Intraflagellar transport and cilium-based signaling

Cilia are specialized structures that not only play diverse roles in cell motility but also transmit signals to the cytoplasm and nucleus to control gene expression, cell function, animal development, and behavior. Cilia are assembled and maintained by the intraflagellar transport (IFT) machinery, which coordinates rapid, bidirectional transport between the cell body and the distal tip of the cilium. A new study (Wang et al., 2006) illuminates the role of IFT in cilium-based signaling during mating in the alga Chlamydomonas.     

Intraflagellar transport

Eukaryotic cilia and flagella, including primary cilia and sensory cilia, are highly conserved organelles that project from the surfaces of many cells. The assembly and maintenance of these nearly ubiquitous structures are dependent on a transport system--known as 'intraflagellar transport' (IFT)--which moves non-membrane-bound particles from the cell body out to the tip of the cilium or flagellum, and then returns them to the cell body. Recent results indicate that defects in IFT might be a primary cause of some human diseases.     

Intraflagellar transport (IFT) during assembly and disassembly of Chlamydomonas flagella

Intraflagellar transport (IFT) of particles along flagellar microtubules is required for the assembly and maintenance of eukaryotic flagella and cilia. In Chlamydomonas, anterograde and retrograde particles viewed by light microscopy average 0.12-microm and 0.06-microm diameter, respectively. Examination of IFT particle structure in growing flagella by electron microscopy revealed similar size aggregates composed of small particles linked to each other and to the membrane and microtubules. To determine the relationship between the number of particles and flagellar length, the rate and frequency of IFT particle movement was measured in nongrowing, growing, and shortening flagella. In all flagella, anterograde and retrograde IFT averaged 1.9 microm/s and 2.7 microm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 mum. The number of flagellar IFT particles was not fixed, but depended on flagellar length. Pauses in IFT particle entry into flagella suggest the presence of a periodic "gate" that permits up to 4 particles/s to enter a flagellum.     

Intraflagellar transport and cilia-dependent diseases

Intraflagellar transport involves the movement of large protein particles along ciliary microtubules and is required for the assembly and maintenance of eukaryotic cilia and flagella. Intraflagellar-transport defects in the mouse cause a range of diseases including polycystic kidney disease, retinal degeneration and the laterality abnormality situs inversus, highlighting the important role that motile, sensory and primary cilia play in vertebrates.     

Intraflagellar transport proteins in ciliogenesis of photoreceptor cells

Background information. The assembly and maintenance of cilia depend on IFT (intraflagellar transport) mediated by molecular motors and their interplay with IFT proteins. Here, we have analysed the involvement of IFT proteins in the ciliogenesis of mammalian photoreceptor cilia. Results. Electron microscopy revealed that ciliogenesis in mouse photoreceptor cells follows an intracellular ciliogenesis pathway, divided into six distinct stages. The first stages are characterized by electron‐dense centriolar satellites and a ciliary vesicle, whereas the formations of the ciliary shaft and the light‐sensitive outer segment discs are features of the later stages. IFT proteins were associated with ciliary apparatus during all stages of photoreceptor cell development. Conclusions. Our data conclusively provide evidence for the participation of IFT proteins in photoreceptor cell ciliogenesis, including the formation of the ciliary vesicle and the elongation of the primary cilium. In advanced stages of ciliogenesis the ciliary localization of IFT proteins indicates a role in IFT as is seen in mature cilia. A prominent accumulation of IFT proteins in the periciliary cytoplasm at the base of the cilia in these stages most probably resembles a reserve pool of IFT molecules for further delivery into the growing ciliary shaft and their subsequent function in IFT. Nevertheless, the cytoplasmic localization of IFT proteins in the absence of a ciliary shaft in early stages of ciliogenesis may indicate roles of IFT proteins beyond their well‐established function for IFT in mature cilia and flagella.     

Intraflagellar transport: keeping the motors coordinated

Intraflagellar transport is a conserved delivery system that services eukaryotic cilia and flagella. Recent work in the nematode Caenorhabditis elegans has identified proteins required for the functional coordination of intraflagellar transport motors and their cargoes.     

