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Correlative light and electron microscopy (CLEM) enables determination of high-resolution structural information for proteins of interest within their biological context through the combination of electron and fluorescence microscopies. Numerous electron microscopy (EM) studies of primary cilia have provided ultrastructural details about these antennal-like organelles that extend from the surface of the cell. The core structure of the cilium includes a microtubule-based axoneme, a basal body derived from the mother centriole, and the ciliary membrane, which is connected to the plasma membrane. The small GTPase Rab8 localizes to the ciliary membrane and is important for ciliogenesis, and Rab11 transports the Rab8 guanine nucleotide exchange factor (GEF) Rabin8 to the mother centriole to activate Rab8-dependent ciliary membrane growth. Some primary cilia have a ciliary pocket membrane (CPM) which is observed as an involution from the plasma membrane to the base of the cilia membrane. The Rab11- and Rab8-assocaited membrane trafficking regulator Eps15 Homology Domain-containing protein 1 (EHD1) and EHD3 also function in early stages of ciliogenesis; however, they localize to the CPM. These ciliary localizations of Rab8 and EHD1 can be resolved using CLEM with conventional fluorescence microscopy and transmission electron microscopy (TEM) imaging. Here, we describe in detail the protocol for this CLEM method applicable for ciliary proteins and proteins in other cellular organelles.

Recent evidence has indicated that caveolins are localized at the base of primary cilia, which are microtubule-based sensory organelles present on the cell surface, and that Caveolin-1 (CAV1) plays important roles in regulating ciliary membrane composition and function. Here we describe methods to analyze the localization and function of CAV1 in primary cilia of cultured mammalian cells. These include methods for culturing and transfecting mammalian cells with a CAV1-encoding plasmid or small interfering RNA (siRNA), analysis of mammalian cells by immunofluorescence microscopy (IFM) with antibodies against ciliary markers and CAV1, as well as methods for analyzing ciliary CAV1 function in siRNA-treated cells by IFM and cell-based signaling assays.

Defects in the gene encoding for the unconventional myosin VIIa leads to human Usher syndrome 1B, the most common form of hereditary combined blindness and deafness. To determine cellular function of myosin VIIa, we have investigated the subcellular localization of myosin VIIa in spacial relation relationship to potentially interacting proteins in mammalian photoreceptor cells. Western blot analysis of the axonemal fraction of photoreceptor cells by Western blot show that myosin VIIa and actin, as well as opsin, were present in the ciliary portion of the photoreceptors. Improved immunoelectron microscopy revealed that in mammalian photoreceptor cells, myosin VIIa was localized at the membrane of the connecting cilium linking photoreceptor inner segments with their outer segments. In addition, actin, the functional partner of myosins, actin, was detected at the ciliary membrane providing considerable evidence that myosin VIIa also interacts with actin filaments in the connecting cilium. Applying the highly-sensitive silver-enhanced indirect nanogold™-labeling for immunoelectron microscopy, intense anti-opsin immunoreactivity was recognized in the membrane of the connecting cilium indicating that rhodopsin trafficing to the outer segment occurs through the ciliary membrane. Co-localization of rhodopsin with the myosin VIIa-actin systems at the membrane also provides evidence for mysion VIIa driven ciliary transport of rhodopsin. Absence of myosin VIIa or presence of non-functioning myosin VIIa may lead to defects in the ciliary transport and cause retinitis pigmentosa in Usher syndrome 1B patients.

As first reported by Jennings (1906) and Mast (1906) Stentor coeruleus swims away from light sources (photodispersal) and collects in the more shaded areas of an unevenly illuminated container. Stentor are sensitive to light because they contain a pigment called stentorin (Moller, 1962; Walker et al., 1979), which releases H+ ions as a result of light absorption (Walker et al., 1981). This rapid proton release is thought to be an initial step in a series of steps which eventuate in photodispersal (Song et al., 1983). This paper summarizes the available evidence describing some of these intervening processes and the physiological mechanisms producing them.

The lipids of isolated cilia and flagella have been analyzed in only a few organisms. There is a substantial number of reports on the ciliary lipids of Tetrahymena. Vast differences in proteins and lipids exist between strains formerly grouped together as T. pyriformis. Thompson’s and Nozawa’s laboratories did their earlier work mainly with T. pyriformis amicronucleate strains W, E, or GL or the sexual strain, formerly known as WH-14 or mating type II, variety 1 and now designated as T. thermophila. These investigators have used the extremely thermotolerant strain, NT-1, in most of their recent studies. Most studies done by Conner and his colleagues have been on the amicronucleate strain, T. pyriformis W. These major contributors to our current understanding of Tetrahymena ciliary membrane lipids have done some of their analyses on isolated membrane preparations; all other workers have analyzed the entire organelle. Smith et al. (1970) and Kennedy and Thompson (1970) were the first to report on lipids of isolated cilia using T. thermophila and T. pyriformis E, respectively. These initial studies were followed by a report on T. thermophila by Jonah and Erwin (1971) who provided more detailed analyses of ciliary lipids, including fatty acid data. Thus, in addition to differences in analytical techniques, species and strain differences must be taken into consideration when apparent discrepancies occur in the Tetrahymena lipid literature

For species whose fertilization is internal, the oviduct is a part of the female genital tract which is the site for a number of different steps required for reproduction, including gamete transport, fertilization, and early embryonic development.

