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Function of septin proteins in cell division of Drosophila melanogaster S2 cells

Name
Anastasiya
Surname
Alekseeva
Scientific organization
Institute of Molecular and Cellular Biology SB RAS and Institute of Chemical Biology and Fundamental Medicine SB RAS
Academic degree
no
Position
PhD student
Scientific discipline
Agricultural & Biological technologies
Topic
Function of septin proteins in cell division of Drosophila melanogaster S2 cells
Abstract
A.L. Alekseeva, E.N. Andreyeva, A.V. Pindyurin, S.A. Fedorova

Drosophila is a convenient model organism for study septin functions. Here, we investigated the role of all five Drosophila septins in mitosis by their subsequent depletion in cultured S2 cells. We found that depletion of either septin did not affect the mitotic index, although depletion of some septins caused cytokinesis defects. The results suggest the existence of mechanism(s) of interdependent regulation of septins.
Keywords
septins, cytokinesis, GTPase, cytoskeleton, S2, Drosophila, RNAi
Summary

Function of septin proteins in cell division of Drosophila melanogaster S2 cells

A.L. Alekseeva1,2*, E.N. Andreyeva1, A.V. Pindyurin1,3, S.A. Fedorova3

1Institute of Molecular and Cellular Biology SB RAS, Novosibirsk, Russia

2Institute of Chemical Biology and Fundamental Medicine SB RAS, Novosibirsk, Russia

3Institute of Cytology and Genetics SB RAS, Novosibirsk, Russia

e-mail: ea463a6864a1aalekseeva@mcb.nsc.ru

*Corresponding author

 

INTRODUCTION: Septins are a conserved family of GTP-binding proteins, which were found in all eukaryotic organisms except plants. Numerous studies have shown that these proteins play a key role in carcinogenesis in vertebrates. Septins have the ability to form long filaments, which undergo significant rearrangements during cell cycle, but their functions are not fully understood. The number of septins varies between organisms. For example, in human there are 13 septin genes encoding >30 protein isoforms. Due to the presence of only 5 septins (Sep1, Sep2, Pnut, Sep4 and Sep5), Drosophila melanogaster is a convenient model organism for study septin functions. Pnut, Sep1 and Sep2 are known to form heteromeric complex, which can undergo polymerization leading to the formation of filaments. The latter were shown to be involved in the formation of the cleavage furrow during cytokinesis. The function of Sep4 and Sep5 is not yet understood. Here, we studied the role of all five Drosophila septins in mitosis by their subsequent depletion by RNAi in cultured S2 cells.

METHODS: Cells were grown in Schneider’s medium (Sigma S0146) supplemented with 10% heat-inactivated fetal bovine serum (FBS, Gibco, 10270-106). After 5-day RNAi treatments, cells were fixed and immunostained to reveal microtubules and centrosomes. The following primary and secondary antibodies were used: mouse anti-α-tubulin (Sigma T5168), rabbit anti-DSpd2 [1] (recognize centrosomes), anti-mouse-FITC (Sigma F8264) and anti-rabbit-Alexa568 (Invitrogen A11036). Western-blot and RT-qPCR analyses were employed to assess the efficiency of RNAi. Mitotic index was defined as the percentage ratio between dividing and total numbers of cells in a population.

RESULTS: We found that depletion of either septin did not affect the mitotic index, although depletion of some septins caused cytokinesis defects. Surprisingly, after depletion of some septins we observed altered amounts of transcripts and proteins of some other septins. For example, knockdown of Pnut increased the amount of Sep4 transcripts and at the same time decreased the amount of Sep1 and Sep2 transcripts. The increased amount of Sep4 transcripts was also detected upon depletion of Sep2, which also caused the decrease of Sep1 and pnut transcripts. Taken together, the obtained results suggest the existence of mechanism(s) of interdependent regulation of septins in cultured Drosophila S2 cells.

REFERENCES:

1. M.G. Giansanti et al. (2008) Drosophila SPD-2 is an essential centriole component required for PCM recruitment and astral-microtubule nucleation. Curr. Biol. 18: 303—309.