Intraflagellar transport is essential for endochondral bone formation

While cilia are present on most cells in the mammalian body, their functional importance has only recently been discovered. Cilia formation requires intraflagellar transport (IFT), and mutations disrupting the IFT process result in loss of cilia and mid-gestation lethality with developmental defects that include polydactyly and abnormal neural tube patterning. The early lethality in IFT mutants has hindered research efforts to study the role of this organelle at later developmental stages. Thus, to investigate the role of cilia during limb development, we generated a conditional allele of the IFT protein Ift88 (polaris). Using the Cre-lox system, we disrupted cilia on different cell populations within the developing limb. While deleting cilia in regions of the limb ectoderm had no overt effect on patterning, disruption in the mesenchyme resulted in extensive polydactyly with loss of anteroposterior digit patterning and shortening of the proximodistal axis. The digit patterning abnormalities were associated with aberrant Shh pathway activity, whereas defects in limb outgrowth were due in part to disruption of Ihh signaling during endochondral bone formation. In addition, the limbs of mesenchymal cilia mutants have ectopic domains of cells that resemble chondrocytes derived from the perichondrium, which is not typical of Indian hedgehog mutants. Overall these data provide evidence that IFT is essential for normal formation of the appendicular skeleton through disruption of multiple signaling pathways.     

Protein particles in Chlamydomonas flagella undergo a transport cycle consisting of four phases

We used an improved procedure to analyze the intraflagellar transport (IFT) of protein particles in Chlamydomonas and found that the frequency of the particles, not only the velocity, changes at each end of the flagella. Thus, particles undergo structural remodeling at both flagellar locations. Therefore, we propose that the IFT consists of a cycle composed of at least four phases: phases II and IV, in which particles undergo anterograde and retrograde transport, respectively, and phases I and III, in which particles are remodeled/exchanged at the proximal and distal end of the flagellum, respectively. In support of our model, we also identified 13 distinct mutants of flagellar assembly (fla), each defective in one or two consecutive phases of the IFT cycle. The phase I-II mutant fla10-1 revealed that cytoplasmic dynein requires the function of kinesin II to participate in the cycle. Phase I and II mutants accumulate complex A, a particle component, near the basal bodies. In contrast, phase III and IV mutants accumulate complex B, a second particle component, in flagellar bulges. Thus, fla mutations affect the function of each complex at different phases of the cycle.     

Intraflagellar transport motors in cilia: moving along the cell's antenna

Intraflagellar transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo transport. I review evidence that kinesin-2 motors physically transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.     

Retrograde Intraflagellar Transport Mutants Identify Complex A Proteins With Multiple Genetic Interactions in Chlamydomonas reinhardtii

The intraflagellar transport machinery is required for the assembly of cilia. It has been investigated by biochemical, genetic, and computational methods that have identified at least 21 proteins that assemble into two subcomplexes. It has been hypothesized that complex A is required for retrograde transport. Temperature-sensitive mutations in FLA15 and FLA17 show defects in retrograde intraflagellar transport (IFT) in Chlamydomonas. We show that IFT144 and IFT139, two complex A proteins, are encoded by FLA15 and FLA17, respectively. The fla15 allele is a missense mutation in a conserved cysteine and the fla17 allele is an in-frame deletion of three exons. The flagellar assembly defect of each mutant is rescued by the respective transgenes. In fla15 and fla17 mutants, bulges form in the distal one-third of the flagella at the permissive temperature and this phenotype is also rescued by the transgenes. These bulges contain the complex B component IFT74/72, but not α-tubulin or p28, a component of an inner dynein arm, which suggests specificity with respect to the proteins that accumulate in these bulges. IFT144 and IFT139 are likely to interact with each other and other proteins on the basis of three distinct genetic tests: (1) Double mutants display synthetic flagellar assembly defects at the permissive temperature, (2) heterozygous diploid strains exhibit second-site noncomplemention, and (3) transgenes confer two-copy suppression. Since these tests show different levels of phenotypic sensitivity, we propose they illustrate different gradations of gene interaction between complex A proteins themselves and with a complex B protein (IFT172).CILIA and flagella are microtubule-based organelles that are found on most mammalian cells. They provide motility to cells and participate in many sensory processes. Defects in or loss of cilia/flagella cause a variety of human diseases that include polycystic kidney disease, retinal degeneration, infertility, obesity, respiratory defects, left–right axis determination, and polydactyly (Fliegauf et al. 2007). Mouse mutants demonstrate that cilia are essential for viability, neural tube closure, and bone development (Eggenschwiler and Anderson 2007; Fliegauf et al. 2007). Cilia and flagella are also present in protists, algae, moss, and some fungi.The assembly and maintenance of cilia and flagella require intraflagellar transport (IFT) (Kozminski et al. 1995). IFT involves the movement of 100- to 200-nm-long protein particles from the basal body located in the cell body to the tip of the flagella using the heterotrimeric kinesin-2 (anterograde movement) (Kozminski et al. 1995) and movement back to the cell body (retrograde movement) using the cytoplasmic dynein complex (Pazour et al. 1999; Porter et al. 1999). IFT particles change their direction of movement as well as their size, speed, and frequency at the ends of the flagella as they switch from anterograde to retrograde movement (Iomini et al. 2001). Biochemical isolation of IFT particles reveals that they are composed of at least 16 proteins and that these particles can be dissociated into two complexes in vitro by changing the salt concentration (Cole et al. 1998; Piperno et al. 1998). Recent genetic and bioinformatics analysis adds at least 7 more proteins to the IFT particle (Follit et al. 2009) (Eggenschwiler and Anderson 2007).

