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A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus

Culture system for the growth of ciliated cells on beads. (A-B) Phase-contrast image of live cells on a bead with contrast set to show cell bodies (A) or cilia (B). (B inset) Enlargement of cilium indicated by arrow. (C) Fluorescent image of bead from A and B labeled with di-8-ANEPPS (di-8). (C inset) Enlargement of cilium indicated by arrow. (D) Fluorescent image of a cell-coated bead fixed and immunolabeled for the ciliary marker acetylated α-tubulin (acet. tub.). (D inset) Enlargement of cilium indicated by arrow. (E) Teflon chamber with medium. Bar = 10 μm (A-D), 2 μm (B-D inset), 1 cm (E).
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Figure 1: Culture system for the growth of ciliated cells on beads. (A-B) Phase-contrast image of live cells on a bead with contrast set to show cell bodies (A) or cilia (B). (B inset) Enlargement of cilium indicated by arrow. (C) Fluorescent image of bead from A and B labeled with di-8-ANEPPS (di-8). (C inset) Enlargement of cilium indicated by arrow. (D) Fluorescent image of a cell-coated bead fixed and immunolabeled for the ciliary marker acetylated α-tubulin (acet. tub.). (D inset) Enlargement of cilium indicated by arrow. (E) Teflon chamber with medium. Bar = 10 μm (A-D), 2 μm (B-D inset), 1 cm (E).

Mentions: The glass-coated beads (G102- < 90, SoloHill Engineering, Ann Arbor, MI USA), were combined with distilled water (to 8 mg/mL) in a scintillation vial and autoclaved. For each milliliter of final chamber volume, 11.6 μL of the bead and water mixture was combined with 88.4 μL of medium in a sterile microcentrifuge tube and incubated at 37°C for at least 1 h. The autoclaved Teflon chambers were placed in a sterile Petri dish and incubated at 37°C for at least 1 h. Cells were dissociated with 0.05% trypsin/ethylenediaminetetraacetic acid (SH3023601, Thermo Fisher Scientific) for about 10 min. The dissociation was ended by the addition of medium with trituration. The cells were centrifuged at 300 × g for 5 min, liquid was removed, and the cells were resuspended in fresh medium with trituration. Cells (final, 2 × 105 cells/mL), medium, and beads (final, 0.09 mg beads/mL, approximately 550 beads/mL) were placed in a depression that had been milled into a Teflon block (Figure1E, 8735K67, McMaster-Carr, Robbinsville, NJ USA). The external dimensions of the block were 76 mm × 26 mm × 13 mm; the dimensions of the depression were 65 mm × 17 mm × 11 mm. We used about 0.5 mL of cell suspension per square centimeter of Teflon chamber floor. The cells and beads were gently shaken for a few seconds every 15 min for the first 90 min of culture. We subsequently dispersed the beads with a cell lifter (70–2180, Biologix, Lenexa, KS USA); this significantly decreased the clumping of the beads. Cell-coated beads were usually used for recording after 10 to 15 days in culture. We used passages 17 to 31. Half of the growth medium was replaced every 2 to 3 days. To replace the medium, the chamber was held at an incline to give the beads time to settle before half of the medium was replaced. Chambers with a siliconized glass (Sigmacote, Sigma-Aldrich, St. Louis, MO USA) bottom were occasionally used, but we did not determine that they were better than Teflon chambers. However, they did permit monitoring of the culture more easily than the opaque Teflon, and at least some of the beads were free of the monolayer. In pilot studies, hollow borosilicate glass beads with a 10 μm diameter (AGSCO Corporation, Wheeling, IL USA) appeared to be engulfed by the cells and no cilia were visible.


A method for measuring electrical signals in a primary cilium.

Kleene NK, Kleene SJ - Cilia (2012)

Culture system for the growth of ciliated cells on beads. (A-B) Phase-contrast image of live cells on a bead with contrast set to show cell bodies (A) or cilia (B). (B inset) Enlargement of cilium indicated by arrow. (C) Fluorescent image of bead from A and B labeled with di-8-ANEPPS (di-8). (C inset) Enlargement of cilium indicated by arrow. (D) Fluorescent image of a cell-coated bead fixed and immunolabeled for the ciliary marker acetylated α-tubulin (acet. tub.). (D inset) Enlargement of cilium indicated by arrow. (E) Teflon chamber with medium. Bar = 10 μm (A-D), 2 μm (B-D inset), 1 cm (E).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3539729&req=5

