Chemical-Free Technique to Study the Ultrastructure of Primary Cilium

A primary cilium is a hair-like structure with a width of approximately 200 nm. Over the past few decades, the main challenge in the study of the ultrastructure of cilia has been the high sensitivity of cilia to chemical fixation, which is required for many imaging techniques. In this report, we demonstrate a combined high-pressure freezing (HPF) and freeze-fracture transmission electron microscopy (FFTEM) technique to examine the ultrastructure of a cilium. Our objective is to develop an optimal high-resolution imaging approach that preserves cilia structures in their best natural form without alteration of cilia morphology by chemical fixation interference. Our results showed that a cilium has a swelling-like structure (termed bulb), which was previously considered a fixation artifact. The intramembrane particles observed via HPF/FFTEM indicated the presence of integral membrane proteins and soluble matrix proteins along the ciliary bulb, which is part of an integral structure within the ciliary membrane. We propose that HPF/FFTEM is an important and more suitable chemical-free method to study the ultrastructure of primary cilia.

HPF/FFTEM technique is a chemical-independent technique that uses rapid freezing and solution-free sample preparation.
The overall protocol for the HPF/FFTEM procedure is straightforward (Fig. 1). While some of the primary cilia are more obvious in one preparation compared with others (Supp Fig. 1), membrane fractures may not necessarily produce primary cilia (Supp Fig. 2). During the fracture of the cells, many fracture configurations are possible (Fig. 2). In most cases, however, a greater magnification is required to confirm Schematic diagram depicting the HPF/FFTEM procedure used in the cilia study. Each of the steps represents a key procedural step and is described in more detail in the Materials and Methods section. Note that the schematic cartoon does not reflect the actual products and mechanics of the instrument.
Scientific RepoRts | 5:15982 | DOi: 10.1038/srep15982 the structure of a cilium. The position of the cilium also dictates the clarity of the image. For example, the grid of the sample support mesh and/or the replica image of the granular cell body could potentially hinder capturing and analyzing the entire cilium. In a different case, the cilium (or microvilli) may not have been replicated throughout the entire ciliary shaft. We term this "angled cilium", in which only a short replica image of the cilium is observed. An optimal image of a cilium can be obtained when the cilium lays flat on the replica plate, and we call this "flat cilium".
Many flat cilia have bulging structures (Fig. 3). These bulging structures or bulbs can have spherical or irregular shapes. The size of the replicated bulbs can range from 80 nm to approximately 1 μ m. The biological functions of these bulbs were recently studied 5 . The significance of the bulb size, however, is still not clear. It is possible that the small bulbs result from a shallow fracture. However, various sizes of the bulb have also been observed during live-cell imaging, indicating that a cell may have the means to regulate the size and appearance of the bulb 5 . Live imaging of cilia can certainly circumvent the concerns of chemical fixation, but image resolution is limited to the wavelength of light used to observe the nanostructure of a cilium. Thus, the use of an electron microscope is preferred for the study of the singularity and nanostructure of a cilium.
Our results indicate that the ciliary bulb shares an intact structure along with the ciliary shaft, and there was no indication that the ciliary bulb is attached separately to the outer ciliary membrane. It is apparent that the bulb bilayer is part of the cilia bilayer. Furthermore, the HPF/FFTEM images show particle indentions along the ciliary membrane, including the ciliary bulb. In a replica of any cell membrane, scattered particles are often visible, indicating the existence of integral membrane proteins. This finding therefore suggests that the ciliary bulb and cilium itself contained integral and/or matrix proteins.
The main challenge with other TEM approaches is the detrimental effect of chemical fixative on cilia structure. Many fixatives, including 4% paraformaldehyde, could dramatically alter ciliary morphology (Fig. 4). In general, shorter and imbalanced cilia are observed when they are chemically fixed. The grossly damaged structure of the cilium makes it extremely difficult to obtain a nicely flat cilium for imaging purposes. We therefore believe that the challenge with many TEM approaches is the inability to obtain cilia with excellent structural integrity.
The freeze-fracture technique was first discovered in the 1950 s for the preparation of samples for electron microscopy 6,7 . Hall and Meryman introduced the sublimation of ice to reveal surface structures by using the combination of freezing and etching techniques. Over the years, the technique started to have more impact on the study of biological ultrastructure 8 . The effectiveness of this technique was further demonstrated when freeze fracture was performed in yeast cells to reveal the three-dimensional ultrastructure of the specimen 9,10 . Since then, the freeze-fracture technique has been a very useful tool some preparations are cleaner than others. Clean preparations for cilia study are those with less granular body. The grid of sample support mesh can also be seen. Incomplete replica image of cilium (angled cilium) can be due to a less optimal angle or position of the cilium during fracture imprint, or it may represent short microvilli. A cilium laying flat on the surface during fracture and imprint would generate a more complete length of cilium, which is termed flat cilium.