TABLE 1

Proteins and gene names for the intraflagellar transport particles in Chlamydomonas, C. elegans, and mouse

Intraflagellar transport and functional analysis of genes required for flagellum formation in trypanosomes

Intraflagellar transport (IFT) is the bidirectional movement of protein complexes required for cilia and flagella formation. We investigated IFT by analyzing nine conventional IFT genes and five novel putative IFT genes (PIFT) in Trypanosoma brucei that maintain its existing flagellum while assembling a new flagellum. Immunostaining against IFT172 or expression of tagged IFT20 or green fluorescent protein GFP::IFT52 revealed the presence of IFT proteins along the axoneme and at the basal body and probasal body regions of both old and new flagella. IFT particles were detected by electron microscopy and exhibited a strict localization to axonemal microtubules 3–4 and 7–8, suggesting the existence of specific IFT tracks. Rapid (>3 μm/s) bidirectional intraflagellar movement of GFP::IFT52 was observed in old and new flagella. RNA interference silencing demonstrated that all individual IFT and PIFT genes are essential for new flagellum construction but the old flagellum remained present. Inhibition of IFTB proteins completely blocked axoneme construction. Absence of IFTA proteins (IFT122 and IFT140) led to formation of short flagella filled with IFT172, indicative of defects in retrograde transport. Two PIFT proteins turned out to be required for retrograde transport and three for anterograde transport. Finally, flagellum membrane elongation continues despite the absence of axonemal microtubules in all IFT/PIFT mutant.     

Intraflagellar transport 20: New target for the treatment of ciliopathies

Cilia are ubiquitous in mammalian cells. The formation and assembly of cilia depend on the normal functioning of the ciliary transport system. In recent years, various proteins involved in the intracellular transport of the cilium have attracted attention, as many diseases are caused by disorders in cilia formation. Intraflagellar transport 20 (IFT20) is a subunit of IFT complex B, which contains approximately 20 protein particles. Studies have shown that defects in IFT20 are associated with numerous system -related diseases, such as those of the urinary system, cardiovascular system, skeletal system, nervous system, immune system, reproductive system, and respiratory system. This review summarizes current research on IFT20.We describe studies related to the role of IFT20 in cilia formation and discuss new targets for treating diseases associated with ciliary dysplasia.     

Intraflagellar transport protein 27 is a small G protein involved in cell-cycle control

BACKGROUND: Intraflagellar transport (IFT) is a motility process operating between the ciliary/flagellar (interchangeable terms) membrane and the microtubular axoneme of motile and sensory cilia. Multipolypeptide IFT particles, composed of complexes A and B, carry flagellar precursors to their assembly site at the flagellar tip (anterograde) powered by kinesin, and turnover products from the tip back to the cytoplasm (retrograde) driven by cytoplasmic dynein. IFT is essential for the assembly and maintenance of almost all eukaryotic cilia and flagella, and mutations affecting either the IFT motors or the IFT particle polypeptides result in the inability to assemble normal flagella or in defects in the sensory functions of cilia. RESULTS: We found that the IFT complex B polypeptide, IFT27, is a Rab-like small G protein. Reduction of the level of IFT27 by RNA interference reduces the levels of other complex A and B proteins, suggesting that this protein is instrumental in maintaining the stability of both IFT complexes. Furthermore, in addition to its role in flagellar assembly, IFT27 is unique among IFT polypeptides in that its partial knockdown results in defects in cytokinesis and elongation of the cell cycle and a more complete knockdown is lethal. CONCLUSION: IFT27, a small G protein, is one of a growing number of flagellar proteins that are now known to have a role in cell-cycle control.