Single-particle imaging reveals intraflagellar transport–independent transport and accumulation of EB1 inChlamydomonasflagella

Abstract

The microtubule (MT) plus-end tracking protein EB1 is present at the tips of cilia and flagella; end-binding protein 1 (EB1) remains at the tip during flagellar shortening and in the absence of intraflagellar transport (IFT), the predominant protein transport system in flagella. To investigate how EB1 accumulates at the flagellar tip, we used in vivo imaging of fluorescent protein-tagged EB1 (EB1-FP) in Chlamydomonas reinhardtii. After photobleaching, the EB1 signal at the flagellar tip recovered within minutes, indicating an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independent of IFT trains, and EB1-FP recovery did not require the IFT pathway. Single-particle imaging showed that EB1-FP is highly mobile along the flagellar shaft and displays a markedly reduced mobility near the flagellar tip. Individual EB1-FP particles dwelled for several seconds near the flagellar tip, suggesting the presence of stable EB1 binding sites. In simulations, the two distinct phases of EB1 mobility are sufficient to explain its accumulation at the tip. We propose that proteins uniformly distributed throughout the cytoplasm like EB1 accumulate locally by diffusion and capture; IFT, in contrast, might be required to transport proteins against cellular concentration gradients into or out of cilia.

The Intraflagellar Transport Machinery

“…The MT plus-end binding protein EB1 localizes to the flagellar tip in Chlamydomonas (Pedersen et al 2003), interacts with IFT172 in coimmunoprecipitation experiments (Pedersen et al 2005), and the flagellar localization of EB1 is abolished in the fla11 ts strain at the restrictive temperature (Pedersen et al 2003). Although these results would suggest a requirement for IFT172 in the anterograde transport of EB1, flagellar tip localization of EB1 does not depend on IFT (Pedersen et al 2003) and has recently been shown to occur by a diffusion and capture mechanism (Harris et al 2016). The underlying molecular mechanism by which IFT172 promotes IFT turnaround at the tip and the potential role for EB1 in this process remain elusive.…”

Cos2/Kif7 and Osm-3/Kif17 regulate onset of outer segment development in zebrafish photoreceptors through distinct mechanisms

“…In Chlamydomonas , it appears as if IFT is important for trafficking cargo such as tubulin under conditions of rapid growth, whereas diffusion can fully accommodate the movement of cargo under conditions of slow turnover or under steady-state conditions (Craft et al, 2015), (Harris et al, 2016). We propose that while osm-3/kif17 functions in initial OS morphogenesis, presumably through early disc assembly, both active transport via kinesin-II and passive transport via diffusion can compensate for loss of osm-3/kif17 (Fig.…”

Intraflagellar transport is required for the maintenance of the trypanosome flagellum composition but not its length

“…In a normal situation, anterograde IFT would return these proteins to the distal tip, hence contributing to both constitution and maintenance of the FLAM8 distal location. Such a principle could be applied to other distal tip proteins (Liu et al, 2010;Saada et al, 2014;Woodward et al, 1995) or IFT-independent mechanisms could also exist, as recently shown for the localization of EB1 at the end of flagellar microtubules in Chlamydomonas (Harris et al, 2016). The motor action of IFT could hence define a micro-domain at the distal end of the flagellum.…”