The auto-inhibitory domain and ATP-independent microtubule-binding region of Kinesin heavy chain are major functional domains for transport in the Drosophila germline.

The major motor Kinesin-1 provides a key pathway for cell polarization through intracellular transport. Little is known about how Kinesin works in complex cellular surroundings. Several cargos associate with Kinesin via Kinesin light chain (KLC). However, KLC is not required for all Kinesin transport. A putative cargo-binding domain was identified in the C-terminal tail of fungal Kinesin heavy chain (KHC). The tail is conserved in animal KHCs and might therefore represent an alternative KLC-independent cargo-interacting region. By comprehensive functional analysis of the tail during Drosophila oogenesis we have gained an understanding of how KHC achieves specificity in its transport and how it is regulated. This is, to our knowledge, the first in vivo structural/functional analysis of the tail in animal Kinesins. We show that the tail is essential for all functions of KHC except Dynein transport, which is KLC dependent. These tail-dependent KHC activities can be functionally separated from one another by further characterizing domains within the tail. In particular, our data show the following. First, KHC is temporally regulated during oogenesis. Second, the IAK domain has an essential role distinct from its auto-inhibitory function. Third, lack of auto-inhibition in itself is not necessarily detrimental to KHC function. Finally, the ATP-independent microtubule-binding motif is required for cargo localization. These results stress that two unexpected highly conserved domains, namely the auto-inhibitory IAK and the auxiliary microtubule-binding motifs, are crucial for transport by Kinesin-1 and that, although not all cargos are conserved, their transport involves the most conserved domains of animal KHCs.

Cytoplasmic streaming in Drosophila oocytes varies with kinesin activity and correlates with the microtubule cytoskeleton architecture.

Cells can localize molecules asymmetrically through the combined action of cytoplasmic streaming, which circulates their fluid contents, and specific anchoring mechanisms. Streaming also contributes to the distribution of nutrients and organelles such as chloroplasts in plants, the asymmetric position of the meiotic spindle in mammalian embryos, and the developmental potential of the zygote, yet little is known quantitatively about the relationship between streaming and the motor activity which drives it. Here we use Particle Image Velocimetry to quantify the statistical properties of Kinesin-dependent streaming during mid-oogenesis in Drosophila. We find that streaming can be used to detect subtle changes in Kinesin activity and that the flows reflect the architecture of the microtubule cytoskeleton. Furthermore, based on characterization of the rheology of the cytoplasm in vivo, we establish estimates of the number of Kinesins required to drive the observed streaming. Using this in vivo data as the basis of a model for transport, we suggest that the disordered character of transport at mid-oogenesis, as revealed by streaming, is an important component of the localization dynamics of the body plan determinant oskar mRNA.

Developmental RNA processing of 3'UTRs in Hox mRNAs as a context-dependent mechanism modulating visibility to microRNAs.

The Drosophila Hox gene Ultrabithorax (Ubx) controls the development of thoracic and abdominal segments, allocating segment-specific features to different cell lineages. Recent studies have shown that Ubx expression is post-transcriptionally regulated by two microRNAs (miRNAs), miR-iab4 and miR-iab8, acting on target sites located in the 3' untranslated regions (UTRs) of Ubx mRNAs. Here, we show that during embryonic development Ubx produces mRNAs with variable 3'UTRs in different regions of the embryo. Analysis of the resulting remodelled 3'UTRs shows that each species harbours different sets of miRNA target sites, converting each class of Ubx mRNA into a considerably different substrate for miRNA regulation. Furthermore, we show that the distinct developmental distributions of Ubx 3'UTRs are established by a mechanism that is independent of miRNA regulation and therefore are not the consequence of miR-iab4/8-mediated RNA degradation acting on those sensitive mRNA species; instead, we propose that this is a hard-wired 3'UTR processing system that is able to regulate target mRNA visibility to miRNAs according to developmental context. We show that reporter constructs that include Ubx short and long 3'UTR sequences display differential expression within the embryonic central nervous system, and also demonstrate that mRNAs of three other Hox genes suffer similar and synchronous developmental 3'UTR processing events during embryogenesis. Our work thus reveals that developmental RNA processing of 3'UTR sequences is a general molecular strategy used by a key family of developmental regulators so that their transcripts can display different levels of visibility to miRNA regulation according to developmental cues.

Drosophila PAT1 is required for Kinesin-1 to transport cargo and to maximize its motility.

Kinesin heavy chain (KHC), the force-generating component of Kinesin-1, is required for the localization of oskar mRNA and the anchoring of the nucleus in the Drosophila oocyte. These events are crucial for the establishment of the anterior-posterior and dorsal-ventral axes. KHC is also essential for the localization of Dynein and for all ooplasmic flows. Interestingly, oocytes without Kinesin light chain show no major defects in these KHC-dependent processes, suggesting that KHC binds its cargoes and is activated by a novel mechanism. Here, we shed new light on the molecular mechanism of Kinesin function in the germline. Using a combination of genetic, biochemical and motor-tracking studies, we show that PAT1, an APP-binding protein, interacts with Kinesin-1, functions in the transport of oskar mRNA and Dynein and is required for the efficient motility of KHC along microtubules. This work suggests that the role of PAT1 in cargo transport in the cell is linked to PAT1 function as a positive regulator of Kinesin motility.