If you are a seller for this product, would you like to suggest updates through seller support? Learn more about Amazon Prime. Read more Read less. Prime Book Box for Kids. About the Author Stephen M. Methods in Cell Biology Book 93 Hardcover: Academic Press; 1 edition December 7, Language: Be the first to review this item Amazon Best Sellers Rank: Related Video Shorts 0 Upload your video. Customer reviews There are no customer reviews yet.
Share your thoughts with other customers.
Following ejaculation, millions of motile sperm use their flagella to swim through the uterus and up into the oviducts. As the sperm cells move through the oviduct, they encounter a flow oriented against their direction of movement generated by oviduct cilia sweeping the ovum from the ovary toward the uterus. Among the nonmotile cilia, the kidney collecting duct and tubule cilia stand out as crucially important.
The wide distribution of cilia throughout the body explains why multiple organ systems are affected in the syndromic ciliary diseases discussed below. What are cilia and where are they found? Olfactory, oviduct, photoreceptor, and kidney cilia reprinted with permission from Kessel and Kardon ; heart cilia reprinted with permission from Willaredt and colleagues ; nodal cilia reprinted with permission from Follit and colleagues ; ependymal cilia reprinted with permission from O'Callaghan and colleagues ; respiratory cilia, reprinted with permission from Rosenbaum and Witman ; sperm on oocyte, micrograph: On the left is a longitudinal representation of the cilium.
On the right are cross sections at different levels, including the axoneme, transition zone, and basal body. The ciliary matrix , akin to the cell cytosol, is the soluble portion of the cilium bounded by the ciliary membrane and not attached to the axoneme.
These include the inner and outer dynein arms, which generate the force needed for ciliary movement; the radial spokes and central microtubule projections, which work together to regulate dynein arm activity; and a dynein regulatory complex that relays mechanical signals from the radial spokes to the dynein arms.
Not surprising for a superstructure of such complexity, the cilium is compositionally complex. Each of the major substructures of the axoneme is, itself, a large, multisubunit complex; for example, the outer dynein arm contains at least 16 different proteins, and the radial spokes contain over 20 different proteins. Even so, most axonemal proteins have not yet been identified with a specific substructure, so many important discoveries are waiting to be made.
The outer doublet microtubules are continuous with the triplet microtubules of the basal body, a cylindrical structure derived from mitotic centrioles. Between the basal body and the axoneme proper is the transition zone , a region that is a major focus of ciliopathy research.
Purchase Cilia: Model Organisms and Intraflagellar Transport, Volume 93 - 1st Edition. Print Book View all volumes in this series: Methods in Cell Biology. Cilia: Model Organisms and Intraflagellar Transport, Volume 93 (Methods in Cell Biology): Medicine & Health Science Books @ bahana-line.com
Several studies have now linked proteins normally found in the transition zone to a function in regulating ciliary protein composition. Despite the fact that cilia are composed of many hundreds of proteins, these proteins are not synthesized within the cilia. Therefore, new proteins incorporated into the growing or steady-state axoneme must be transported into cilia from their sites of synthesis in the cell body; many of these proteins are known to be incorporated into the axoneme at the ciliary tip Rosenbaum and Witman The question of how cells deliver axonemal proteins from the cell body to the ciliary tip was answered in part in with the description of intraflagellar transport IFT , a bidirectional movement of large particles along the doublet microtubules just beneath the flagellar membrane.
IFT is evolutionarily highly conserved and is required for the assembly of almost all cilia, including both primary and motile cilia, in every organism in which it has been studied. The locomotives for anterograde trains—the trains moving toward the tip—are members of the kinesin family of microtubule motors; retrograde trains are carried back to the cell body by another microtubule motor, a specialized cytoplasmic dynein. Both anterograde and retrograde IFT trains carry cargo, including both axonemal and membrane-associated proteins.
