Geometric constrains for detecting short actin filaments by cryogenic electron tomography
© Kudryashev et al 2010
Received: 7 September 2009
Accepted: 5 March 2010
Published: 5 March 2010
Polymerization of actin into filaments can push membranes forming extensions like filopodia or lamellipodia, which are important during processes such as cell motility and phagocytosis. Similarly, small organelles or pathogens can be moved by actin polymerization. Such actin filaments can be arranged in different patterns and are usually hundreds of nanometers in length as revealed by various electron microscopy approaches. Much shorter actin filaments are involved in the motility of apicomplexan parasites. However, these short filaments have to date not been visualized in intact cells. Here, we investigated Plasmodium sporozoites, the motile forms of the malaria parasite that are transmitted by the mosquito, using cryogenic electron tomography. We detected filopodia-like extensions of the plasma membrane and observed filamentous structures in the supra-alveolar space underneath the plasma membrane. However, these filaments could not be unambiguously assigned as actin filaments. In silico simulations of EM data collection and tomographic reconstruction identify the limits in revealing the filaments due to their length, concentration and orientation.
PACS Codes: 87.64.Ee
Here, we aimed at visualizing actin filaments in Plasmodium berghei sporozoites using cryogenic electron tomography , which has readily revealed filaments in various cells types [29–32]. Plasmodium berghei is a rodent malaria parasite and sporozoites are the forms transmitted by the mosquito during a bite. Sporozoites move in a stick and slip fashion assisted by the formation and turnover of discrete adhesion sites, which regulate the overall speed of the parasites in an actin-dependent fashion . We have used cryogenic electron tomography of sporozoites to report new features of the microtubule cytoskeleton and of the subpellicular network associated with the inner face of the IMC [7, 34]. All these approaches imply that in principle the actin filaments should be detectable in the tomograms of sporozoites as well.
Only few studies report the presence of actin filaments in apicomplexan parasites despite the clear indication that actin is important for parasite motility and host cell invasion [5, 39]. In Toxoplasma, actin filaments were first reported from high-resolution low-voltage field emission scanning EM studies . A subsequent study showed that actin filaments are rate limiting for T. gondii tachyzoites motility and the filaments were visualized as parallel arrangements in freeze-dried platinum replicas . As expected filaments were not detected after application of cytochalasin D, an F-actin depolymerizing drug, while randomly arranged filaments could be found after application of jasplakinolide, an inhibitor of actin filament disassembly, which leads to disordered actin filament arrays [41, 42]. As the core machinery that drives parasite motility and invasion is conserved between Toxoplasma and Plasmodium, it appears likely that filaments are arranged in a similar fashion in T. gondii tachyzoites and Plasmodium sporozoites . We detected sparse filamentous structures of 20-200 nm length in the supra-alveolar space of P. berghei sporozoites. However, due to their undulating shape and rare appearance we cannot categorize them unambiguously as actin filaments. The question raises why cryogenic electron tomography, an imaging method that is exceptionally suitable for analyzing F-actin in whole intact eukaryotic cells [29–32] and has also revealed filaments in bacteria [38, 43–46] failed to visualize actin in Plasmodium sporozoites.
In the following paragraphs we discuss four possible reasons for this failure. One possibility is that the regulation of filament formation and its turnover, and thus filament length varies between the two apicomplexan parasites. Although little is known about parasite or stage specific effects of actin-binding proteins  an interesting recent study showed that the beta-subunit of the actin capping protein is important for ookinete and essential for sporozoite motility, while being dispensable for merozoite invasion . Also, a number of trans-membrane proteins of the TRAP family that play important roles in parasite motility are unique to the respective parasites and parasite forms [23, 49, 50].
A second possibility could be the reported shortness of actin filaments [9–11, 18]. Indeed, simulation experiments showed that actin filaments could be short enough to fall below the detection limit of cryo-electron tomography. Simulation experiments showed that short filaments will be most reliably detected if oriented in the direction of the electron beam and perpendicularly to the tilt axis (Figure 7 and 8). In our tomograms this ideal situation would be given if filaments link the IMC with the plasma membrane (Figure 7). However we found no such connectors, suggesting that either the filaments are less than 30 nm long, or we underestimated the level of noise for our simulations, or that this somewhat unexpected orientation does indeed not occur in sporozoites. Simulation experiments also showed that filaments oriented in the expected way in parallel to the IMC and plasma membrane are harder to detect if they are not oriented in the direction of the electron beam. This was the case for the filament-like structures described in figure 4, which could have thus been tempting to accept as true actin filaments. Such filaments, oriented perpendicular to the beam can give rise to the appearance of wrongly (90° tilted) oriented filaments that only appear in the direction of the electron beam when high noise is applied for the models (Figure 7). Lastly, amongst a high concentration of densities of different shapes it would be very hard to detect single filaments (Figures 7 and 8). For these reasons we cannot unambiguously define the detected filamentous structures as filaments, whether made from actin or other proteins. In contrast, long filaments can be readily detected by eye and by surface rendering algorithms. Obviously, both types of filaments are equally affected by the noise in tomograms; however, long filaments are suggestively easier to detect and trace. This "eye catching effect" is similar to what had been extensively studied in the early days of electron microscopy. Then, studies comparing point-to-point, line-to-line or plane-to-plane resolution in EM images indicated that the latter yielded the highest accuracy. Clearly, for reliable detection one would ideally employ independent techniques for tracking the continuity of objects.
