Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans

Robert O'Hagan, Maureen M. Barr

Research output: Chapter in Book/Report/Conference proceedingChapterResearchpeer-review

2 Citations (Scopus)

Abstract

Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules. Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM- 3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

Original languageEnglish
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages107-122
Number of pages16
DOIs
StatePublished - 1 Jan 2016

Publication series

NameMethods in Molecular Biology
Volume1454
ISSN (Print)1064-3745

Fingerprint

Cilia
Caenorhabditis elegans
Green Fluorescent Proteins
Kymography
Molecular Motor Proteins
TRPP Cation Channels
Axoneme
Retinal Cone Photoreceptor Cells
Retinal Rod Photoreceptor Cells
Chlamydomonas reinhardtii
Vertebrate Photoreceptor Cells
Flagella
Sensory Receptor Cells
Dendrites
Eukaryota
Microtubules
Retina
Microscopy
Maintenance

Keywords

  • Caenorhabditis elegans
  • Cell biology
  • In vivo
  • KLP-6
  • Kinesin-2
  • Kinesin-3
  • Kymograph
  • Male
  • OSM-3
  • PKD- 2
  • Polycystins
  • Sensory non-motile cilia

Cite this

O'Hagan, R., & Barr, M. M. (2016). Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans. In Methods in Molecular Biology (pp. 107-122). (Methods in Molecular Biology; Vol. 1454). Humana Press Inc.. https://doi.org/10.1007/978-1-4939-3789-9_8
O'Hagan, Robert ; Barr, Maureen M. / Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans. Methods in Molecular Biology. Humana Press Inc., 2016. pp. 107-122 (Methods in Molecular Biology).
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O'Hagan, R & Barr, MM 2016, Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans. in Methods in Molecular Biology. Methods in Molecular Biology, vol. 1454, Humana Press Inc., pp. 107-122. https://doi.org/10.1007/978-1-4939-3789-9_8

Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans. / O'Hagan, Robert; Barr, Maureen M.

Methods in Molecular Biology. Humana Press Inc., 2016. p. 107-122 (Methods in Molecular Biology; Vol. 1454).

Research output: Chapter in Book/Report/Conference proceedingChapterResearchpeer-review

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N2 - Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules. Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM- 3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

AB - Intraflagellar Transport (IFT) is driven by molecular motors that travel upon microtubule-based ciliary axonemes. In the single-celled alga Chlamydomonas reinhardtii, movement of a single anterograde IFT motor, heterotrimeric kinesin-II, is required to generate two identical motile flagella. The function of this canonical anterograde IFT motor is conserved among all eukaryotes, yet multicellular organisms can generate cilia of diverse structures and functions, ranging from simple threadlike non-motile primary cilia to the elaborate cilia that make up rod and cone photoreceptors in the retina. An emerging theme is that additional molecular motors modulate the canonical IFT machinery to give rise to differing ciliary morphologies. Therefore, a complete understanding of the trafficking of ciliary receptors, as well as the biogenesis, maintenance, specialization, and function of cilia, requires the characterization of motor molecules. Here, we describe in detail our method for measuring the motility of proteins in cilia or dendrites of C. elegans male-specific CEM ciliated sensory neurons using time-lapse microscopy and kymography of green fluorescent protein (GFP)-tagged motors, receptors, and cargos. We describe, as a specific example, OSM- 3::GFP puncta moving in cilia, but also include (Fig. 1) with settings that have worked well for us measuring movement of heterotrimeric kinesin-II, IFT particles, and the polycystin TRP channel PKD-2.

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KW - Cell biology

KW - In vivo

KW - KLP-6

KW - Kinesin-2

KW - Kinesin-3

KW - Kymograph

KW - Male

KW - OSM-3

KW - PKD- 2

KW - Polycystins

KW - Sensory non-motile cilia

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U2 - 10.1007/978-1-4939-3789-9_8

DO - 10.1007/978-1-4939-3789-9_8

M3 - Chapter

T3 - Methods in Molecular Biology

SP - 107

EP - 122

BT - Methods in Molecular Biology

PB - Humana Press Inc.

ER -

O'Hagan R, Barr MM. Kymographic analysis of transport in an individual neuronal sensory cilium in caenorhabditis elegans. In Methods in Molecular Biology. Humana Press Inc. 2016. p. 107-122. (Methods in Molecular Biology). https://doi.org/10.1007/978-1-4939-3789-9_8