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

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.

FIGURE 1:

The cellular distribution of endogenous and FP-tagged EB1 is similar. (A) Schematic presentation of the EB1-FP expression vector. The sequence for either GFP or NG were integrated into the genomic DNA encompassing the EB1 gene, including its endogenous promoter (pro) and terminator (term). The selectable marker gene aphVIII was present on the same plasmid. The arrows indicate the orientation of the genes. (B) Western blot analysis of whole cells and isolated flagella of wild type (control) and strains expressing EB1-GFP or EB1-NG probed with antibodies to EB1, and as loading controls, to IC78 and α-tubulin. The flagellar samples were 70 times more concentrated than the whole-cell samples (i.e., ∼140 flagella/cell). (C) Flagellar extracts from a EB1-GFP–expressing strain and a wild-type control were incubated with anti-GFP beads and the depleted extract (Unbound), the bound fraction (Eluate), and the original extract (Input), were analyzed by SDS–PAGE and Western blotting with anti-EB1. Note that endogenous EB1 copurifies with EB1-GFP. (D) Silver staining of the eluate obtained from a strain expressing EB1-GFP by GFP affinity purification. (E) DIC (a), TIRF (b), and the corresponding merged image (c) of live EB1-GFP cells. Scale bar: 3 μm. (F) Schematic representation (left) and live images of a focal series through a EB1-NG cell. Arrowheads in a, flagellar tips; arrows in d, punctae of EB1-NG in a posterior region of the cell. Scale bar: 3 μm.

FIGURE 2:

Fluorescent EB1 localizes to comets in the cell body. (A and B) Individual frames (A) and corresponding kymogram (B) from a recording of EB1-NG comets in the cell body. The comet marked by arrowheads moved from the flagella-bearing cell apex to its posterior, presumably tracking the tip of an elongating MT. Scale bar: 2 μm. (B) Kymogram of the comet marked in A. Dashed lines indicate time points corresponding to the frames in A. Arrowheads with A and P, anterior and posterior of the cell. Scale bar: 1 μm and 5 s. (C) Histogram depicting the distribution of the velocities of EB1-NG comets. (D, a–e) Single frames from a video depicting EB1-NG loss, presumably during catastrophic MT shortening. As described in other species, some residual EB1 remains attached to the length of the MTs in the C. reinhardtii cell body. The MT labeled by arrowheads is initially capped by EB1-NG (T0) and then retreats with time (T19–T37 in seconds). Scale bar: 2 μm. (f) Kymogram corresponding to a; the arrow indicates the trace corresponding to the EB1 signal labeled in a. (g) Kymogram showing growth and retreat of an EB1-NG comet. Arrowhead, elongation; arrows, catastrophe. Scale bars: 1 μm and 10 s. See Supplemental Movie S1.

FIGURE 3:

EB1 at the flagellar tip is rapidly exchanged independent of/unaided by IFT. (A and B) Individual frames (A) and corresponding kymogram (B) from a FRAP experiment demonstrating the exchange of EB1-NG at the tips of steady-state flagella. (A) Images taken before (pre) and at various time points (0–320 s) after photobleaching of the flagellar tip using a spot laser (position indicated by the dashed red circle). The dashed white box indicates the area used for FRAP analysis. In the kymogram (B), the flagellar tip and base and the bleaching step are indicated. Scale bars: 1 μm and 20 s. (C) Quantitative analysis of a FRAP experiment. The recovery of fluorescence (in arbitrary units, a.u.) at the flagellar tip was measured after photobleaching of the entire flagellum. The signal recovers to prebleach strength in ∼3 min. Arrowhead, bleaching step. (D) Kymograms depicting recovery of EB1-GFP in flagella of fla10-1 cells maintained at 22°C and 32°C. The base of the flagella (B) and the distal tip (T) are marked. Scale bars: 2 μm and 10 s. (E and F) Mean recovery rates of wild-type (E) and fla10-1 (F) cells expressing EB1-NG and EB1-GFP, respectively. Cells were analyzed at the permissive (22°C) and restrictive (32°C) temperatures for IFT in fla10-1. Error bars indicate the SD. The differences in the rates of fla10-1 and the control strain are likely to be caused by differences in the microscope settings. (G) Merged kymogram from simultaneous imaging of EB1-NG (green) and IFT20-mCherry (red) in flagella. Note that EB1 and IFT20 move independent of each other. The base of the flagellum (base) and the distal tip (tip) are marked. Scale bars: 2 μm and 10 s.

FIGURE 4:

Growing flagellar tips attract more EB1. (A) TIRF image of two EB1-NG cells, one with steady-state (arrows) and one with regenerating (arrowheads) flagella. Scale bar: 3 μm. (B) Mean fluorescence intensity of EB1-NG in the tip region of steady-state (SS; n = 15) and regenerating (REG; n = 17) flagella. (243 a.u., SD 113 a.u.; n = 17 vs. 90 a.u., SD 43 a.u., n = 15); Error bars indicate SD. Significance: p ≤ 0.0001. (C) TIRF image showing EB1-NG in the flagella of a long-short cell; the long flagellum is marked by an arrow, the short one by an arrowhead. Scale bar: 3 μm. (D) Bar graph showing the mean fluorescence intensity of EB1-NG at the tips of long (n = 12) and short (n = 12) flagella of long-short cell. Error bars indicate SD. Significance: p ≤ 0.01.