In further analogy to freight trains, some IFT trains include specialized cars that are adapted for carrying specific types of cargo. Although the specifics of IFT function have been somewhat elusive, some details have emerged over the last 15 years. In addition, it is now abundantly clear that IFT has important cargo transport functions other than transporting axonemal precursors for ciliary assembly. These pathways play crucial roles during embryonic development, which explains why many ciliopathies caused by defects in IFT are congenital developmental disorders rather than degenerative diseases.
In addition, for these pathways to function properly, the signaling receptors must be included or excluded from the ciliary membrane at specific times and under precise sets of conditions. Therefore, the regulation of ciliary membrane composition, which occurs at the basal body—transition zone region, is of special importance for human health.
To date, all diseases caused by defective cilia are due to mutations in the nuclear genome; as a result, all are inherited and many are manifested in the embryo or newborn. Below we discuss some of the most well characterized ciliopathies. Because cilia are so widespread in the human body, defects in cilia frequently cause syndromes—that is, collections of symptoms in which multiple tissues are affected by a single underlying cause.
Patients with PCD have chronic bronchitis and sinusitis because their cilia fail to clear mucus and inhaled bacteria out of their airways. The male patients are infertile, because of impaired motility of the sperm flagellum. About half of the patients have situs inversus due to impaired motility of the nodal cilia that initiate left—right asymmetry in the early embryo. When this flow is missing or abnormal, it is a matter of chance whether this signaling cascade is initiated on the left or right side; therefore, half of PCD patients have normal left—right asymmetry, and half have situs inversus.
Reprinted with permission from Hildebrandt and Zhou Reprinted with permission from Huber and Cormier-Daire Reprinted with permission from Aldahmesh and colleagues Reprinted with permission from Forsythe and Beales As more and more proteins that generate motility in the cilium have been identified, this has facilitated the discovery of the genes that cause PCD when they are mutated.
Most of these genes encode proteins of the outer dynein arms, the radial spokes, the dynein regulatory complex, and the central microtubule projections, as well as proteins necessary for preassembly of the dynein arms, which occurs in the cytoplasm. In general, mutations specifically affecting ciliary motility and causing PCD lead to a different set of symptoms than those affecting nonmotile cilia, which are discussed below. Polycystic kidney disease, or PKD, is the most common life-threatening inherited disease in humans, and it affects The disease comes in two major forms: Ultimately, this leads to end-stage renal failure.
The first hint of a connection between PKD and cilia came from the finding that homologues of the human proteins polycystin-1 and polycystin-2, which, together, account for most cases of ADPKD, are localized to the sensory cilia involved in the mating behavior of the nematode Caenorhabditis elegans Barr and Sternberg However, at that time, the function of the primary cilium was not known, and why an inability to form these cilia in the kidney would lead to ARPKD was not clear.
Because polycystin-1 and polycystin-2 interact to form a receptor-channel complex that acts at an early step in a signaling pathway that controls kidney epithelial cell differentiation and proliferation, it was concluded that the primary cilium was functioning as a sensory antenna, displaying these two PKD proteins to the environment and relaying signals from them to the cell body. Mutation of either of these two ciliary proteins, or an inability to assemble the cilium as a whole, as in the mouse model, results in the defective ciliary signaling that leads to PKD.
Therefore, PKD is a disease of the cilium. The entire outer segment is a modified cilium. Many of these proteins are likely to be dependent on IFT for their movement into the outer segment. Subsequent studies have documented similar results for mice lacking other IFT-particle proteins.
Therefore, it seems likely that photoreceptor cells are exquisitely sensitive to perturbations in the transport of proteins to the outer segment and respond by initiating apoptosis to commit cell suicide, thereby ensuring the elimination of defective cells from the retina. As was noted above, the connecting cilium of photoreceptor cells is thought to be a greatly elongated ciliary transition zone, and proteins that localize to the transition zone in other cell types localize to the connecting cilium of photoreceptor cells.
