Abnormal expression of cytoskeletal protein in mkrn1 exS

While we were doing various experiments, we found interesting phenomena.

First, we found abnormalities in ACTIN expression in ovary lysate. In the case of mkrn1^exS, the amount and size of ACTIN were larger than control. (Figure. 12A)

Second, we found that the expression of TUBULIN in the ovary lysate is abnormal. In the case of mkrn1^exS, the amount of TUBULIN was slightly smaller than control. (Figure. 12B)

Thus, we concluded that the expression of cytoskeletal protein in mkrn1^exS is abnormal.

Figure 12. Abnormal Expression of cytoskeletal proteins in mkrn1^exS Adult flies were collected at 3^rd day after hatched. Ovary extracts were prepared for immunoblot using anti-ACTIN antibodies and anti-ß-TUBULIN antibodies. (A) Determination of actin protein expression in fly ovaries. (B) Determination of the ß-tubulin protein expression in fly ovaries. C means control and exS means mkrn1^exS.

DISCUSSION

Recent studies have shown that mutations in the makorin3(mkrn3) gene cause precocious puberty. Precocious puberty occurs when the maturation is abnormally early age.

In order to investigate the effect of mkrn3 on mammals and to conduct in vivo experiments, the experiment was carried out using mkrn1, a orthologous of human mkrn3.

The mkrn1 gene was non-specifically removed using a P-element to create a mutation. (Figure. 1) In order to determine whether the mutation affects growth and maturation as well as humans, we examined the various states of drosophila to see what changes in their growth and maturation. In case of mkrn1^exS, pupation time took longer than the control and pupa length was longer. The mkrn1^exS females were heavier and consumed much food than the other flies . (Figure. 3) After then, we found that the mkrn1 mutant female lost reproductive ability. (Table . 2)

From these results, we found that mkrn1 mutation affects the growth and maturation of female drosophila and causes loss of reproductive capacity.

The loss of reproductive capacity is due to the cause that vitellogenesis does not occur in mkrn1^exS. (Figure. 5) When vitellogenesis does not occur, the follicle cell breaks down. (Figure. 6)

The vitellogenesis plays an important role in the developmental stage,

The InR/TOR signaling pathway was checked through phosphorylated

Therefore, we thought that abnormal InR/TOR pathway is not the cause of vitellogenesis does not occur. This is because vitellogenesis does not occur when the InR/TOR pathway is reduced. (D. Drummond-Barbosa, et al., 2001.)

In Drosophila melanogaster, when the Notch signaling pathway is activated, it inhibits the expression of CUT protein by Hindsight.

(Patrick J et al., 2014). In the case of control flies, the CUT protein is expressed in the st.6 of egg chamber, but mkrn1^exS is not expressed in st.6. (Figure. 9)

In the case of Hindsight, there is no difference in expression pattern but there is a difference in expression level. More amounts of Hnt are

Cyclin B is expressed until st.5, which is the pre -stage of M/E switch operation. However, it can be seen that an abnormally large number of follicle cell is expressed Cyc B in mkrn1^exS. (Figure. 11). Thus, we concluded that mutant drosophila does not have a normal cell cycle regulator, which may cause the M/E switch not to occur normally.

However, it is not yet clear whether this is the direct cause of vitellogenesis does not occur.

We could find an interesting phenomenon during the experiment. It is an abnormal expression of the cytoskeletal protein. First, the ACTIN size was larger in mkrn1^exS and the expression amount was larger. In the case of TUBULIN, the mutant expresses a relatively small amount. (Figure 11). Through this result, we thought that this is not a direct cause of vitellogenesis does not occur. However, we thought that the reason for the abnormality of ACTIN, which is related to follicle cell migration (Tânia Ferreira et al., 2014), and tubulin, which is needed to form polar cells (Alexandre D et al., 2012), is that vitellogenesis did not occur.

Altogether, these results reveal that mkrn1^exS affects Drosophila growth. And mkrn1 is required for drosophila oogenesis. However, exact mechanism is unknown yet.

CONCLUSION

We have successfully generated mutants of mkrn1 by P-element excision mutagenesis. MKRN1 protein strongly expressed in ovary.

(Figure. 1,2)

To determine the effect of this mutant line on growth and maturation, we crossed male and female of homozygous and heterozygous mutant each other. We were able to conclude that male homozygous drosophila's fertility is not affected by the excision of mkrn1, but female homozygous mkrn1 mutants lose their fertility. (Table. 2)

To determine whether a change in mkrn1 mutation causes similar effects on drosophila as it does in mammals. We checked pupation time, pupa length and weight. mkrn1^exS has longer pupation time than control, and has longer pupa length than control. mkrn1^exS female fly is heavier than other flies. Also, we found that mkrn1^exS much more consumed food than other flies. (Figure. 3)

Vitellogenesis does not occur in mkrn1^exS flies, resulting in the defects in the development of egg chambers. Ovaries from mkrn1^exS flies demonstrated diminished follicle cells around the time when vitellogenesis normally occur. (Figuer. 4,5)

To test if insulin signaling is affected in mkrn1 mutants, we have examined relative level of pAKT. In mkrn1^exS, AKT phosphorylation higher than control. This result is because PTEN decreased in mkrn1^exS.

CUT and Hindsight are related to Notch signaling. We observed that patterns of CUT expression is different between control and mkrn1^exS. In case of control, the expression level in st.6 decreased but is still expressed. In mkrn1^exS, CUT is not expressed in st.6. (Figure. 9)

There is no difference in Hnt expression pattern, but expression level is different. In the case of mkrn1^exS, expression level is higher than control.

(Figure. 10)

We checked the Cyclin B, a cell cycle regulator. There is no difference in Cyclin B expression pattern. However, in mkrn1^exS, more follicle cells expressed Cyclin B than control. (Figure. 11)

In addition, we found that the expression of the cytoskeletal protein was abnormal. In the case of mkrn1^exS, the amount of ACTIN and size are larger than control. Also, the expression amount of TUBULIN is slightly smaller than control. (Figure. 12)

Altogether, these results reveal that mkrn1^exS affects Drosophila growth. And mkrn1 is required for drosophila oogenesis. However, exact mechanism is unknown yet.

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-국문요 약 -

계속해서 실험을 진행하기 위하여 동형접합 돌연변이 유전자를 가

Notch 신호전달 경로는 초파리의 follicle 세포의 세포주기에 변화