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Targeting of ASH Domain-Containing Proteins to the Centrosome

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Cilia

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1454))

Abstract

A growing number of studies have used new generation technologies to characterize the protein constituents of cilia and centrosomes. This has led to the identification of a vast number of candidate ciliary or centrosomal proteins, whose subcellular localization needs to be investigated and validated. Here, we describe a simple and inexpensive method for analyzing the subcellular localization of candidate cilium- or centrosome-associated proteins, and we illustrate the utility as well as the pitfalls of this method by applying it to a group of ASH (ASPM, SPD-2, Hydin) domain-containing proteins, previously predicted to be cilia- or centrosome-associated proteins based on bioinformatic analyses. By generating plasmids coding for epitope-tagged full-length (FL) or truncated versions of the ASH domain-containing proteins TRAPPC8, TRAPPC13, NPHP4, and DLEC1, followed by expression and quantitative immunofluorescence microscopy (IFM) analysis in cultured human telomerase-immortalized retinal pigmented epithelial (hTERT-RPE1) cells, we could confirm that TRAPPC13 and NPHP4 are highly enriched at the base of primary cilia, whereas DLEC1 seems to associate specifically with motile cilia. Results for TRAPPC8 were inconclusive since epitope-tagged TRAPPC8 fusion proteins were unstable/degraded in cells, emphasizing the need for combining IFM analysis with western blotting in such studies. The method described should be applicable to other candidate ciliary or centrosomal proteins as well.

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Abbreviations

ASH:

ASPM, SPD-2, Hydin

ATCC:

American-type culture collection

DAPI:

4′ 6-Diamidino-2-phenylindole, dihydrochloride

DLEC1:

Deleted in lung and esophageal cancer 1

DMEM:

Dulbecco’s modified Eagle’s medium

DTT:

Dithiothreitol

ECL:

Enhanced chemiluminescence

EDTA:

Ethylenediaminetetraacetic acid

EGFP:

Enhanced GFP

FAP:

Flagella-associated protein

FL:

Full length

GFP :

Green fluorescent protein

hTERT-RPE1 :

Human telomerase-immortalized retinal pigmented epithelial

IFM:

Immunofluorescence microscopy

LB:

Luria-Bertani

LDS:

Loading sample buffer

MFI:

Mean fluorescence intensity

MSP:

Major sperm protein

NPHP4:

Nephronophthisis 4

OCRL:

Oculocerebrorenal syndrome of Lowe

PACT:

Pericentrin-AKAP450 centrosomal targeting

PBS:

Phosphate-buffered saline

RT:

Room temperature

siRNA:

Small interfering RNA

TBS-T:

Tris-buffered saline with Tween-20

TPR:

Tetratricopeptide

TRAPP:

Transport protein particle

TRAPPC:

TRAPP complex

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Acknowledgments

This work was supported by the University of Copenhagen Excellence Programme for Interdisciplinary Research and the Danish Council for Independent Research (1331-00254). S.K.M. was partially supported by a Ph.D. fellowship from the Department of Biology, University of Copenhagen. We thank Sophie Saunier for the gift of human NPHP4 cDNA, Søren L. Johansen for technical assistance, Kenneth B. Schou for assistance with DLEC1 and NPHP4 PCR and cloning, and Louise Lindbæk for assistance with image analysis.

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Authors and Affiliations

  1. Department of Biology, University of Copenhagen, August Krogh Building, Universitetsparken 13, 2100, Copenhagen, OE, Denmark

    Patricia Verdier, Stine K. Morthorst & Lotte B. Pedersen

Authors
  1. Patricia Verdier
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  2. Stine K. Morthorst
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  3. Lotte B. Pedersen
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Corresponding author

Correspondence to Lotte B. Pedersen .

Editor information

Editors and Affiliations

  1. Albert Einstein College of Medicine, Yeshiva University, Bronx, New York, USA

    Peter Satir

  2. Department of Biology, University of Copenhagen, Copenhagen, Denmark

    Søren Tvorup Christensen

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Verdier, P., Morthorst, S.K., Pedersen, L.B. (2016). Targeting of ASH Domain-Containing Proteins to the Centrosome. In: Satir, P., Christensen, S. (eds) Cilia. Methods in Molecular Biology, vol 1454. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3789-9_2

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  • DOI: https://doi.org/10.1007/978-1-4939-3789-9_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3787-5

  • Online ISBN: 978-1-4939-3789-9

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