Interestingly, defects in these proteins seem to have a high probability of causing blindness. Inasmuch as the transition zone appears to form a barrier to the entry of nonciliary proteins into the cilium and may also be the site at which ciliary proteins are sorted to gain access to the cilium, it is likely that these functions are particularly important in photoreceptor cells. This may explain why the transition zone is so elongated in these cells and why the cells are so sensitive to defects in the sorting or trafficking of proteins to the outer segment. In disorders such as Leber's congenital amaurosis, in which degeneration of the photoreceptor cells is progressive whereas other organs are spared, there is a window between diagnosis and the complete loss of the photoreceptors during which the disorder could be amenable to gene therapy.
Although significant challenges remain, efforts are now underway to develop the methodology for similar rescue of photoreceptor cells affected by defects in ciliary proteins such as CEP As the group of ciliopathy-associated phenotypes has become more well defined, several additional syndromes have been identified as ciliopathies. These range in severity from disorders that include one or two common ciliopathy phenotypes that are not acutely life threatening to more severe lethal disorders combining multiple different symptoms. Skeletal malformations such as those in JATD are relatively common among ciliopathies and are shared to varying degrees by different syndromes.
They also highlight the importance of specific cilia-dependent signaling pathways for the development of the skeleton. As was mentioned above, hedgehog signaling is dependent on IFT in cilia and is required for normal skeletal patterning during development. The receptor protein for this pathway is found in the ciliary membrane, and once hedgehog ligand binds to this receptor, a complex series of events occurs involving the export of the receptor from the cilium, the trafficking of transcriptional regulatory proteins through the cilium and to the nucleus, and their ultimate activation of the downstream genes involved in normal tissue patterning Goetz and Anderson Another disorder with polydactyly as a common feature is BBS.
Although the exact mechanism of disease development is unclear, at least some of the clinical manifestations of BBS are likely to involve specific signaling defects. A number of other syndromic ciliopathies are caused by mutations in IFT-associated genes. However, IFT-A may be particularly closely connected with these disorders because causative mutations for these diseases have now been identified in the genes encoding all known IFT-A proteins. An interesting possible clue about a specific function for the IFT protein is that skeletal ciliopathies caused by other IFT-A mutations generally do not include retinal degeneration.
Some syndromic ciliopathies also have distinctive central nervous system CNS abnormalities as hallmarks of the diseases. One of the most well-studied examples is Joubert syndrome, which presents with extremely variable multiorgan involvement. Many of these defects, including retinal degeneration, polydactyly, and kidney disease are shared with other ciliopathies.
However, the diagnostic feature of Joubert syndrome is the presence of the molar tooth sign —a striking tooth-shaped abnormal growth of tissue visible in evaluations of the brain created through magnetic resonance imaging. Ninety percent of MKS cases have an occipital encephalocele, a protrusion of brain tissue and associated protective membranes through a hole in the back of the skull. MKS also includes the prenatal development of enlarged cystic kidneys, enlarged liver, and polydactyly Salonen and Paavola The combination of these multiple severe defects prior to birth explains the almost exclusively prenatal and perinatal lethality characteristic of MKS.
Pedigree analysis combined with modern DNA-sequencing technologies have led to the identification of a number of new candidate ciliopathy genes in recent years. Research in which immortalized human cells and primary cell culture from ciliopathy patients were used has also been important for answering some human-specific questions and for testing hypotheses generated by work in other organisms.
However, for obvious ethical reasons, many types of questions related to ciliopathy mechanisms cannot be answered directly by research on humans. In addition, the growth of tissue culture cells is expensive, and there are technical limitations on what can be accomplished with research on human cells. For instance, classical genetic analysis is not available in cell lines.
These cells cannot be mated, and, until very recently, no tools were available to easily make targeted gene knockouts in cell lines. In addition, the biochemical analysis of human cilia has so far been extremely challenging because of the need to grow large numbers of cells and the difficulty of isolating cilia in large quantities and high purity.
