LARP6/PIH1D3 Complex as a Possible Cause of Primary Ciliary - - PowerPoint PPT Presentation

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LARP6/PIH1D3 Complex as a Possible Cause of Primary Ciliary - - PowerPoint PPT Presentation

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LARP6/PIH1D3 Complex as a Possible Cause of Primary Ciliary Dyskinesia Summer Research Fellowship 2017 Ryan Earwood Kato Lab CILIA Background Krisch, 2014 Ke & Yang, 2014 Benmerah, et al , 2015

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LARP6/PIH1D3 Complex as a Possible Cause of Primary Ciliary Dyskinesia

Summer Research Fellowship 2017 Ryan Earwood – Kato Lab

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CILIA

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Background

Ke & Yang, 2014 Krisch, 2014 Benmerah, et al, 2015
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Ciliopathies

Radiopaedia Physiopaedia Gleeson, 2011
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PIH1D3

  • X-Linked form of human PCD
  • Contains CS domain
  • Localizes to apical cytoplasm
  • Leads to dysfunctional motile

cilia

Olcese et al, 2017
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PIH1D3 interacted with LARP6

WB: HA WB: Flag

  • xlLARP6-2HA

Flag-xlPIH1D3

  • +

+

  • +

+

input IP: HA

  • A. mammalian cells
  • B. Xenopus embryos
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Expression profiles PIH1D3

animal view A V a p a p

Stages 9 14 20 25 30 dorsal view lateral view

A: Animal pole; V: Vegetal pole; a: anterior; p: posterior

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PIH1D3 knockdown induced swelling phenotype

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PIH1D3 knockdown compromised the structure of pronephros

Control MO PIH1D3 MO

Hoechst: nucleus α-tubulin: cilia Lectin: tubular epithelial cells mRFP: plasma membrane

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Control MO LARP6 MO

a-tub memGFP a-tub memRFP

A B

PIH1D3 knockdown induced ciliary defects in the multi-ciliated epidermal cells

α-tubulin: cilia mRFP: plasma membrane mGFP: plasma membrane mRFP: plasma membrane

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PIH1D3 and LARP6 controls ciliogenesis of the multi-ciliated cells together

Control MO LARP6 MO – 7.5ng PIH1D3 MO – 5ng LARP6 MO/PIH1D3 MO

α-tubulin: cilia mRFP: plasma membrane mGFP: plasma membrane (control)

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Proximity Ligation Assay

1° Antibody 2° Antibody Ligation Amplification

PIH1D3 LARP6

Sigma: Life Sciences
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PIH1D3 and Larp6 formed granules in the multi- ciliated cells

HA-Larp6/Flag-PIH1D3

Hoechst: nucleus mGFP: plasma membrane Cy3: PIH1D3/LARP6 complex

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Expression profiles

a: anterior; p: posterior

Larp6 PIH1D3

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LARP6/PIH1D3 Localization

nucleus PIH1D3 Cilia Larp6

Olcese et al., 2017
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Summary

  • PIH1D3 is involved in kidney development.
  • PIH1D3 synergizes of Larp6 function in MCC

ciliogenesis.

  • PIH1D3 and LARP6 form distinct granules in

MCCs.

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Proposed model

LARP6/PIH1D3 Complex Centrosome/Basal Body

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Reference List

  • Benmerah, Alexandre & Durand, Bénédicte & Giles, Rachel & Harris, Tess & Kohl, Linda &

Laclef, Christine & Meilhac, Sigolene & Mitchison, Hannah & B Pedersen, Lotte & Roepman, Ronald & Swoboda, Peter & Ueffing, Marius & Bastin, Philippe. (2015). The more we know, the more we have to discover: An exciting future for understanding cilia and ciliopathies.

  • Cilia. 4. 5. 10.1186/s13630-015-0014-0.
  • Duolink kit by Sigma: Life Science
  • Gleeson, Joseph. (2011). A systems-biology approach to understanding the ciliopathy
  • disorders. Genome medicine. 3. 59. 10.1186/gm275.
  • https://www.physio-pedia.com/Polycystic_Kidney_Disease
  • https://radiopaedia.org/cases/situs-inversus-9
  • Krisch, J. A. (2014, December 10). Why Scientists Are Blaming Cilia for Human
  • Disease. (https://www.scientificamerican.com/article/why-scientists-are-blaming-cilia-for-

human-disease/)

  • Manojlovic, Zarko et al. “La-Related Protein 6 Controls Ciliated Cell Differentiation.” Cilia 6

(2017): 4. PMC. Web. 8 Oct. 2017.

  • Olcese C, Patel MP, Shoemark A, et al. X-linked primary ciliary dyskinesia due to mutations

in the cytoplasmic axonemal dynein assembly factor PIH1D3. Nature Communications. 2017;8:14279. doi:10.1038/ncomms14279.

  • Yi-Ni Ke, Wan-Xi Yang, Primary cilium: an elaborate structure that blocks cell division? Gene,

Volume 547, Issue 2, 1 September 2014, Pages 175-185, ISSN 0378-1119, https://doi.org/10.1016/j.gene.2014.06.050

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Kato lab

Diana Perez Mia Stroud Yoichi Kato*

Florida Sate University

Branko Stefanovic* Ruth Didier Hao Wang

Acknowledgements Funding

FSU COM Summer Research Fellowship NIH

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Future directions

  • Identify the role of granules including

LARP6/PIH1D3 complex in ciliogenesis.

  • Identify RNAs that interact with LARP6 in

ciliogenesis and test if they are included in LARP6/PIH1D3 complex.

  • Screen for patients with mutations in LARP6.