FIGURE 5:

Recovery of EB1-NG is not linked to tubulin exchange at the flagellar tip. (A–C) Gallery of individual frames (A), corresponding kymograms (B), and signal quantification (C) of a two-color FRAP experiment. (A) The distal flagellar region of a cell coexpressing EB1-NG and mCherry-α-tubulin was bleached using a laser spot that was moved along the flagellum (indicated by the dashed region), and FRAP was analyzed over several minutes (T0–T900 s). Top row, EB1-NG; bottom, mCherry-α-tubulin; arrowheads, flagellar tip. Scale bar: 1 μm. (B) Single-channel kymograms corresponding to A. The bleaching step and the orientation of the flagella are indicated. Scale bars: 1 μm and 10 s. (C) Quantification of the fluorescence intensity of EB1-NG (green) and mCherry-α-tubulin (red) corresponding to the photobleaching experiment depicted in A and B. The prebleach fluorescence intensity was set to 100 for both proteins; EB1-NG recovered rapidly and completely, while only traces of mCherry-α-tubulin were recovered even after 15 min of observation. See Supplemental Figure S4 for a similar experiment.

FIGURE 6:

Differential mobility of EB1-NG explains its accumulation at the flagellar tip. (A) Gallery of kymograms depicting diffusion of EB1-NG in flagella. Open arrows, particles with reduced mobility near the flagellar tip; open arrowheads, bleaching events; white arrows, EB1-NG particles preferably moving in one direction along the flagellar shaft; white arrowhead, reappearance of photobleached EB1-NG as it is infrequently observed for NG-tagged proteins. Note the reduced mobility of particles approaching the tip (compare c with d and e), transiently stationary EB1-NG at the tip (a, b, d, and e), and the differences in the time EB1-NG remains trapped at the tip. Scale bars: (a and b) 1 μm and 2 s; (c–e) 1 μm and 1 s. (B) Mean-square displacement vs. time plots for EB1-NG particles moving in the flagellar shaft (open squares; n = 41) or tip segment (filled squares; n = 14).

FIGURE 7:

Modeling EB1 distribution in flagella. The flagellum was modeled as a 12-μm-long line with a 1-μm-long low-mobility region (diffusion coefficient 0.06 μm²s⁻¹) at the tip and assuming a diffusion coefficient of 1.06 μm²s⁻¹ for the flagellar shaft of 11 μm. One hundred particles were released at the base of the line at T0. (A) Individual frames from a simulation. The line on the left indicates the position of the flagellar shaft and the low mobility region. See corresponding Supplemental Movie S7. (B) Plot of the fraction of particles in the tip segment vs. time based on the simulation described in A. Green line, expected share of particles in 1/12 of the flagellum assuming random distribution of the particles. (C) Left, to explore to what extent the geometry of the tip will result in an accumulation of particles, we performed a simulation similar to A but using the same particle diffusion coefficient along the entire flagellar length. Note the minimal accumulation of particles at the flagellar ends in this maximum-intensity projection over ∼1000 frames. Right: A similar maximum-intensity projection but using the conditions described in A. (D) Plot of the fraction of particles in the tip segment vs. time based on the single diffusion-coefficient simulation described in C. Green line as in B. (E) Histogram showing the distribution of dwell times in the tip region based on the simulation described in A.

FIGURE 8:

The role of diffusion and IFT in flagellar protein transport. The saturation of the orange background color indicates protein concentration, with dark colors specifying high concentrations. (A) Proteins with similar concentrations in the cell body cytoplasm and the ciliary matrix might not require IFT for transport. An example is EB1, which accumulates locally by being captured onto microtubule plus-ends. (B) Proteins in which the concentration in the ciliary matrix exceeds that in the cell body cytoplasm require IFT to be concentrated in the ciliary compartment. An example is tubulin, which enters cilia by diffusion and by IFT (Craft et al., 2015). IFT of tubulin and the tubulin concentration in the ciliary matrix are elevated during ciliary growth, presumably to allow for an efficient elongation of the axoneme. (C) Many proteins are abundant in the cell body but efficiently excluded from flagella. If such proteins are able to enter cilia by diffusion, IFT might function as a scavenger, exporting such proteins from cilia. An example in phospholipase D, which is retained to > 98% in the cell body of C. reinhardtii and removed from cilia in an IFT- and BBSome-dependent manner (Lechtreck et al., 2013a). In summary, IFT functions in moving proteins against concentration gradients into and out of cilia. While this is likely to apply to small proteins, which are able to diffuse through the transition zone, larger proteins might depend on IFT to pass through the transition zone.