low FRET signal.

Question 23

How was FRET used with the YIC protein construct to detect localisation of RanGTP?

Answer 23

• The YIC was made up of a central importin binding protein, YFP on one end and CFP on the other.
• Importin preferentially binds RanGTP than its binding protein.
• When importin is bound to the construct (low RanGTP), the fluorescent proteins can’t come into close proximity / interact.
• High RanGTP = high FRET signal.

Question 24

How was it discovered that RanGTP is required and sufficient for spindle assembly?

Answer 24

• Depleting RanGTP with excess RanGAP or RanT24N (inhibits Rcc1) inhibited spindle assembly close to the DNA beads.
• Increasing RanGTP with excess Rcc1 or RanQ69L-GTP (cannot hydrolyse GTP) promoted spindle assembly everywhere - even away from the DNA beads.

Question 25

How does RanGTP induce spindle assembly?

Answer 25

It removes importin from SAFs, which activates them.

Question 26

How were spindle assembly factors identified?

Answer 26

• Adding importin to bind SAFs in Xenopus extract.
• Removing the importin bound to SAFs (removing SAFs). Spindle assembly inhibited!
• Adding HeLa nuclear extract to depleted Xenopus extract. Spindle assembly can occur again ie HeLa extract contains SAFs!
• Repeated fractionation of the HeLa nuclear extract.
• Removal of any fractions not able to promote spindle assembly in the depleted Xenopus extract.
• Finally just 1 fraction that promoted spindle assembly remained.

Question 27

What is the name of the SAF?

Answer 27

TPX2.

Question 28

How was immunoprecipitation and western blotting used to validate TPX2 as a SAF in Xenopus?

Answer 28

1. Validated TPX2 binds importin by pulling down TPX2 (+ binding proteins) with TPX2 Ab, then using importin Abs on gel to show importin was also pulled down.
2. Validated importin is removed from TPX2 by RanGTP using an excess of RanQ69L-GTP to produce excess RanGTP. Importin was no longer bound by TPX2 (not bound by importin Abs on TPX2 pull down gel).

Question 29

How was immunodepletion and fluorescence staining used to validate TPX2 as a SAF in Xenopus?

Answer 29

1. A high concentration of TPX2 Abs were used to deplete it from the extract. Staining of tubulin and DNA showed in the presence of DNA beads, without TPX2 spindles did not assemble.
2. To prove it was TPX2 and not a TPX2 binding protein, recombinant TPX2 was introduced into the depleted extract and spindles were able to assemble.

Spindles are still formed in mock depletions where Abs against other elements of the extract were used.

Question 30

What spindle assembly proteins, other than TPX2, are activated by RanGTP in Xenopus?

Answer 30

• NuMA (spindle pole associated)
• NuSAP (spindle MT associated)
• HURP (KT associated)