Abstract
The dynamic organization of signaling cascades inside primary cilia is key to signal propagation. Yet little is known about the dynamics of ciliary membrane proteins besides a possible role for motor-driven Intraflagellar Transport (IFT). To characterize these dynamics, we imaged single molecules of Somatostatin Receptor 3 (SSTR3, a GPCR) and Smoothened (Smo, a Hedgehog signal transducer) in the ciliary membrane. While IFT trains moved processively from one end of the cilium to the other, single SSTR3 and Smo underwent mostly diffusive behavior interspersed with short periods of directional movements. Statistical subtraction of instant velocities revealed that SSTR3 and Smo spent less than a third of their time undergoing active transport. Finally, SSTR3 and IFT movements could be uncoupled by perturbing either membrane protein diffusion or active transport. Thus ciliary membrane proteins move predominantly by diffusion, and attachment to IFT trains is transient and stochastic rather than processive or spatially determined. DOI:http://dx.doi.org/10.7554/eLife.00654.001.
Highlights
Membrane proteins explore cellular space by diffusion or motor-driven transport on the cytoskeleton, but the relative contributions of these processes in specific cellular contexts have not been directly assessed
Biotinylated Somatostatin receptor 3 (SSTR3) and Smo molecules were labelled on the cell surface by extracellular addition of a low concentration (50 pM) of monovalent streptavidin conjugated to Alexa647 fluorescent dye (Figure 1A)
By following the movements of single membrane proteins in the primary cilium, we find that previous models of cargo loading onto intraflagellar transport (IFT) trains are no longer tenable
Summary
Membrane proteins explore cellular space by diffusion or motor-driven transport on the cytoskeleton, but the relative contributions of these processes in specific cellular contexts have not been directly assessed. A prototypical system to study how proteins explore cellular space is the primary cilium, a surface-exposed compartment consisting of a microtubule-based axoneme ensheathed within a plasma membrane protrusion (Satir and Christensen, 2007). The requirement of the IFT machinery for ciliary assembly, and the bidirectional movements of IFT trains in mature cilia have been substantiated in every model system tested (Pedersen and Rosenbaum, 2008) and it is generally accepted that soluble and membrane-embedded cargo explore the ciliary space by loading onto IFT trains at one end of the primary cilium and unloading at the other end (Qin et al, 2004; Pedersen et al, 2006). There is limited direct experimental evidence to support this model of IFT-mediated transport of ciliary membrane proteins
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