Poc1B and Sas-6 Function Together during the Atypical Centriole Formation in Drosophila melanogaster
Abstract
:1. Introduction
2. Methods
2.1. Transgenic Flies
Reference | Name in Text | Name in Ref | Description |
[37] | Ana2GFP | pUbq-Ana2-GFP | Ana2 cDNA tagged at the C-terminus with GFP. Expressed from an ubiquitin promoter. |
[37] | Sas-6GFP | pUbq-GFP-Sas6 | Sas-6 cDNA tagged at the N-terminus with GFP. Expressed from an ubiquitin promoter. |
[6] | gPoc1BGFP | Poc1A/B-GFP | Poc1 gene tagged at the end of the last poc1B exon with GFP. Expressed from a poc1 promoter. |
[6] | Poc1BGFP | UAS-Poc1B-GFP | Poc1B cDNA tagged at the C-terminus with GFP. Expressed from a UAS promoter. |
[37] | Sas-6RFP | Ubq-RFP-Sas-6 | Sas-6 cDNA tagged at the N-terminus with RFP. Expressed from an ubiquitin promoter. |
VDRC ID-108219 | Poc1RNAi | Poc1-RNAi | RNAi knockdown of Poc1RNAi |
[6] | Poc1W87X | Poc1W87X | Poc1 truncation mutant |
[25] | Sas-4GFP | Sas-4-GFP | Sas4 gene tagged at the end of the last exon with GFP. Expressed from a sas-4 promoter. |
[38] | Bam-Gal4 | P{bam-GAL4:VP16} | GAL4 line expressing GAL4 from a Bam promoter. |
Bloomington stock #8092 | Poc1 def | Df(3R)ED5066 | A chromosomal deletion that is also missing the poc1 gene |
[39] | ana2 719 | ana2 | Ana2 truncation at R175 |
Bloomington stock #12119 | sas-4s2214 | sas-4 | P-element insertion in the Sas-4 codding leading to a strong loss-of-function |
[13] | Ana1GFP | Ana1-GFP | Ana1 gene tagged at the end of the last exon with GFP. Expressed from an Ana1 promoter |
[13] | gSas-6GFP | gSas-6-GFP | Sas-6 gene tagged at the end of the last exon with GFP. Expressed from a Sas-6 promoter |
[13] | Ana1tdtomato | Ana1-tdtomato | Ana1 gene tagged at the end of the last exon with Tdtomato. Expressed from an Ana1 promoter |
This study | Poc1BΔCGFP | Poc1B cDNA lacking VLDVSIESGSDLCSCSARL DEISKLLDSIDERVRRLEGIYSL amino acid tagged at the C-terminus with GFP. Expressed from a UAS promoter. | |
All fly stocks were cultured on standard media at 25 °C. |
2.2. Transgenic Flies Functional Test References
Ubq-Ana2-GFP and Ubq-GFP-Sas-6 “The homo-oligomerisation of both Sas-6 and Ana2 is required for efficient centriole assembly in flies” [40] |
gPoc1B-GFP and UAS-Poc1B-GFP “Centriole Remodeling during Spermiogenesis in Drosophila” [6] |
gSas-6-GFP “Self-assembling SAS-6 Multimer Is a Core Centriole Building Block” [41] |
Ana1-tdtomato “Drosophila Asterless the ortholog of vertebrate Cep152 is essential for centriole duplication” [13] |
2.3. Testes Fluorescence Microscopy
2.4. Antibodies
2.5. Tissue Processing and Transmission Electron Microscopy
2.6. Super-Resolution Microscopy
2.7. Statistical Methods
2.8. Reagent and Data Availability
3. Results
3.1. Poc1B Is Recruited during the Initial Stage of the PCL Formation
3.2. Poc1B Is Downstream to Ana-2 or Sas-6 during PCL Initiation
3.3. Poc1B Overexpression Induces Increase Cellular and PCL Sas-6 Level
3.4. Poc1B and Sas-6 Are Localized in the PCL Core
3.5. Ana2 and Sas-6 Overexpression Can Induce PCL-Like Structure That Contains Poc1
3.6. Poc1 Proteins Are Essential for Proper Formation of PCL-Like Structure
3.7. Poc1B and Sas-6 Co-Overexpression Is Sufficient to Induce PCL-Like Structures
3.8. The Coiled Coil Domain of Poc1B Is Essential to Induce Ectopic Particles
4. Discussion
- Poc1B and Sas-6 were recruited during the initial stage of PCL formation.
- Poc1B and Sas-6 were localized in the PCL core.
- Poc1B recruitment was dependent on Sas-6.
- Poc1 mutation increased Sas-6 localization to the PCL.
- Poc1B overexpression increased Sas-6 in the testes and PCL.
- Overexpression of Ana2 and Sas-6 induced the formation of atypical particles in PCL-like structures that include Poc1 proteins.
- Poc1 proteins were essential for forming stable PCL-like structures.
- Overexpression of Poc1B and Sas-6 induced the formation of PCL-like structures.
- The C-terminal coiled coil of Poc1B was essential for formation of ectopic practical.
4.1. PCL Could Be Homologous to the Intermediate Structure during Pro-Centriole Formation
4.2. Poc1B May Be a Component of the Cartwheel Inner Density
4.3. Poc1B May Have Multiple Functions in Centriole Assembly
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Jo, K.H.; Jaiswal, A.; Khanal, S.; Fishman, E.L.; Curry, A.N.; Avidor-Reiss, T. Poc1B and Sas-6 Function Together during the Atypical Centriole Formation in Drosophila melanogaster. Cells 2019, 8, 841. https://doi.org/10.3390/cells8080841
Jo KH, Jaiswal A, Khanal S, Fishman EL, Curry AN, Avidor-Reiss T. Poc1B and Sas-6 Function Together during the Atypical Centriole Formation in Drosophila melanogaster. Cells. 2019; 8(8):841. https://doi.org/10.3390/cells8080841
Chicago/Turabian StyleJo, Kyoung H., Ankit Jaiswal, Sushil Khanal, Emily L. Fishman, Alaina N. Curry, and Tomer Avidor-Reiss. 2019. "Poc1B and Sas-6 Function Together during the Atypical Centriole Formation in Drosophila melanogaster" Cells 8, no. 8: 841. https://doi.org/10.3390/cells8080841