Because of the limitations of working with human cell lines, nonhuman model organisms have been crucial in the discovery of ciliopathies and the subsequent explosion of ciliopathy research. These different model organisms each have their own advantages, which are highlighted below. Importantly, some can be grown cheaply and easily and are ideal for use in the teaching laboratory.
Model organisms used in ciliopathy research. Reprinted with permission from Pazour and colleagues The WT is on the left, and a bbs4 mutant exhibiting obesity is on the right. It was originally identified in the green algae Chlamydomonas and has been discovered throughout the evolutionary tree. The IFT machinery is widely conserved and acts to establish, maintain, and disassemble cilia and flagella.
Understanding the role of IFT in cilium signaling and regulation requires a methodology for observing it directly. Here we describe current methods for observing the IFT process in mammalian primary cilia through the generation of fluorescent protein fusions and their expression in ciliated cell lines. The observation protocol uses high-resolution time-lapse microscopy to provide detailed quantitative measurements of IFT particle velocities in wild-type cells or in the context of genetic or other perturbations.
Direct observation of IFT trafficking will provide a unique tool to dissect the processes that govern cilium regulation and signaling. Cilia are microtubule-based projections that are present in organisms throughout the evolutionary tree. The motile forms, which in some organisms are referred to as flagella, are found in many single-celled organisms and tissues in metazoans and serve to drive the motion of fluid at the cellular surface. Primary or nonmotile cilia are found in metazoans where they are thought to act as a specialized extracellular signal transduction apparatus and as a privileged subcellular compartment for specific biochemical reactions.
Motile cilia have microtubule motor dynein-mediated interconnections between the outer doublets that promote ATP-dependent sliding, which, in turn, induces a bending motion of the entire structure and their characteristic whip-like motion. As a result, these cilia are not motile with the lone exception being the nodal cilia that determine left—right symmetry in the early embryo and have been thought to be a vestigial feature of the many cell types on which they are found [reviewed in Gerdes et al.
Cilia are dynamic structures that are assembled and maintained by a specialized, internal transport machinery termed intraflagellar transport IFT. Originally identified in the green algae Chlamydomonas , components of the IFT machinery have been found to be required for cilia formation in all ciliated organisms Cole et al.
The dynamicity of the cilium, as observed decades ago in classic deflagellation experiments in Chlamydomonas Rosenbaum et al. Anterograde activities deliver IFT particles to the tip of cilium and retrograde activities return them back to the cell body. Both motilities are tightly regulated to keep cilia intact and maintain them at their genetically specified lengths [ Dentler ; Marshall and Rosenbaum ; Marshall et al.
The disruption of IFT leads to defects in cilia assembly and maintenance, resulting in various human diseases, including polycystic kidney disease, first shown by Pazour et al. Primary cilia assembly and maintenance via intraflagellar transport. Primary cilia, often found on the apical surface of tubular or ductal epithelia, are formed and maintained via intraflagellar transport.
The delivery and removal of proteins is a dynamic process constantly turning over the axonemal and membrane components. The original observation of IFT was based on high-resolution differential interference contrast DIC imaging in the flagella of Chlamydomonas Kozminski et al. The dynamic motion of small membrane bulges supported the hypothesis that material required for flagellar assembly and maintenance may be transported along flagellum itself.
Subsequent electron microscopy and biochemical studies Cole et al. These provided a number of key insights based on where these proteins are localized in the complex architecture of the flagellum and through genetic perturbation their role in building and maintaining flagella. The identification of proteins of the IFT machinery has also enabled a flurry of activity in other model systems.
Homologues in other unicellular organisms Davidge et al. However the molecular description of the IFT particles provided a unique opportunity to make live observations through fusions of IFT proteins to fluorescent proteins. Fluorescent proteins have now been derived from many species since their original discovery in the jellyfish, Aequorea victoria, and expression in other systems Chalfie et al.
