Intraflagellar transport drives flagellar surface motility
- University of California, Berkeley, United States
- University of California, San Francisco, United States
- University of Texas Southwestern Medical Center, United States
- Albert Einstein College of Medicine, United States
Figures
Figure 1 with 1 supplement
IFT transports FMG1-B.
(A) (Top) Simultaneous tracking of anti-FMG1-B beads (red) and IFT27-GFP (green). (Bottom) Kymographs show that bead motility colocalizes with IFT trains during processive runs. Between the runs, …
Figure 1—figure supplement 1
Additional examples of simultaneous tracking of bead motility and IFT.
(A–C) Three additional examples for colocalization and correlated movement of IFT trains (IFT27 GFP) and beads. (D) Two-dimensional Gaussian fitting reveals the position of the bead and IFT …
Figure 2 with 4 supplements
Dynein-1b drives gliding motility.
(A) (Left) Kymograph of a gliding IFT20-GFP cell. A single retrograde IFT train transiently pauses (red arrow) and initiates the gliding movement of the cell toward the paused train. (Right) A …
Figure 2—figure supplement 1
Three additional examples for kymographs of gliding IFT27-GFP cells.
(A–C) Multiple retrograde IFT trains pause in the leading flagellum prior to gliding motility.
Figure 2—figure supplement 2
The procedure for Fourier space direction analysis.
(A) The original kymograph of an IFT27-GFP cell during gliding motility. (B) Magnitude of the fast Fourier transform of the kymograph. (C) Mask with colors assigned to different slopes. (D) The …
Figure 2—figure supplement 3
Lag time between IFT pausing and initiation of gliding motility.
(A) A kymograph of IFT trains in a uniflagellate gliding cell. (B) A schematic representing the timing and trajectories of paused IFT trains in the gliding cell shown in (A). Arrows show the time …
Figure 2—figure supplement 4
Three examples for kymograph of gliding uniflagellate IFT27-GFP cells.
(A)–(C) Multiple retrograde IFT trains pause prior to gliding motility.
Figure 3 with 1 supplement
Ciliobrevin D inhibits dynein-1b and stops gliding motility.
(A) Kymographs of IFT20-GFP cells treated with varying concentrations of ciliobrevin D. Images were acquired 5 min after addition of ciliobrevin D. (B and C) Frequency and speed of retrograde and …
Figure 3—figure supplement 1
Additional example for kymographs of IFT20-GFP cells treated with varying concentrations of ciliobrevin D.
https://doi.org/10.7554/eLife.00744.014
Figure 4
Mechanisms of reversal in gliding motility.
(A) The average intensity of all the frames in the first 60 s of Video 5 of an IFT27-GFP pf18 cell, showing the path of the gliding flagella. (B) To monitor IFT trains while the cells alter their …
Figure 5 with 1 supplement
Ca2+ is required for the pausing of IFT trains at flagellar adhesion sites.
(A) (Left) Kymograph of an IFT27-GFP cell adhering both of its flagella in the presence of 0.34 mM free Ca2+. Pausing (middle) and moving (right) IFT trains were split into separate kymographs by …
Figure 5—figure supplement 1
The analysis of IFT pausing in the presence and absence of free Ca2+ in media.
(A) Two additional examples for kymographs of IFT27-GFP cells adhering both flagella to the surface in the presence of 0.34 mM free Ca2+. Pausing (middle) and moving (right) IFT trains were split to …
Figure 6 with 4 supplements
Stall force measurements on single IFT trains.
(A) Schematic representation of combined optical trapping of bead motility and fluorescent tracking of IFT. (B) Simultaneous tracking of IFT27-GFP and bead motility. At t = 3 s, the microscope stage …
Figure 6—figure supplement 1
Three additional examples of simultaneous bead trapping and IFT27-GFP fluorescence tracking.
(A) A single anterograde IFT train stalls directly underneath the trapped bead for 20 s. (B and C) The bead shows multiple runs and escapes the trap. The optical trapping beam was on throughout the …
Figure 6—figure supplement 2
The offset between the bead position and IFT trains.
(A) Schematic for an in vitro fixed-trap assay with a single motor bound to a microtubule. (B) Schematic for the optical trap assay of Chlamydomonas bead motility (roughly to scale). In both (A and B…
Figure 6—figure supplement 3
Additional examples for bead motility under fixed trap.
(Top) Displacement records of bead motility in fla10ts cells show successive runs, stalling and releasing events along the anterograde direction at permissive temperatures. (Bottom) The bead …
Figure 6—figure supplement 4
The effect of antibody concentration on peak forces measurements.
(A) The fraction of beads moving along the flagellar surface as a function of the antibody concentration on the bead surface. (B) The fraction of stall, release and escape events along the …
Figure 7 with 1 supplement
Viscous drag of the membrane slows down the motility of IFT trains that carry beads.
(A) The average speed of FMG1-B antibody-coated beads and IFT trains. Coating of the coverslip surface with 0.7 mg/ml polylysine led to a ∼20% reduction in the speed of IFT motility (two tailed t-tes…
Figure 7—figure supplement 1
Measurement of viscous drag on bead movement along the surface of the flagellar membrane.
(A) A polystyrene bead was trapped by the laser beam and brought on top of a surface-immobilized flagellum. After the bead was bound to FMG-1B, the trap was oscillated 500 nm back and forth along …
Figure 8
Force measurements on temperature-sensitive mutants.
(A) Peak force histograms for IFT movement in pf1 fla10ts cells at 22°C and 34°C. Stalling events are less common than the release of the bead. The frequency of anterograde IFT trains is reduced …
Author response image 1
PSD response curve as a function of bead-trap separation. The linear range of the PSD is ±150 nm. PSD signal decreases as the bead separation exceeds 200–300 nm.
Author response image 2
Simultaneous tracking of IFT27-GFP and bead motility. At t = 3 s, the microscope stage was moved to bring the flagellum underneath the trapped bead. An IFT train stalls at the trap position (t = 5–8 …
Videos
Video 1
Simultaneous imaging of bead motility and IFT.
The left channel shows fluorescent beads coated with anti-FMG1-B. One bead displays bidirectional movement along the flagellum. The middle channel shows the movement of IFT trains within the …
Video 2
Pausing of IFT trains during gliding motility.
A, Gliding motility of an IFT20-GFP cell on a glass surface. A single IFT train tethered to the surface (arrow) immediately preceding the initiation of gliding motility. The size of the window is …
Video 3
Gliding motility of uniflagellate WT and dhc1b-3 cells.
Uniflagellate WT and dhc1b-3 Chlamydomonas cells glide with the flagellum in the lead. The size of the whole window is 47.0 × 11.9 µm. The data was collected at 10 frames/s under a bright-field …
Video 4
IFT motility at different concentrations of ciliobrevin D.
IFT27-GFP pf18 cells were immobilized on a glass coverslip. 0–150 µM ciliobrevin D was added to the cell culture and the movies were recorded at 10 frames/s within 2–10 min of drug treatment.
Video 5
Reversal of direction in gliding motility.
Gliding motility of an IFT27-GFP cell on a glass surface. The cell changes the direction and speed of gliding motility either by raising one of its flagella or by the pausing of one or more IFT …
