Electron-tomographic analysis of intraflagellar transport particle trains in situ

Abstract

Intraflagellar transport (IFT) is the bidirectional movement of multipolypeptide particles between the ciliary membrane and the axonemal microtubules, and is required for the assembly, maintenance, and sensory function of cilia and flagella. In this paper, we present the first high-resolution ultrastructural analysis of trains of flagellar IFT particles, using transmission electron microscopy and electron-tomographic analysis of sections from flat-embedded Chlamydomonas reinhardtii cells. Using wild-type and mutant cells with defects in IFT, we identified two different types of IFT trains: long, narrow trains responsible for anterograde transport; and short, compact trains underlying retrograde IFT. Both types of trains have characteristic repeats and patterns that vary as one sections longitudinally through the trains of particles. The individual IFT particles are highly complex, bridged to each other and to the outer doublet microtubules, and are closely apposed to the inner surface of the flagellar membrane.

Figure 1.

TEM micrographs of flat-embedded flagella from WT C. reinhardtii cells containing IFT trains. (a) Low-magnification view of longitudinally sectioned flagella (arrowheads) and cell bodies (cb). (b) Four cross sections of WT flagella containing IFT trains. The arrows show some cross-sectioned IFT trains (IFT), located between the flagellar membrane and the axoneme and close to outer dynein arms (ODA). Arrowheads indicate the links between the IFT trains and B tubule of MT doublets. (c) A longitudinal section of a flat-embedded flagellum from a WT cell. The section is cut through the central core of the flagellum at the level of the central pair apparatus (cp). Two of the nine MT doublets (md) are visible beside the central pair. Underneath the flagellar membrane (m), IFT trains with two different morphologies are present: short trains are labeled with white arrowheads, whereas longer trains are marked with black arrowheads, and their extremities delineated with short black lines.

Figure 2.

TEM micrographs of in situ IFT trains in flat-embedded flagella from WT and fla14 mutant cells of C. reinhardtii. WT flagella display two types of trains, each with a characteristic length and ultrastructure. (a–d) Four short IFT trains (framed in red lines). (e) A magnified view of panel d showing that these trains are electron dense and compact, with an internal 16-nm repeat of small “lollipop-like particles.” (f–i) Four long IFT trains (framed in red lines). At higher magnification (l), these trains are less compact than short trains and are characterized by an arc-shaped pattern, not seen in short trains. (m, n, and o) Three long IFT trains from fla14 mutant cells, which lack retrograde IFT. These trains are characterized by a structure and a repeat very similar to those observed in long trains from WT cells.

Figure 3.

Length distribution of IFT trains in WT and fla14 flagella. (a) Table of the number of clearly identifiable and measurable long and short IFT trains in WT and fla14 flagella. (b and c) Histograms of the length distributions of IFT trains in flagella of WT (b) and fla14 (c) cells. The arrows mark the mean length (±SD) of the long (grey) and short (black) IFT trains.

Figure 4.

TEM images of cross sections of flagella from flat-embedded pf28 mutant C. reinhardtii cells, which lack outer dynein arms. (a) Cross section of pf28 flagella showing IFT trains located underneath the flagellar membrane (m). The arrowheads indicate the two links connecting the bilobed IFT particle to the B tubule of a MT doublet (md). The same structures can be observed in b–e (arrowheads). The IFT particle also shows links to the flagellar membrane (white lines). cp, central pair; g, glycocalyx (a membrane overlay composed of hydroxyproline-rich glycoproteins, which surrounds the C. reinhardtii flagella). (b–e) Cross sections showing bilobed IFT particles. Each IFT particle has connections with the flagellar membrane on one side and connections to the B tubule of the MT doublet on the other side (arrowheads). (f and g) Two consecutive serial cross sections of a flagellum from a pf28 mutant cell showing two side-by-side IFT trains on adjacent doublet MTs (arrows). (g) Adjacent IFT trains can occasionally contact each other laterally, forming wider structures that can extend for ∼70 nm over two neighboring MT doublets.

Figure 5.

Tomographic sections of flagella from fla14 mutant C. reinhardtii. (a and b) Approximately 13-nm-thick tomographic sections along the central planes of the axonemes. The central pair complex (cp), with its lateral projections, and the MT doublets (md) are visible. In the upper part of the pictures, two long IFT trains are visible in the space between the flagellar membrane (m) and the MT doublets (md). Notice the bulges of IFT trains accumulated in the space between the flagellar membrane and the MT doublets in the bottom part of panels a and b (white ovals), below the axonemes. White arrowheads indicate the links of IFT trains to the MT doublet. Black arrowheads indicate the links between the IFT particles and the flagellar membrane. The white circle in panel a shows one of the zones that has been selected from the tomogram for particle averaging so that each volume contains a tract of the MT doublet, three contiguous IFT particles, and a portion of the flagellar membrane. The inset shows a close up view of the structures in the white circle. Notice the association of IFT particles with the flagellar membrane. g, glycocalyx. (c–f) Montage of 0.66-nm-thick individual planes of tomograms reconstructed from flagella of fla14 cells. Planes were cut with the same orientation used in panels a and b, and colors were assigned to different flagellar structures using semiautomatic segmentation. Yellow, axonemal MTs; red, IFT trains; white, flagellar membrane; green, glycocalyx. (c and d) These sectioning levels show the links of IFT trains with the flagellar membrane and reveal a structural pattern very similar to that shown in Fig. 6, panel 1. (e and f) In fla14 flagella, IFT trains are frequently visible on top of each other, giving rise to multiple layers of IFT trains (arrows). All the images are oriented so that the tip of flagellum points to the left of the plate. Video 1 shows a tomographic reconstruction related to panel a.