The first fluorescent protein fusion movement of IFT was described by Orozco et al. Since that time a number of systems have been used to observe IFT imaging via fluorescent protein fusions including Chlamydomonas Mueller et al. Central to these observations has been the ability to dissect particle velocities as well as distinctions between anterograde and retrograde motility.
While at this writing this technique is in its infancy, we expect it to be a core technique for understanding the complex process of cilium assembly, regulation, and signaling. Here we describe current techniques for the construction of mammalian cell lines stably expressing IFT-fluorescent protein fusions, high-resolution time-lapse imaging, and analysis of IFT particle transport. Generation of cell lines stably expressing fluorescent protein fusions to cilium proteins has been an important element of high-quality mammalian IFT protein imaging.
Transient transfection can result in high levels of expression that can itself interfere with cilium assembly but also results in high cellular background, such that primary cilia are not easily distinguished from the cell body by widefield fluorescence microscopy. A variety of methods exist to generate stably expressing cell lines: Here we describe a method of retroviral gene transfer we have used successfully to generate a number of murine inner medullary collecting duct IMCD cell lines stably expressing fluorescent proteins fusions to IFT and other cilium proteins.
The major steps are: Under optimal conditions this entire process requires 4—6 weeks. Murine inner medullary collecting duct IMCD cell line or parental cell line of choice. A variety of modern molecular reagents now permit rapid amplification and cloning of cDNA sequences into fluorescent protein fusions, so we leave the details of isolation of the cDNA of interest and cloning the fusion sequence open to the preference of the reader. However, there are a few guidelines we have used for the choice of cDNAs and the fluorescent protein. In many cases we have chosen to use cDNAs from the same species as the cell lines of interest e.
While the proteins, especially the IFT proteins, are strongly conserved, this choice eliminates cross-species issues when considering possible mislocalization problems that can arise. This becomes of particular importance if the fusion cDNA is being used to replace the endogenous protein, either in a cell line generated from genetically modified mice or in the context of an RNA interference RNAi -mediated knockdown. Fluorescent proteins FPs should be chosen for the imaging equipment available. The most popular FP is EGFP, the enhanced version of the green fluorescent protein codon-optimized for mammalian expression and available in a variety of reading frames.
The spectral characteristics of EGFP excitation maximum near nm are excellent for most fluorescence illuminators as well as confocal imaging with laser-based excitation. Another popular set of FPs are those with fluorescence emission in red and have taken many forms including mCherry and Tag-RFP being the most popular monomeric forms. Many of these are also compatible with a variety of fluorescence light sources. Once the fusion cDNA has been generated, transient transfection can be used to test cilium localization; however, as described above, this can result in overexpression and difficulty in cilium localization.
Our retroviral vector was constructed from a modified pBABE vector Morgenstern and Land, containing the blasticidin resistance gene expressed from a SV40 promoter that is independent of the viral long terminal repeat promoter. A packaging cell line derived from the human embryonic kidney cell line that expresses the MoMLV gag and pol genes GP but lacking a viral envelope gene was used to generate retroviral particles via cellular expression of a VSV-G amphotropic envelope protein.
First, this MoMLV-derived retrovirus has high stability for long-term storage.
Such viruses can infect a broad spectrum of cell types of mammalian as well as nonmammalian origin. After incubation of this mixture and addition of 1 ml of growth medium the transfection cocktail was immediately spread dropwise over GP cells and gently agitated to ensure uniform mixing. Approximately 10 h post-transfection, the transfection growth medium was gently replaced with 6 ml of fresh growth medium.
The growth medium containing the retroviral particles was collected 48 h after medium change. The retroviral supernatant was gently collected using ml syringe and filtered through a 0. The retroviral supernatant was ready for immediate use. An important parameter in choosing the parental cell line is the robust production of long primary cilia in culture.
Some cells require extensive culture after reaching confluence to produce primary cilia, whereas others produce cilia at high percentage even before the onset of confluence. The retrovirus integrates its viral genome, encoding the gene of interest, into the host genome after the infected cell has undergone nuclear envelope breakdown and mitosis.