Single-cell
metabolite analysis plays an important role in biological
study. While mass spectrometry is a powerful tool for identification
and quantitation of metabolites, the low absolute analyte amounts
in single cell and difficulty in sampling represent significant challenges
in single cell analysis. In this study, we developed an effective
method with a simple sampling procedure for analyzing single cells.
A single cell was driven to a capillary tip through electro-migration,
followed by releasing the cell contents through electroporation, into
a sealed small volume (∼1.5 pL) to prevent dilution. Subsequent
mass spectrometry analysis was performed directly with nanoelectrospray
ionization. This method was applied for analyzing a variety of cells
and monitoring the metabolic changes in response to perturbed cell
culturing conditions. This method opens a new avenue for easy and
rapid analysis of single cells with high sensitivity.
Intraflagellar transport (IFT) is required for ciliary assembly and maintenance. While disruption of IFT may trigger ciliary disassembly, we show here that IFT mediated transport of a CDK-like kinase ensures proper ciliary disassembly. Mutations in flagellar shortening 2 (FLS2), encoding a CDK-like kinase, lead to retardation of cilia resorption and delay of cell cycle progression. Stimulation for ciliary disassembly induces gradual dephosphorylation of FLS2 accompanied with gradual inactivation. Loss of FLS2 or its kinase activity induces early onset of kinesin13 phosphorylation in cilia. FLS2 is predominantly localized in the cell body, however, it is transported to cilia upon induction of ciliary disassembly. FLS2 directly interacts with IFT70 and loss of this interaction inhibits its ciliary transport, leading to dysregulation of kinesin13 phosphorylation and retardation of ciliary disassembly. Thus, this work demonstrates that IFT plays active roles in controlling proper ciliary disassembly by transporting a protein kinase to cilia to regulate a microtubule depolymerizer.
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