A second cytoplasmic dynein heavy chain (cDhc) has recently been identified in several organisms, and its expression pattern is consistent with a possible role in axoneme assembly. We have used a genetic approach to ask whether cDhc1b is involved in flagellar assembly in Chlamydomonas. Using a modified PCR protocol, we recovered two cDhc sequences distinct from the axonemal Dhc sequences identified previously. cDhc1a is closely related to the major cytoplasmic Dhc, whereas cDhc1b is closely related to the minor cDhc isoform identified in sea urchins, Caenorhabditis elegans, and Tetrahymena. The Chlamydomonas cDhc1b transcript is a low-abundance mRNA whose expression is enhanced by deflagellation. To determine its role in flagellar assembly, we screened a collection of stumpy flagellar (stf) mutants generated by insertional mutagenesis and identified two strains in which portions of the cDhc1b gene have been deleted. The two mutants assemble short flagellar stumps (<1-2 micrometer) filled with aberrant microtubules, raft-like particles, and other amorphous material. The results indicate that cDhc1b is involved in the transport of components required for flagellar assembly in Chlamydomonas.

Microtubules, purified by cycles of assembly and disassembly in vitro, are composed of tubulin and several microtubule-associated proteins (MAPs). When the MAPs were separated from the tubulin by phosphocellulose chromatography, the tubulin by phosphocellulose chromatography, the tubulin no longer assembled at 37 degrees C as measured by turbidity. If the MAPs and tubulin were recombined and warmed to 37 degrees C, microtubules assembled. MAPs stimulated tubulin assembly by affecting both the initiation and elongation processes. The effect on initiation was indicated by results showing an increase in initial rate and a decrease in average microtubule length as the MAP:tubulin ratio was increased. The initiation and elongation activities of the MAPs at 4 degrees C during which time the initiating activity decreased while the ability to affect the total amount of assembly remained constant. The decrease in initiating ability was correlated with the loss of the two major components of the MAP fraction, MAPs 1 and 2.

Tight junctions between brain microvessel endothelial cells (BMECs) maintain the blood-brain barrier. Barrier breakdown is associated with brain tumors and central nervous system diseases. Tumor cell-secreted vascular endothelial growth factor (VEGF) increases microvasculature permeability in vivo and is correlated with the induction of clinically severe brain tumor edema. Here we investigated the permeability-increasing effect and tight junction formation of VEGF. By measuring [(14)C]sucrose flux and transendothelial electrical resistance (TER) across BMEC monolayer cultures, we found that VEGF increased sucrose permeability and decreased TER. VEGF also caused a loss of occludin and ZO-1 from the endothelial cell junctions and changed the staining pattern of the cell boundary. Western blot analysis of BMEC lysates revealed that the level of occludin but not of ZO-1 was lowered by VEGF treatment. These results suggest that VEGF increases BMEC monolayer permeability by reducing occludin expression and disrupting ZO-1 and occludin organization, which leads to tight junction disassembly. Occludin and ZO-1 appear to be downstream effectors of the VEGF signaling pathway.

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.

Two structures on the distal ends of Chlamydomonas flagellar microtubules are described. One of these, the central microtubule cap, attaches the distal ends of the central pair microtubules to the tip of the flagellar membrane. In addition, filaments, called distal filaments, are observed attached to the ends of the Atubules of the outer doublet microtubules. Inasmuch as earlier studies suggested that flagellar elongation in vivo occurs principally by the distal addition of subunits and because it has been shown that brain tubulin assembles in vitro primarily onto the distal ends of both central and outer doublet microtubules, the presence of the cap and distal filaments was quantitated during flagellar resorption and elongation. The results showed that the cap remains attached to the central microtubules throughout flagellar resorption and elongation. The cap was also found to block the in vitro assembly of neurotubules onto the distal ends of the central microtubules. Conversely, the distal filaments apparently do not block the assembly of neurotubules onto the ends of the outer doublets. During flagellar elongation, the distal ends of the outer doublets are often found to form sheets of protofilaments, similar to those observed on the elongating ends of neurotubules being assembled in vitro. These results suggest that the outer doublet microtubules elongate by the distal addition of subunits, whereas the two central microtubules assemble by the addition of subunits to the proximal ends.KEY WORDS flagella microtubules microtubule assembly 9 Chlamydomonas membranes It has been shown by pulse labeling and light and electron microscope autoradiography that flagella (21, 22) and flagellar microtubules (34) assemble in vivo principally by the addition of subunits to their distal ends. These observations were made by amputating the flagella of certain protozoans and algae, allowing them to regenerate their flagella to half-length, and then adding a radioactive precursor, such as tritiated leucine, to the cells until flagellar regeneration was completed. After autoradiography, most of the incorporated radioactive precursor was observed to be in the distal (tip) half of the flagellum (21, 22) or flagellar microtubules (34), indicating that elongation oc-