William L. Dentler

Microtubule-membrane interactions in cilia and flagella.

Publisher Summary This chapter discusses the microtubule-membrane interactions in cilia and flagella. Cilia and eukaryotic flagella are specialized organelles that project from the cell surface. They are responsible for the movements of whole cells and for the movements of materials across cell surfaces. All eukaryotic cilia and flagella possess the same uniform substructure of nine doublet microtubules (the outer doublets) surrounding two single central microtubules. The microtubule-membrane bridges, along the long axis of the doublet microtubule, are the second major sites to which membranes are attached. These bridges are responsible for the attachment of the doublet microtubules to the ciliary necklace and ciliary granule plaques at the ciliary base, to accessory fibers in sperm, protozoans, and ctenophores, to extraciliary structures such as mastigonemes, and to adjacent ciliary or plasma membranes as in ctenophores, mussel gill laterofrontal cilia, and in trypanosomes. The morphological studies of cilia in a wide variety of organisms revealed that the microtubules comprising the axonemes are attached to the membrane where they are enveloped.

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-μm and 0.06-μm 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 μm/s and 2.7 μm/s, respectively, but retrograde IFT was significantly slower in flagella shorter than 4 μm. 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.

Regulation of flagellar length in Chlamydomonas

The length of eukaryotic cilia and flagella depends on the cell cycle-regulated assembly and disassembly of at least 9 doublet and 2 central microtubules, their associated proteins, and the surrounding membrane. In light-synchronized Chlamydomonas cells, flagella assembled to 10-14 microm in length near the beginning of the light period and they disassembled prior to cell division, during the dark period. Flagella on light-synchronized pf18 Chlamydomonas mutants grew to 10-12 microm near the beginning of the light period but shortened by 50% or more by the end of the light period. Flagellar length was cell-cycle regulated: when flagella were amputated at various times during the light period, new flagella regenerated to the lengths of control cells at that time of the light cycle. The later in the cycle pf18 cells were deflagellated, the shorter were the regenerated flagella. Flagellar shortening was not affected, in either pf18 or wild-type (wt) cells, by inhibitors of protein synthesis or of microtubule assembly, so flagellar length cannot depend on protein turnover. Shortening in pf18 was attenuated by Li+, which stimulated flagellar growth in wt cells, by red light, by protein kinase inhibitors, and by the Ca2+ channel blockers La3+ and Cd2+. Shortening was increased by cAMP, Na+, K+, and EGTA. Ca2+-CAM blockers did not affect pf18 shortening but they increased shortening in wt and fa1 cells. We propose that flagellar length is regulated by a signal transduction pathway that is sensitive to Ca2+ levels and red light.

Linkages between Microtubules and Membranes in Cilia and Flagella

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.

Defective flagellar assembly and length regulation in LF3 null mutants in Chlamydomonas

Four long-flagella (LF) genes are important for flagellar length control in Chlamydomonas reinhardtii. Here, we characterize two new null lf3 mutants whose phenotypes are different from previously identified lf3 mutants. These null mutants have unequal-length flagella that assemble more slowly than wild-type flagella, though their flagella can also reach abnormally long lengths. Prominent bulges are found at the distal ends of short, long, and regenerating flagella of these mutants. Analysis of the flagella by electron and immunofluorescence microscopy and by Western blots revealed that the bulges contain intraflagellar transport complexes, a defect reported previously (for review see Cole, D.G., 2003. Traffic. 4:435–442) in a subset of mutants defective in intraflagellar transport. We have cloned the wild-type LF3 gene and characterized a hypomorphic mutant allele of LF3. LF3p is a novel protein located predominantly in the cell body. It cosediments with the product of the LF1 gene in sucrose density gradients, indicating that these proteins may form a functional complex to regulate flagellar length and assembly.

Paclitaxel-induced microtubule stabilization causes mitotic block and apoptotic-like cell death in a paclitaxel-sensitive strain of Saccharomyces cerevisiae

Wild‐type Saccharomyces cerevisiae tubulin does not bind the anti‐mitotic microtubule stabilizing agent paclitaxel. Previously, we introduced mutations into the S. cerevisiae gene for β‐tubulin that imparted paclitaxel binding to the protein, but the mutant strain was not sensitive to paclitaxel and other microtubule‐stabilizing agents, due to the multiple ABC transporters in the membranes of budding yeast. Here, we introduced the mutated β‐tubulin gene into a S. cerevisiae strain with diminished transporter activity and developed the first paclitaxel‐sensitive budding yeast strain. In the presence of paclitaxel, cytoplasmic microtubules were stable to cold depolymerization. Paclitaxel‐treated cells showed evidence of a mitotic block, with an increase in large‐budded cells and cells with a 2N DNA content and DNA fragmentation, identified by FACS analysis and the TUNEL assay. In the presence of paclitaxel, the number of dead cells in cultures increased three‐fold and cells containing reactive oxygen species were present. We conclude that paclitaxel blocks mitosis in this strain, leading to an apoptotic‐like cell death. This strain will also be useful in further studies of the effect of microtubule dynamics on various cellular processes in S. cerevisiae. Copyright

Asymmetrical microtubule capping structures in frog palate cilia.