Obviously, failing to detect actin filaments for example due to limited length does not rule out their presence. In the future, technical improvements such as implementation of phase plate tomography  and better detectors, as well as improved algorithms for data mining and analysis could lead to a shift in detecting structures in noisy tomograms and circumvent the size limit apparently needed for the detection of short actin filaments. In this respect it is curious to note the presence of long filopodia-like projections from the apical end of some sporozoites, which intuitively suggest that they might be caused by the polymerization of actin filaments in the supra-alveolar space. Much larger extensions were present at the apical end of T. gondii tachyzoites when the F-actin stabilizing drug jasplakinolide was applied . The bundles of long actin filaments were detected in such extensions. We failed to detect such structures in Plasmodium sporozoites when applying jasplakinolide (unpublished data) further suggesting that the way actin polymerization is controlled in these two parasites might differ in some important details. The material present within these extensions appeared similar with that in the supra-alveolar space. This would suggest that this structure is not strictly associated with the IMC, but is either soluble or associated to the plasma membrane, although such an association could be transient or unstable. However, no structural connectors could be found between the material and either the IMC or the plasma membrane, suggesting that the material is likely not tightly membrane bound. On the other hand the possible connecting molecules might be too rare or beyond the resolution of the method used in this study.
A third reason for not detecting filaments is the possibility that sporozoites imaged by cryogenic electron tomography were not motile at the time of freezing. If actin filaments only form at sites of adhesion to the substrate and are not formed anywhere else, this could lead to the absence of filaments in non-motile parasites, such as the ones that are not adhered to the surface of the EM grid. As we anticipated this problem, we aimed at establishing a correlative approach to first visualize motile sporozoites with the light microscope at ambient temperatures and then correlate these to images later recorded by cryo-light microscopy  and cryo-electron microscopy after plunge freezing the samples. This strategy allows correlation between light and electron microscopy for well adhering cells . Unfortunately, sporozoites often appeared to be displaced from the substrate during blotting right before plunge-freezing and could rarely be located by cryo-light or the cryo-electron microscope at the same sites as before freezing . However, even if we would have been successful in localizing motile sporozoites, filaments might only form at the contact sites of sporozoites to the grid, which unfortunately is the place yielding the lowest resolution in cryo-electron tomograms.
Lastly, it might be possible that during the blotting step just prior to plunge-freezing or during plunge-freezing itself the integrity of the plasma membrane might have been damaged. This could have led to a rapid depolymerization of the already short filaments. Indeed, we recently showed membrane leakage to occur in fibroblasts during blotting .
One possibility to circumvent these problems would be to image sporozoites lacking the sporozoite specific protein SPECT, which is needed for transmigration of cells [53, 54]. These sporozoites efficiently invade cells and it might be possible to trap sporozoites during the process of invasion without displacing them during blotting as they are intimately associated with the host cell. However, the additional plasma membrane and cytoplasm of the host cell that surrounds the parasite during this step  might increase the thickness of the ice layer thus decreasing the quality of the tomograms, making it harder to reveal actin filaments. Indeed, we recently described the difficulty of obtaining full tomographic tilt series from the thicker central regions of isolated sporozoites . To circumvent this problem, cryo-electron microscopy of vitrified sections (CEMOVIS) could be applied, which allows tomography of rapidly frozen and cryo-sectioned samples [55, 56]. Alternatively, merozoites could be imaged in the process of invading an erythrocyte ghost [57, 58]. However, even if actin filaments would be revealed, it would still be interesting to image actin filaments during sporozoite gliding as filaments might well be differently arranged during motility and host cell invasion.
Instead of using electron microscopy, it might also be feasible to identify actin filaments using optical nanoscopy methods [59, 60], especially as these can now be performed with classic fluorescent proteins . However, the maximum resolution of about 25 nm that these approaches routinely achieve might still be too little for identification of short actin filaments. It would be interesting for such an approach to first apply a similar simulation analysis as we present in Figures 7 and 8 prior to performing extensive imaging and image analysis. The use of proteins that bind only to F-actin but not to G-actin  could also be helpful in analyzing actin filaments during migration and invasion, possibly in combination with high resolution total internal reflection fluorescence microscopy .