Cilia and flagella of eukaryotes are generally long, whiplike appendages extending from the cell body; historically, the term flagellum has been used when these structures are present singly or in small numbers, whereas the term cilium has been used when the structures occur in larger numbers. Typically, a flagellum propagates nearly symmetrical bends from the base to the tip of the organelle, causing the fluid in which it is beating to flow parallel to the flagellar axis (Fig. 1). Cilia generally move with an asymmetrical beat consisting of an effective and a recovery stroke. During the effective stroke a large bend is formed at the base of the cilium, causing the cilium to slice rapidly through the medium; this is followed by the recovery stroke, during which the bend is propagated along the ciliary shaft until the cilium returns to the position it held before the beginning of the effective stroke. The result of this beat pattern is that fluid is moved parallel to the cell surface in the direction of the effective stroke (Fig. 1). Cilia and flagella are very similar if not identical in terms of their internal structures and mechanisms of movement; indeed, the flagella of many organisms can beat with both flagellar and ciliary type waveforms. Consequently, most of my general comments about these organelles may be applied equally well to either cilia or flagella. The eukaryotic flagellum should not be confused with the prokaryotic flagellum, which is a completely different structure having a different protein composition and a different mechanism of movement (see Macnab, 1987a,b, for reviews). The prokaryotic flagellum is an extracellular appendage, whereas the eukaryotic flagellum is an intracellular organelle, surrounded by an extension of the cell’s plasma membrane. It is, of course, the interesting and frequently unique properties of ciliary and flagellar membranes that are the raison d’etre of this book.

Tetrahymena cells (Fig. 1) have upwards of 600 cilia depending upon the species and the stage in the cell cycle (Nanney, 1971). Of these, about 20% are in the oral apparatus (Williams and Bakowska, 1982). The cilia are easily detached (less so the oral cilia) by the application of mild shearing forces after various chemical treatments (reviewed by Nozawa, 1975). This, combined with the fact that some species will approach 106 cells/ml before entering stationary phase, has made Tetrahymena cilia a favored material for biochemical investigation. Studies of the surface membrane of Tetrahymena cilia will be reviewed here with an emphasis on biogenesis.

In comparison with ciliary and flagellar axonemes, relatively little is known about the structure, composition, or function of ciliary membranes or about the bridge structures that link the membranes to the microtubules. Bridges link the membrane to the microtubules at the ciliary bases, along the length of the axoneme, and to the distal tips of the doublet and central pair microtubules (Fig. 1). The role of each type of bridge structure in ciliary function is not understood but they appear to anchor the membrane to the microtubules, bind proteins or other structures to the external surface of the membrane, and may be involved with the transport of proteins up and down the flagellum. The capping structures linking the distal tips of the microtubules to the membrane may be responsible for the initiation and regulation of microtubule growth. This chapter will review some aspects of microtubule-membrane interactions in cilia and flagella with an expectation that the mechanisms regulating these interactions will provide insight into ciliary and flagellar function as well as aspects of microtubule function and assembly in the cytoplasm.

Vertebrate photoreceptors provide an excellent system for investigating the organization and maintenance of the membrane domains and cell polarity necessary for sensory function. In particular, the connecting cilium of photoreceptors appears to play an important role in photoreceptor organization. This specialized region corresponds structurally to the transition zone of motile cilia. As a consequence, studies of the structure and function of the photoreceptor connecting cilium are likely to provide insight into the function of the transition zone of motile and sensory cilia found more generally in eukaryotic cells. The purpose of this chapter is to review our current understanding of both the structure and function of the photoreceptor connecting cilium with emphasis on features that it may share with other cilia. The principal focus of recent research is on the role of the connecting cilium in the generation and maintenance of the polarized structure of photoreceptors. Details of the many recent advances in photoreceptor development, molecular biology, transduction, and membrane turnover are beyond the scope of this chapter, but are discussed in a series of recent reviews (Papermaster and Schneider, 1982; Bok, 1985; Besharse, 1982, 1986; Besharse et al., 1988; Liebman, 1987; Applebury and Hargrave, 1986; Adler, 1986).

