Abstract. The unidirectional movements of the microtubule-associated motors, dyneins, and kinesins, provide an important mechanism for the positioning of cellular organelles and molecules. An intriguing possibility is that this mechanism may underlie the directed transport and asymmetric positioning of morphogens that influence the development of multicellular embryos. In this report, we characterize the Drosophila gene, Dhc64C, that encodes a cytoplasmic dynein heavy chain polypeptide. The primary structure of the Drosophila cytoplasmic dynein heavy chain polypeptide has been determined by the isolation and sequence analysis of overlapping cDNA clones. Drosophila cytoplasmic dynein is highly similar in sequence and structure to cytoplasmic dynein isoforms reported for other organisms. The Dhc64C dynein transcript is differentially expressed during development with the highest levels being detected in the ovaries of adult females. Within the developing egg chambers of the ovary, the dynein gene is predominantly transcribed in the nurse cell complex. In contrast, the encoded dynein motor protein displays a striking accumulation in the single cell that will develop as the oocyte. The temporal and spatial pattern of dynein accumulation in the oocyte is remarkably similar to that of several maternal effect gene products that are essential for oocyte differentiation and axis specification. This distribution and its disruption by specific maternal effect mutations lends support to recent models suggesting that microtubule motors participate in the transport of these morphogens from the nurse cell cytoplasm to the oocyte. M ICROTUB U LES provide the architectural framework on which many cellular organelles are transported. The microtubule polymer is an intrinsically polar structure resulting from the asymmetric, head-to-tail polymerization of the orb tubulin heterodimer (Amos et al., 1976;Luduena et al., 1977). Consequently, the two ends of a microtubule can be distinguished by their tendency to gain (the plus end) or lose (the minus end) tubulin subunits. At a basic level the regulation of microtubule-based transport within cells is determined by the polarity of microtubules and their associated motor proteins. This has been emphasized in recent years by demonstrations that microtubule motors use the energy derived from ATP hydrolysis to translocate in a single direction along the microtubule lattice (for reviews see Mclntosh and Porter, 1989;Goldstein, 1991;Vallee, 1993;Bloom, 1992;Walker and Sheetz, 1993). The depletion of endogenous nucleotides from cytoplasmic extracts increases the affinity between motors and microtubules and has allowed the purification of microtubule motors from a variety of organisms and cell types (Mclntosh and M.-G. Li and M. McGrail have contributed equally to this work.

Sequence comparisons and structural analyses show that the dynein heavy chain motor subunit is related to the AAA family of chaperone-like ATPases. The core structure of the dynein motor unit derives from the assembly of six AAA domains into a hexameric ring. In dynein, the first four AAA domains contain consensus nucleotide triphosphate-binding motifs, or P-loops. The recent structural models of dynein heavy chain have fostered the hypothesis that the energy derived from hydrolysis at P-loop 1 acts through adjacent P-loop domains to effect changes in the attachment state of the microtubule-binding domain. However, to date, the functional significance of the P-loop domains adjacent to the ATP hydrolytic site has not been demonstrated. Our results provide a mutational analysis of P-loop function within the first and third AAA domains of the Drosophila cytoplasmic dynein heavy chain. Here we report the first evidence that P-loop-3 function is essential for dynein function. Significantly, our results further show that P-loop-3 function is required for the ATP-induced release of the dynein complex from microtubules. Mutation of P-loop-3 blocks ATP-mediated release of dynein from microtubules, but does not appear to block ATP binding and hydrolysis at P-loop 1. Combined with the recent recognition that dynein belongs to the family of AAA ATPases, the observations support current models in which the multiple AAA domains of the dynein heavy chain interact to support the translocation of the dynein motor down the microtubule lattice.

The maize genome has been shown to contain six glutamine synthetase (GS) genes with at least four different expression patterns. Noncoding 3' gene-specific probes were constructed from all six GS cDNA clones and used to examine transcript levels in selected organs by RNA gel blot hybridization experiments. The transcript of the single putative chloroplastic GS2 gene was found to accumulate primarily in green tissues, whereas the transcripts of the five putative GS1 genes were shown to accumulate preferentially in roots. The specific patterns of transcript accumulation were quite distinct for the five GS1 genes, with the exception of two closely related genes.