In conclusion, by combining cryogenic electron tomography with in silico modeling we defined limits within which short filaments can be visualized in cells and discuss possible ways of circumventing these limits.
4. Materials and methods
4.1. Parasites and light microscopy
Plasmodium berghei (strain Nk65) sporozoites expressing cytoplasmic GFP were isolated from infected Anopheles stephensi salivary glands and imaged in serum free RPMI or phosphate buffered saline containing 3% bovine serum albumin . Sporozoites were then transferred either on EM grids (see below) or onto glass slides. Imaging was performed on an inverted Axiovert 200 M Zeiss microscope in an air-conditioned imaging suite at room temperature (24°C). Images were collected with a Zeiss Axiocam HRm every 1 second using the Axiovision 4.6 software and 63× objective lens (NA 1.40). Images were processed using ImageJ and figures assembled using the Adobe Creative suite package.
4.2. Cryo-electron tomography
was performed essentially as described before [7, 34]. Plasmodium berghei (strain Nk65) sporozoites were transferred onto EM carbon grids and incubated for 5-40 min. After removal of excess liquid by blotting with a filter paper, grids were rapidly plunged into liquid ethane and stored in liquid nitrogen. Grids were mounted in a Gatan cryo-holder (model: 626) and investigated using a cryo-electron microscope (FEI - CM 300 or FEI - Polara G2, both operating at the accelerating voltage of 300 keV, equipped with TWIN objective lens, field emission gun (FEG) and Gatan post column energy filter). The tilt series of low dose images (with a cumulative dose of under 10 000 electrons/nm2) were recorded on a 2048 pixel Gatan CCD camera, at a magnification of 43,000 (0,82 nm/pixel), and an objective lens defocus between -5 and -15 μm. We generally aimed at covering an angular range of -60° to 60° with 2° increment and filtered at zero energy loss. The recordings from the center of sporozoites frequently did not yield a full angle coverage . For this study we used a subset from a total of 50 tomograms of tilt images aligned using fiducial gold markers. These reconstructions were calculated by weighted back-projection using the 'EM-image processing package' . For visualization tomograms were filtered using non-linear anisotropic diffusion . Visualization, volume rendering, and segmentation were performed using the Amira package (TGS Europe S.A., France). Quantitative analysis of tomograms was performed with the TOM toolbox for Matlab .
4.3. Simulations of image formation
Filaments were generated with 5 nm diameter and three different lengths. The lengths of filaments were normally distributed with a standard deviation of the half the length around the average of 24, 48 and 72 nm, respectively. Filaments were iteratively placed in random locations at random orientations between walls (IMC and the plasma membrane) spaced 30 nm away from each other (Figure 8). Filaments were placed such that they did not overlap with the walls and the other filaments. Iterative placement of filaments finished with reaching two final volumes: 5% ("high concentration", Figure 8, top) and 0.7% ("low concentration", Figure 8, bottom) of the total volume available between the membranes. For the simulation the volume of the voxel was 0.823 nm3 as in most of the actual tomograms.
The initial volume was tilted with 2° steps from -60° to +60° degrees; at every step the projection was acquired.
To every projection a randomly generated white noise was added in Fourier space. The amount of noise was σprojection/σnoise0.5 ("low noise") and 0.15 ("high noise"); σ is the standard deviation from the mean, which was set to 0 for both the noise and the projection.
Convolution with the contrast transfer function (CTF) in Fourier space. Parameters used for generation of the CTF function with "tom_ctf" from the TOM toolbox : defocus: -10 μm, accelerating voltage: 300 keV, pixel size: 0.822 nm2; for all other parameters the default values were used.
Back projection of the projections to a three-dimensional volume (tomogram) customized in the TOM toolbox.
Volume rendered visualization using Amira 4. The threshold levels were calculated in a way that the number of pixels in the volume after reconstruction was the same as in the original volume. Views in figure 8, the top and middle columns in figure 7 are from the plane of the plasma membrane towards the IMC and perpendicular to the direction of the electron beam. Views in the third column in figure 7 are along the IMC and the plasma membrane in the direction of the electron beam.
We thank Diana Scheppan for mosquito infections, Stefan Bohn for help in data collection, Jürgen Plitzko and Günter Pfeiffer for help with electron microscopy as well as Stephan Hegge, Markus Ganter, Stephan Schmitz and Sylvia Münter for discussions and reading the manuscript. The work was funded by grants from the German Federal Ministry for Education and Research (BMBF, BioFuture) to FF and the German Research Foundation (DFG, SPP 1128) to FF and MC. Support from the Max Planck Society, the Medical School and the cluster of excellence CellNetworks at the University of Heidelberg as well as the Institute for Computational Modeling of the Siberian Branch of the Russian Academy of Sciences is acknowledged. FF is a member of the European Network of Excellence BioMalPar.
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