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.

A microtubule-based transport of protein complexes, which is bidirectional and occurs between the space surrounding the basal bodies and the distal part of Chlamydomonas flagella, is referred to as intraflagellar transport (IFT). The IFT involves molecular motors and particles that consist of 17S protein complexes. To identify the function of different components of the IFT machinery, we isolated and characterized four temperature-sensitive (ts) mutants of flagellar assembly that represent the loci FLA15, FLA16, and FLA17. These mutants were selected among other ts mutants of flagellar assembly because they displayed a characteristic bulge of the flagellar membrane as a nonconditional phenotype. Each of these mutants was significantly defective for the retrograde velocity of particles and the frequency of bidirectional transport but not for the anterograde velocity of particles, as revealed by a novel method of analysis of IFT that allows tracking of single particles in a sequence of video images. Furthermore, each mutant was defective for the same four subunits of a 17S complex that was identified earlier as the IFT complex A. The occurrence of the same set of phenotypes, as the result of a mutation in any one of three loci, suggests the hypothesis that complex A is a portion of the IFT particles specifically involved in retrograde intraflagellar movement.

Resonance Raman spectra of myoglobins reconstituted with hemes isotopically substituted at the central iron atom or the pyrrole nitrogen atoms have been recorded to address the issue of whether the strong line at ~220 cm"1 is the iron-histidine stretching mode or the iron-pyrrole nitrogen stretching mode. The frequency of the line at 220 cm'1 is 1.7 cm"1 lower in myoglobin reconstituted with the 57Fe heme than it is in the 54Fe-substituted heme. No large shifts were detected in any other Raman lines. When myoglobin reconstituted with 15N-substituted pyrrole nitrogens in the heme is compared to the unsubstituted myoglobin no large change is detected in the line at 220 cm"1, but the frequency of the line at 243 cm"1 is 1.5 cm"1 lower. In comparing myoglobin buffered in D20 to that buffered in H20 only the line at 220 cm"1 changes frequency (1.4 cm"1). From these isotopic substitution studies, we conclude that the line at ~220 cm"1 in myoglobin is the iron-histidine stretching mode. The mode at ~243 cm"1 has a significant contribution from the pyrrole nitrogens, and it is likely an out-of-plane pyrrole tilting mode. The 54Fe-57Fe isotope shift of 1.7 cm"1 in the 220-cm"1 line is smaller than predicted for a diatom oscillator of the iron and the histidine. We conclude that the iron-histidine stretching mode is either mixed with an internal mode of the histidine and/or mixed with skeletal modes of the porphyrin macrocycle.Resonance Raman scattering has been applied extensively to 7 AT&T Bell Laboratories.

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Human neutrophils (PMNs) express two receptors for the Fc domain of IgG: the transmembrane FcγRIIA, whose cytosolic sequence contains an immunoreceptor tyrosine-based activation motif, and the GPI-anchored FcγRIIIB. Cross-linking of FcγRIIIB induces cell activation, but the mechanism is still uncertain. We have used mAbs to cross-link selectively each of the two receptors and to assess their signaling phenotypes and functional relation. Cross-linking of FcγRIIIB induces intracellular Ca2+ release and receptor capping. The Ca2+ response is blocked by wortmannin and by N,N-dimethylsphingosine, inhibitors of phosphatidylinositol 3-kinase and sphingosine kinase, respectively. Identical dose-response curves are obtained for the Ca2+ release stimulated by cross-linking FcγRIIA, implicating these two enzymes in a common signaling pathway. Wortmannin also inhibits capping of both receptors, but not receptor endocytosis. Fluorescence microscopy in double-labeled PMNs demonstrates that FcγRIIA colocalizes with cross-linked FcγRIIIB. The signaling phenotypes of the two receptors diverge only under frustrated phagocytosis conditions, where FcγRIIIB bound to substrate-immobilized Ab does not elicit cell spreading. We propose that FcγRIIIB signaling is conducted by molecules of FcγRIIA that are recruited to protein/lipid domains induced by clustered FcγRIIIB and, thus, are brought into juxtaposition for immunoreceptor tyrosine-based activation motif phosphorylation and activation of PMNs.