Paramecium is a large unicell: of the most commonly studied species, P. tetraurelia can reach 150-200 µm in length, while P. caudatum may exceed 300 µm. The cell body of both species supports 5000-6000 cilia, which are individually covered by a membrane that collectively accounts for about half of the total cell surface area. The ciliary and somatic membranes are physically contiguous, but their biochemical compositions are quite distinct. How this segregation is achieved is an interesting problem in itself (for more details, the reader is referred to chapters in this volume by Bouck et al., Williams, Chailley et al., and Kaneshiro), but it ensures a functional specialization of the two surfaces. A primary role of the ciliary membrane is in providing a barrier between the locomotory apparatus of the cilium (the axoneme) and the external environment, but it is also involved in establishing physical contact between mating-competent cells. This cell agglutination reaction is discussed in some detail by Watanabe (this volume). Also, there is evidence to suggest that cilia may be sensitive to chemicals in the environment (Preston and Usherwood, 1988). However, it is perhaps in its role as an ion-selective filter that the ciliary membrane is most important to the day-to-day survival of the unicell.

When a forward-moving protozoan collides with a mechanical obstacle at its anterior end, it moves backward for a while, then resumes forward movement in a direction different from that before the collision. Thus protozoans avoid mechanical obstacles standing in their way in order to continue forward locomotion. The transient locomotor response has been termed an avoiding reaction (Jennings, 1906).

In 1972 Oschman and Wall observed the presence of electron-dense deposits (EDDs) along the plasma membranes of intestinal cells (1). They systematically tested the components of their fixative solutions and concluded that the combination of glutaraldehyde and millimolar concentrations of several divalent cations formed these deposits. Using what has become known as the Oschman and Wall fixation technique, most commonly employing glutaraldehyde and CaCl2, several workers have since reported the presence of similar EDDs which often occur at specific sites where Ca2+ action has been implicated. Examples of these reports are given in Table 1. The deposits characteristically form on the cytoplasmic side of surface membranes. X-ray microprobe analyses show that deposits contain Ca2+ (6,7,9,12,14,15,18). Sr2+, Ba2+, Co2+, Mn2+, Fe2+, Zn2+, Ni2+, and Mg2+ can also produce deposits but all monovalent and trivalent cations tested do not (1–3,11,13,14). Other Ca2+ salts such as Ca(NO3) produce EDDs, hence it is the cation and not the anion which is important for deposit formation (14). Deposits can be classified under at least two different groups—those associated with membranes and those contained within organelles with no apparent associations with membranes. In some cases membrane deposit sites are associated with sites known to contain intramembranous particles (IMPs) within the membrane bilayer and fibrillar material attached to the bilayer (8,11,14).

Although there has been a long standing debate on the number of phyletic Unes of photoreceptor structure evolution (Eakin, 1982; Salvini-Plawen, 1982; Vanfleteren, 1982), there seems to be a growing consensus that photoreceptor organelles may have arisen anew several times from photosensitive ciliated ectodermal cells (Salvini-Plawen and Mayr, 1977; Burr 1984). Modifications to a simple photosensitive cell leading to a more complex “eye” appear to be a “...polyphyletic reaction to respective selective pressures...” (Salvini-Plawen, 1982). This process has led to an extraordinarily diverse array of photoreceptor types. To generate this diversity of photoreceptors, modifications were presumably functionally adaptive to a particular group of animals. This basic premise implies that there is a positive correlation between the degree of structural complexity and photoreceptor function (Salvini-Plawen and Mayr, 1977), revealed either by its photophysiology or by the behavior of the animal. Yet, for many groups of simple invertebrates, and particularly cnidarians, there has been insufficient information to begin to examine this correlation.

Calcium influx through voltage-dependent Ca channels leads to a transient increase in intracellular Ca, which can initiate a number of biological processes, such as muscle contraction, synaptic transmission, secretion, ciliary motility, enzyme activation, growth and development. The electrical activity promoted by Ca channels is the most variable and widespread form of excitation. Ca channels are ubiquitous, from unicellular organisms to mammals, and Ca-dependent electrogenesis results in membrane depolarizations of wide range in amplitude and duration. Currents through Ca channels are diverse in their properties, and they can have great impact on the Ca homeostasis in cells. During the influx of Ca ions through open channels the initially low intracellular Ca activity of about 0.1 uM may increase several orders of magnitude within milliseconds.

Protozoa form a group of unicellular, (acellular), prokaryotes at the animal end of a broad spectrum of organisms, the other extremity being occupied by unicellular algae such as Desmids and Diatoms. Borderline groups, such as the fungus-like Mycetozoa (Myxomycetes) or the green flagellates lie in the no-man’s land, forever to be disputed by protozoologists and phycologists.

The overt behavior of many single celled eukaryotic organisms, as well as most sperm cells and some multicellular organisms, is, in large measure, swimming behavior determined by the physiological parameters controlling a single cell organelle, the cilium.1 The eukaryotic cilium is considerably more complex than the prokaryotic flagellum, both biochemically and structurally, but the larger diameter of the cilium permits the single living organelle to be used in physiological experiments.