Capping structures at the distal tips of frog palate cilia are attached to the A- and central pair microtubules by electron-dense plug structures similar to those found in protozoan cilia and flagella and in epithelial cilia from invertebrates and vertebrates (W.L. Dentler, 1980, J. Cell Sci. 42, 207-220; W.L. Dentler and E.L. LeCluyse, 1982, Cell Motil. 2, 549-573). The caps in frog palate cilia are composed of a proximal shelf, to which doublets Nos. 1-3, 8, 9 and the central microtubules are attached and a larger distal cap to which doublets Nos. 4-7 are bound. The smaller proximal shelf is positioned to one side of the cilium and gives the cap an asymmetrical appearance. Striated ciliary rootlets attached to the basal bodies are also described. The smaller cap is placed on the same side of all cilia on the palate relative to both the direction of the effective stroke and the position of the rootlets. These results confirm that capping structures are common to most, if not all, cilia and provide direct evidence that they are precisely positioned on specific microtubules.

A Role for the Membrane in Regulating Chlamydomonas Flagellar Length

Flagellar assembly requires coordination between the assembly of axonemal proteins and the assembly of the flagellar membrane and membrane proteins. Fully grown steady-state Chlamydomonas flagella release flagellar vesicles from their tips and failure to resupply membrane should affect flagellar length. To study vesicle release, plasma and flagellar membrane surface proteins were vectorially pulse-labeled and flagella and vesicles were analyzed for biotinylated proteins. Based on the quantity of biotinylated proteins in purified vesicles, steady-state flagella appeared to shed a minimum of 16% of their surface membrane per hour, equivalent to a complete flagellar membrane being released every 6 hrs or less. Brefeldin-A destroyed Chlamydomonas Golgi, inhibited the secretory pathway, inhibited flagellar regeneration, and induced full-length flagella to disassemble within 6 hrs, consistent with flagellar disassembly being induced by a failure to resupply membrane. In contrast to membrane lipids, a pool of biotinylatable membrane proteins was identified that was sufficient to resupply flagella as they released vesicles for 6 hrs in the absence of protein synthesis and to support one and nearly two regenerations of flagella following amputation. These studies reveal the importance of the secretory pathway to assemble and maintain full-length flagella.

Microtubule-membrane interactions in ctenophore swimming plate cilia

The cilia in ctenophore swimming plates are organized into long rows and the cilia within each of the rows are connected to one another by interciliary bridges. The interciliary bridges form a type of intracellular junction and are periodically spaced at 15 nm intervals along the long axis of a cilium. The bridges bind adjacent cilia together even after dissolution of the ciliary membrane by non-ionic detergent. Interciliary bridges are attached to the compartmenting lamellae, which are paracrystalline structures composed of spherical particles which are periodically attached to the outer doublet microtubules at the sites to which the microtubule-membrane bridges are bound. It is proposed that the compartmenting lamellae are modifications of the ciliary microtubule-membrane bridge found in other eukaryotic cilia and that it is associated with a junctional complex that binds adjacent cilia together in swimming plates.

Recording and Analyzing IFT in Chlamydomonas Flagella

The transport of materials to and from the cell body and tips of eukaryotic flagella and cilia is carried out by a process called intraflagellar transport, or IFT. This process is essential for the assembly and maintenance of cilia and flagella: in the absence of IFT, cilia cannot assemble and, if IFT is arrested in ciliated cells, the cilia disassemble. The major IFT complex proteins and the major motor proteins, kinesin-2 and osm-3 (which transport particles from the cell body to ciliary tips) and cytoplasmic dynein 1b (which transports particles from ciliary tips to the cell body) have been identified. However, we have little understanding of the structure of the IFT particles, the cargo that these particles carry, how cargo is loaded and unloaded from the particles, or how the motor proteins are regulated. The focus of this chapter is to provide methods to observe and quantify the movements of IFT particles in Chlamydomonas flagella. IFT movements can be visualized in paralyzed or partially arrested flagella using either differential interference contrast (IFT) microscopy or, in cells with fluorescently tagged IFT components, with fluorescence microscopy. Methods for recording IFT movements and analyzing movements using kymograms are described.