Figure 6.

Sectioning of the 3D density map obtained by aligning and averaging IFT particles from fla14 mutant cells. The density map was obtained by averaging 15 tomographic volumes from a single train, each one containing three contiguous IFT particles as shown in the inset of Fig. 5 a. The images 1–6 were produced by serial sectioning along the z axis of the cubic volume shown on the left. To illustrate the spatial orientation of sectioning planes in the model, a cross section of it is shown on the frontal face of the cube and a longitudinal section is shown on the top face. The cutting planes, represented by the white lines on the cube, are numbered from 1 to 6, proceeding along the z axis from the top to the bottom of the 3D map. Each section is 0.66 nm thick. The glycocalyx, the flagellar membrane (m), the IFT, and the MT doublet (md) have been manually segmented using the same color coding shown in Fig. 5 (green, glycocalyx; white, membrane; red, IFT particles; yellow, MT doublet). This kind of rendering revealed fine structural details of IFT particles with patterning variations depending on the sectioning level of the 3D density map. Panels are oriented so that the tip of flagellum is oriented downward.

Figure 7.

Four different views of a surface-rendering representation of the 3D model of IFT particles in fla14 flagella. Colors have been assigned to subvolumes of the density map corresponding to different flagellar structures using the same color coding shown in Figs. 5 and 6 (white, flagellar membrane; red, IFT particles; yellow, MT doublets). In this and subsequent figures, the images are oriented so the flagella point down or to the right. (a) The white lines highlight the festooned profile of the IFT particles. The same wave pattern is seen in Fig. 6, panel 1, and Fig. 2 (f–o). (b) Cross-section view of the same model oriented so that the tip of flagellum points toward the reader. The white arrows indicate the position of the links between each IFT particle and the B subtubule of the MT doublet (B). A, A subtubule of the MT doublet. (c) Three pairs of IFT particles lying on the flagellar membrane (white wire net pattern) as seen from the interior of the axoneme. Oblique links (d) connect neighboring particles (white ovals) along each strand. Links (l), oriented parallel to the longitudinal axis of IFT trains, connect the heads of adjacent particles. The position of the links between IFT particles and the B subtubule of the MT doublet are labeled with a k, and may be the kinesin-2 motors. (d) The pairs of IFT particles as seen from the exterior of the flagellum. The yellow net behind them represents the outer surface of an MT doublet. 1, 2, 3, and 4 indicate the contact points between IFT particles and the flagellar membrane. Video 2 shows a rotation of the surface-rendered model.

Figure 8.

Comparison of sections of IFT trains from a tomogram and the 3D model. (a) Schematic representation of part of a tomogram from a fla14 flagellum. The selected volume contains part of an IFT train, located in the space between the flagellar membrane (m) and an MT doublet (md). (b and d) Virtual sections (∼15 nm thick) obtained by sectioning the reconstructed volume tangentially to the axoneme, between the flagellar membrane and the MT doublet (as shown in panel a), thus sectioning the IFT train longitudinally. The white arrows indicate the IFT particles aligned along the train. (c and e) Individual planes of IFT particles from fla14 flagella maps after tomographic reconstruction and particle averaging. The planes are cut with the same orientation and at levels comparable to those in panels b and d. Notice the strong similarity of IFT sectioning contours between panels b and c and panels d and e. Colored masks are used to highlight the similarity of the structures in panels b and c (yellow masks) and d and e (red masks), and the similarity between the particles composing each IFT unit. The arrowheads in panels d and e indicate the link between the top and the bottom IFT particles.

Figure 9.

Serial sectioning of tomograms obtained from flat-embedded flagella of WT and pf28 mutant C. reinhardtii cells showing short IFT trains. The ∼5-nm-thick virtual sections are cut along the longitudinal axis of the axoneme. The three selected cutting planes show ultrastructural features of the short IFT trains that are routinely observed in tomograms obtained from both WT and pf28 flagella. (a–c) Sections of a short train from a WT flagellum. (d–f) Sections of a short train from a pf28 flagellum. Panels a and d show the association of IFT trains with the flagellar membrane. Projections linking the IFT trains to the MT doublet, possibly cytoplasmic dynein 1b because these are retrograde IFT trains, are also visible at this sectioning level. Sections b and e are cut through the axial core of the IFT train, and show the presence of a rodlike structure running parallel to the longitudinal axis of the MT doublet with no internal periodicity. Panels c and f show sections of IFT trains cut through the lollipop-like particles visible along the IFT trains with a 16-nm repeat. (g–i) Colored diagrams superimposed onto panels d–f to highlight the ultrastructural features shown in a–f. White, flagellar membrane; red, IFT train; yellow/brown, MT doublet.

Figure 10.

Surface rendering of a short retrograde IFT train from a WT flagellum tomogram. The density map was semiautomatically segmented, and colors have been assigned as in previous models (white, flagellar membrane [m]; red, IFT particles [IFT]; yellow, MT doublets [md]). (a) Longitudinal view of the model. The IFT train is located in the space between the flagellar membrane and the MT doublet. The two ends of the IFT train have different appearances, which suggests the presence of a polarity in the retrograde train. (b) The retrograde IFT train as seen from the interior of the axoneme. (c) The IFT train as seen from the exterior of the axoneme. Note in b and c the asymmetry between the head and the tail ends, and the presence of repeating structures along the train. Black arrows in each frame indicate the flagellar tip orientation.