in studying the ultrastructure of lipid structures, fats and oils, membrane lipids, non-lipid lamellae, dry lipids thermotropic states, lyotropic states, lateral phase separation in lipid bilayers and biological membranes, non-lamellar lipid structures, single micelles, aggregates of micelles, bicontinuous cubic phases of type II, biological membranes and surface views or membrane splitting 8 . Shown here is a typical ciliary bulb, which indicates that the bulb membrane is continuous from the cilia bilayer and that particle indentions are observed in the ciliary membrane and bulb. Arrow indicates a cilium to be analyzed The HPF/FFTEM technique is certainly appropriate for all of these studies. Furthermore, we believe that the HPF/FFTEM technique is particularly suitable for studies associated with cytoskeletal-based cellular structures, such as microvilli, kinocilia, photoreceptor cilia, motile and non-motile cilia, and various junctional proteins or receptors whose structural activities depend on intermediate filaments. Our study, examining the nanostructure of the ciliary membrane using a chemical-free technique, is only a beginning. With rapid development of new ways to adopt the freeze-fracture technique and accessibility to image-reconstruction, we could potentially obtain 3D topology, cytoskeletal structure and protein localization within a cilium using TEM in the near future. Certainly, immunogold protein localization can be achieved with freeze-fracture replica. This method used to be very time-consuming, requiring five days to prepare one group of samples because the freeze fracturing allowed retrieval of only one of the two membrane fracture faces. With recent advancements in methodology, freeze fracture replica immunogold-labeling in biological samples has overcome many of the challenging issues associated with two apposed membrane fractures. The new approach offers an unambiguous identification of the membrane side and may become a standard and more reliable procedure 12 . Furthermore, freeze fracture can also be used to study cytoskeletal proteins. Coupled with deep etching or freeze etching, freeze fracture has been used to capture both microtubules and actin filaments 13,14 . Without doubt, the relatively very old procedure of freeze fracturing offers easy access to the interiors of samples that can A nice, smooth elongated cilium was observed in the controls (without treatment). Treatment with 4% paraformaldehyde for 5 minutes grossly damages the structural integrity of the cilium. After treatment, the cilium seems to be weakened and is not projecting up straight. All images were taken from the side-view with high-resolution differential interference contrast (DIC) microscope (Nikon TiU). Cells were then grown on collagen-coated tungsten wire to allow cilia to orient outward for visualization, as previously described 18 . otherwise only be reached by exceedingly difficult procedures with chemical fixation. The visibility of the ciliary membrane with freeze fracturing certainly provides a unique angle and is more advantageous.
A wide range of variations of FFTEM-related techniques has also been developed over the past half century and is still widely used for biological materials and some other soft-matter materials, such as liquid crystals. FFTEM is often considered to be a traditional or even "old" technique compared with some of the new developments, such as cryo-EM of vitreous sections (CEMOVIS) and freeze substitution. However, some of the advantages that FFTEM can provide are often found to be unique and no other alternative technique is comparable. For example, its unique surface sensitivity was found to be extremely useful in studies of complex liquid crystal systems 15 . For biological materials, one of the strengths of FFTEM is that the frozen membranes may tend to fracture along the central hydrophobic core, which makes FFTEM a valuable tool for the study of cilia. On the other hand, CEMOVIS and freeze substitution are both ultramicrotomy-based techniques. It is generally difficult to obtain similar information without extra effort, such as serial sectioning and 3D reconstruction.
Of note is that many other FFTEM techniques require various chemicals and cryoprotectants that can deteriorate the biological samples easily. On the other hand, other cryo-TEM techniques use plunge-frozen thin solution films and require blotting of the excessive solution with filter paper. In addition to a shorter sample processing time, the HPF/FFTEM technique is thus superior by avoiding the use of cryo-TEM and chemical fixation throughout the process. Like other imaging techniques, a high level of expertise is needed to implement the HPF/FFTEM protocol. However, the HPF/FFTEM protocol is simple enough that if trained properly, a new trainee should be able to perform the technique independently in a very short time.
In summary, we suggest that the ultrastructure of the mammalian primary cilium is best studied with no chemical fixative. The use of the HPF/FFTEM approach offers various advantages to the study of the ultrastructure of a cilium. Technically, the same cryo-fixation procedure can also be used for other TEM techniques, namely CEMOVIS (or cryo-TEM on thin specimens prepared by focused ion beam) and freeze substitution. Compared with the latter two, the HPF/FFTEM technique provides a much simpler process and is more focused on the membrane structure and ultrastructure of primary cilia. HPF/FFTEM also involves an inexpensive procedure to reveal the ultrastructure of the ciliary bulb without the use of any chemical fixation and to maintain the cilia morphology in its best natural form. This technique is therefore an indispensable tool to study the etiology of cilia-related diseases by offering high-resolution of the ultrastructure image of primary cilia. We propose that HPF/FFTEM is an important and more reliable chemical-free method for studying the ultrastructure of primary cilium, which is sensitive to chemical fixation.
Prepare 2% Formvar solution by dissolving 0. C. Note that cells on the gold-plated flat carriers can only be observed using upright or surgical microscope. Alternatively, to ensure cell growth on the gold-plated flat carrier, one of the gold-plated flat carriers can be transferred into a clean petri dish and incubated in trypsin to observe detached cells under a regular cell culture microscope.