Single particle imaging reveals IFT-independent transport and accumulation of EB1 in Chlamydomonas flagella

The microtubule (MT) +-end tracking protein EB1 is present at the tip of cilia and flagella; 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 http://www.molbiolcell.org/content/suppl/2015/11/30/mbc.E15-08-0608v1.DC1 Supplemental Material can be found at: 2 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 indicative for an exchange with unbleached EB1 entering the flagella from the cell body. EB1 moved independently 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.