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Doerner, Julia F

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Doerner

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Julia F

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Doerner, Julia F

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2013 - 20152

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Author's Publications: search.filters.isAuthorOfPublication.879082af-4e0d-4e5d-ae02-1ec0bc611754

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    Ion channels and calcium signaling in motile cilia
    (eLife Sciences Publications, Ltd, 2015) Doerner, Julia F; Delling, Markus; Clapham, David
    The beating of motile cilia generates fluid flow over epithelia in brain ventricles, airways, and Fallopian tubes. Here, we patch clamp single motile cilia of mammalian ependymal cells and examine their potential function as a calcium signaling compartment. Resting motile cilia calcium concentration ([Ca2+] ~170 nM) is only slightly elevated over cytoplasmic [Ca2+] (~100 nM) at steady state. Ca2+ changes that arise in the cytoplasm rapidly equilibrate in motile cilia. We measured CaV1 voltage-gated calcium channels in ependymal cells, but these channels are not specifically enriched in motile cilia. Membrane depolarization increases ciliary [Ca2+], but only marginally alters cilia beating and cilia-driven fluid velocity within short (~1 min) time frames. We conclude that beating of ependymal motile cilia is not tightly regulated by voltage-gated calcium channels, unlike that of well-studied motile cilia and flagella in protists, such as Paramecia and Chlamydomonas. DOI: http://dx.doi.org/10.7554/eLife.11066.001
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    Controlled delivery of bioactive molecules into live cells using the bacterial mechanosensitive channel MscL
    (2013) Doerner, Julia F; Febvay, Sebastien; Clapham, David
    Bacterial mechanosensitive channels are some of the largest pores in nature. In particular, MscL, with a pore diameter > 25 Å, allows passage of large organic ions and small proteins. Functional MscL reconstitution into lipids has been proposed for applications in vesicular-based drug release. Here we show that these channels can be functionally expressed in mammalian cells to afford rapid controlled uptake of membrane impermeable molecules. We first demonstrate that MscL gating in response to increased membrane tension is preserved in mammalian cell membranes. Molecular delivery is controlled by adopting an established method of MscL charge-induced activation. We then determine pore size limitations using fluorescently labeled model cargoes. Finally, we activate MscL to introduce the cell-impermeable bi-cyclic peptide phalloidin, a specific marker for actin filaments, into cells. We propose that MscL will be a useful tool for gated and controlled delivery of bioactive molecules into cells.