Figure 1: Culture system for the growth of ciliated cells on beads. (A-B) Phase-contrast image of live cells on a bead with contrast set to show cell bodies (A) or cilia (B). (B inset) Enlargement of cilium indicated by arrow. (C) Fluorescent image of bead from A and B labeled with di-8-ANEPPS (di-8). (C inset) Enlargement of cilium indicated by arrow. (D) Fluorescent image of a cell-coated bead fixed and immunolabeled for the ciliary marker acetylated α-tubulin (acet. tub.). (D inset) Enlargement of cilium indicated by arrow. (E) Teflon chamber with medium. Bar = 10 μm (A-D), 2 μm (B-D inset), 1 cm (E).
Mentions: The glass-coated beads (G102- < 90, SoloHill Engineering, Ann Arbor, MI USA), were combined with distilled water (to 8 mg/mL) in a scintillation vial and autoclaved. For each milliliter of final chamber volume, 11.6 μL of the bead and water mixture was combined with 88.4 μL of medium in a sterile microcentrifuge tube and incubated at 37°C for at least 1 h. The autoclaved Teflon chambers were placed in a sterile Petri dish and incubated at 37°C for at least 1 h. Cells were dissociated with 0.05% trypsin/ethylenediaminetetraacetic acid (SH3023601, Thermo Fisher Scientific) for about 10 min. The dissociation was ended by the addition of medium with trituration. The cells were centrifuged at 300 × g for 5 min, liquid was removed, and the cells were resuspended in fresh medium with trituration. Cells (final, 2 × 105 cells/mL), medium, and beads (final, 0.09 mg beads/mL, approximately 550 beads/mL) were placed in a depression that had been milled into a Teflon block (Figure1E, 8735K67, McMaster-Carr, Robbinsville, NJ USA). The external dimensions of the block were 76 mm × 26 mm × 13 mm; the dimensions of the depression were 65 mm × 17 mm × 11 mm. We used about 0.5 mL of cell suspension per square centimeter of Teflon chamber floor. The cells and beads were gently shaken for a few seconds every 15 min for the first 90 min of culture. We subsequently dispersed the beads with a cell lifter (70–2180, Biologix, Lenexa, KS USA); this significantly decreased the clumping of the beads. Cell-coated beads were usually used for recording after 10 to 15 days in culture. We used passages 17 to 31. Half of the growth medium was replaced every 2 to 3 days. To replace the medium, the chamber was held at an incline to give the beads time to settle before half of the medium was replaced. Chambers with a siliconized glass (Sigmacote, Sigma-Aldrich, St. Louis, MO USA) bottom were occasionally used, but we did not determine that they were better than Teflon chambers. However, they did permit monitoring of the culture more easily than the opaque Teflon, and at least some of the beads were free of the monolayer. In pilot studies, hollow borosilicate glass beads with a 10 μm diameter (AGSCO Corporation, Wheeling, IL USA) appeared to be engulfed by the cells and no cilia were visible.

Bottom Line: In 47% of attempts, suction resulted in a seal with high input resistance.In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cancer and Cell Biology, University of Cincinnati, PO Box 670521, Cincinnati, OH 45267-0521, USA.

ABSTRACT

Background: Most cells in the body possess a single primary cilium. These cilia are key transducers of sensory stimuli, and defects in cilia have been linked to several diseases. Evidence suggests that some transduction of sensory stimuli by the primary cilium depends on ion-conducting channels. However, the tiny size of the cilium has been a critical barrier to understanding its electrical properties. We report a novel method that allows sensitive, repeatable electrical recordings from primary cilia. Adherent cells were grown on small, spherical beads that could be easily moved within the recording chamber. In this configuration, an entire cilium could be pulled into a recording microelectrode.

Results: In 47% of attempts, suction resulted in a seal with high input resistance. Single channels could be recorded while the cilium remained attached to the cell. When the pipette was raised into the air, the cell body was pulled off at the air-bath interface. The pipette retained the cilium and could then be immersed in various solutions that bathed the cytoplasmic face of the membrane. In excised cilia, ionic currents through ciliary channels were modulated by cytoplasmic Ca(2+) and transmembrane voltage.

Conclusions: Ciliary recording is a direct way to learn the effects of second messengers and voltage changes on ciliary transduction channels.

No MeSH data available.


Related in: MedlinePlus