RESUMEN
Primary cilia are solitary, non-motile extensions of the centriole found on nearly all nucleated eukaryotic cells between cell divisions. Only â¼200-300 nm in diameter and a few micrometres long, they are separated from the cytoplasm by the ciliary neck and basal body. Often called sensory cilia, they are thought to receive chemical and mechanical stimuli and initiate specific cellular signal transduction pathways. When activated by a ligand, hedgehog pathway proteins, such as GLI2 and smoothened (SMO), translocate from the cell into the cilium. Mutations in primary ciliary proteins are associated with severe developmental defects. The ionic conditions, permeability of the primary cilia membrane, and effectiveness of the diffusion barriers between the cilia and cell body are unknown. Here we show that cilia are a unique calcium compartment regulated by a heteromeric TRP channel, PKD1L1-PKD2L1, in mice and humans. In contrast to the hypothesis that polycystin (PKD) channels initiate changes in ciliary calcium that are conducted into the cytoplasm, we show that changes in ciliary calcium concentration occur without substantially altering global cytoplasmic calcium. PKD1L1-PKD2L1 acts as a ciliary calcium channel controlling ciliary calcium concentration and thereby modifying SMO-activated GLI2 translocation and GLI1 expression.
Asunto(s)
Canales de Calcio/metabolismo , Señalización del Calcio , Cilios/metabolismo , Proteínas Hedgehog/metabolismo , Orgánulos/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/química , Células Cultivadas , Citoplasma/metabolismo , Femenino , Proteínas Hedgehog/deficiencia , Proteínas Hedgehog/genética , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Masculino , Proteínas de la Membrana/química , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Nucleares/metabolismo , Receptores de Superficie Celular/química , Receptores de Superficie Celular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptor Smoothened , Proteína con Dedos de Zinc GLI1 , Proteína Gli2 con Dedos de ZincRESUMEN
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 labelled 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.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Canales Iónicos/metabolismo , Animales , Células CHO , Células COS , Línea Celular , Cricetinae , Electrofisiología , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Técnica del Anticuerpo Fluorescente , Células HeLa , Humanos , Canales Iónicos/genética , Faloidina/metabolismoRESUMEN
Transient receptor potential melastatin-like 7 (TRPM7) is a channel protein that also contains a regulatory serine-threonine kinase domain. Here, we find that Trpm7-/- T cells are deficient in Fas-receptor-induced apoptosis and that TRPM7 channel activity participates in the apoptotic process and is regulated by caspase-dependent cleavage. This function of TRPM7 is dependent on its function as a channel, but not as a kinase. TRPM7 is cleaved by caspases at D1510, disassociating the carboxy-terminal kinase domain from the pore without disrupting the phosphotransferase activity of the released kinase but substantially increasing TRPM7 ion channel activity. Furthermore, we show that TRPM7 regulates endocytic compartmentalization of the Fas receptor after receptor stimulation, an important process for apoptotic signaling through Fas receptors. These findings raise the possibility that other members of the TRP channel superfamily are also regulated by caspase-mediated cleavage, with wide-ranging implications for cell death and differentiation.
Asunto(s)
Apoptosis , Canales Catiónicos TRPM/metabolismo , Receptor fas/metabolismo , Animales , Caspasas/metabolismo , Línea Celular , Endocitosis , Humanos , Canales Iónicos/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Nanoparticle (NP)-mediated drug delivery typically relies on cargo release to occur passively or in response to environmental stimuli. Here we present a delivery method based on light-activated disruption of intracellular vesicles after internalization of biofunctionalized mesoporous silica nanoparticles loaded with cargo. This method combines the power of targeted delivery with the spatiotemporal control of light activation. As an example, we delivered a cell-impermeable fluorescent compound exclusively to the cytosol of multidrug resistant cancer cells in a mixed population.