Abstract
Doublecortin X (DCX), known to be essential for neuronal migration and cortical layering in the developing brain, is a 40 kDa microtubule (MT)-associated protein. DCX directly interacts with MTs via its two structured doublecortin (DC) domains, but the dynamics of this association and the possible regulatory roles played by the flanking unstructured regions remain poorly defined. Here, we employ quantitative fluorescence recovery after photobleaching (FRAP) protocols in living cells to reveal that DCX shows remarkably rapid and complete exchange within the MT network but that the removal of the C-terminal region significantly slows this exchange. We further probed how MT organization or external stimuli could additionally modulate DCX exchange dynamics. MT depolymerisation (nocodazole treatment) or stabilization (taxol treatment) further enhanced DCX exchange rates, however the exchange rates for the C-terminal truncated DCX protein were resistant to the impact of taxol-induced stabilization. Furthermore, in response to a hyperosmotic stress stimulus, DCX exchange dynamics were slowed, and again the C-terminal truncated DCX protein was resistant to the stimulus. Thus, the DCX dynamically associates with MTs in living cells and its C-terminal region plays important roles in the MT-DCX association.
Highlights
Microtubules (MTs), the cytoskeletal polymers of tubulin, are critical contributors to cell mechanics, protein trafficking, signaling events and cell migration[1]
Whilst those studies reveal the positioning of Doublecortin X (DCX) on the MT lattice, highlighting DCX binding to the corner of four neighboring tubulin dimers[10], they present a static view of this interaction
Our choice was the COS-1 cell line because analyses in these cells would not be confounded by endogenous DCX and studies of cytoskeleton organisation and regulation are facilitated by the large, well-spread cytoplasm of these cultured cells[12]
Summary
Microtubules (MTs), the cytoskeletal polymers of tubulin, are critical contributors to cell mechanics, protein trafficking, signaling events and cell migration[1]. We have exploited quantitative fluorescence recovery after photobleaching (FRAP) protocols to reveal unanticipated rapid dynamics in the association of GFP-labelled DCX with MTs in living cells.
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