The remodeling of the actin cytoskeleton is essential for cell migration, cell division, and cell morphogenesis. Actin-binding proteins play a pivotal role in reorganizing the actin cytoskeleton in response to signals exchanged between cells. In consequence, actin-binding proteins are increasingly a focus of investigations into effectors of cell signaling and the coordination of cellular behaviors within developmental processes. One of the first actin-binding proteins identified was filamin, or actin-binding protein 280 (ABP280). Filamin is required for cell migration (Cunningham et al. 1992), and mutations in human α-filamin (FLN1; Fox et al. 1998) are responsible for impaired migration of cerebral neurons and give rise to periventricular heterotopia, a disorder that leads to epilepsy and vascular disorders, as well as embryonic lethality. We report the identification and characterization of a mutation in Drosophila filamin, the homologue of human α-filamin. During oogenesis, filamin is concentrated in the ring canal structures that fortify arrested cleavage furrows and establish cytoplasmic bridges between cells of the germline. The major structural features common to other filamins are conserved in Drosophila filamin. Mutations in Drosophila filamin disrupt actin filament organization and compromise membrane integrity during oocyte development, resulting in female sterility. The genetic and molecular characterization of Drosophila filamin provides the first genetic model system for the analysis of filamin function and regulation during development.

Variations in subunit composition and modification have been proposed to regulate the multiple functions of cytoplasmic dynein. Here, we examine the role of the Drosophila ortholog of tctex-1, the 14-kDa dynein light chain. We show that the 14-kDa light chain is a bona fide component of Drosophila cytoplasmic dynein and use P element excision to generate flies that completely lack this dynein subunit. Remarkably, the null mutant is viable and the only observed defect is complete male sterility. During spermatid differentiation, the 14-kDa light chain is required for the localization of a nuclear "cap" of cytoplasmic dynein and for proper attachment between the sperm nucleus and flagellar basal body. Our results provide evidence that the function of the 14-kDa light chain in Drosophila is distinct from other dynein subunits and is not required for any essential functions in early development or in the adult organism. INTRODUCTIONThe minus-end-directed microtubule motor cytoplasmic dynein has been implicated in a variety of cellular processes, including nuclear envelope breakdown, mitotic spindle assembly and orientation, chromosome movements, intracellular trafficking of organelles and mRNAs, and intraflagellar transport (reviewed in Karki and Holzbaur, 1999). The heavy chain subunit of dynein is known to provide ATPase and microtubule binding functions, and although more than one cytoplasmic dynein heavy chain has been identified, the major cytoplasmic dynein motor contains a homodimer of a single heavy chain. It remains unclear how this single cytoplasmic dynein motor is targeted to distinct organelles and cellular processes. One hypothesis is that the accessory intermediate, light intermediate, and light chain subunits of cytoplasmic dynein mediate its functional specialization. Consistent with this idea, in some eukaryotes these subunits are encoded by multiple genes that are differentially expressed and/or alternatively spliced as distinct transcripts in different tissues and cells (Gill et al., 1994;Pfister et al., 1996a;Bowman et al., 1999;Susalka et al., 2000;Tynan et al., 2000;Tai et al., 2001). In addition, the posttranslational modification of subunits may contribute to the heterogeneity of subunit composition in the dynein complex . Although the mutational analysis of the cytoplasmic dynein heavy chain has revealed a range of motor functions, the functional contribution of other individual subunits is not well understood.The present study addresses the function of the 14-kDa dynein light chain. This light chain was first identified as a cytoplasmic dynein subunit in mammalian brain (King et al., 1996a) and as an axonemal dynein subunit within the specialized inner arm dynein of the Chlamydomonas flagella (Harrison et al., 1998). In Drosophila, a molecular study of the 14-kDa light chain gene reported defective male fertility for hypomorphic alleles, but the nature of the mutations left unresolved the significance of the 14-kDa light chain in cytoplasmic dynein (Caggese et al., 2001). Sequence analysis...