Abstract
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.
Highlights
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)
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
Summary
2. Freeze-fracture ring for the freeze-fracture carrier (Leica 16706867). 3. Freeze-fracture carrier for Leica EM PACT2 high-pressure freezer (Leica 16706866). 4. Freeze-fracture bayonet pod to lock carrier and ring (Leica 16707829). 5. High-pressure freezer with rapid transfer system (Leica EM PACT2). I. Prepare 2% Formvar solution by dissolving 0.9 g of Formvar powder in 45 mL of ethylene dichloride. Incubate solution in 37 °C water bath. Prepare 50 μ M collagen solution by dissolving collagen in cold PBS solution. Check and adjust pH to 7.4 if not dissolved. These solutions are for cell culture use only. No additional solution is needed for HPF/FFTEM sample preparation and processing
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