The Drosophila egg chamber provides a useful model for examining mechanisms by which cell fates are specified and maintained in the context of a complex tissue. The egg chamber is also an excellent model for understanding the mechanism by which cytoskeletal filaments are organized and the critical interplay between cytoskeletal organization, polarity establishment, and cell fate specification. Previous work has shown that Egalitarian (Egl) is required for specification and maintenance of oocyte fate. Mutants in egl either completely fail to specify an oocyte, or if specified, the oocyte eventually reverts back to nurse cell fate. Due to this very early role for Egl in egg chamber maturation, it is unclear whether later stages of egg chamber development also require Egl function. In this report, we have depleted Egl at specific stages of egg chamber development. We demonstrate that in early-stage egg chambers, Egl has an additional role in organization of oocyte microtubules. In the absence of Egl function, oocyte microtubules completely fail to reorganize. As such, the localization of microtubule motors and their cargo is disrupted. In addition, Egl also appears to function in regulating the translation of critical polarity determining messenger RNAs (mRNAs). Finally, we demonstrate that in midstage egg chambers, Egl does not appear to be required for microtubule organization, but rather for the correct spatial localization of oskar, bicoid, and gurken mRNAs.

While it is well known that CD4+ T cells and B cells collaborate for antibody production, our group previously reported that CD8+ T cells downregulate alloantibody responses following transplantation. However, the exact mechanism involved in CD8+ T cell-mediated downregulation of alloantibody remains unclear. We also reported that alloantibody production is enhanced when either perforin or FasL is deficient in transplant recipients. Here, we report that CD8+ T cell-deficient transplant recipient mice (high alloantibody producers) exhibit an increased number of primed B cells compared to wild-type transplant recipients. Furthermore, CD8+ T cells require FasL, perforin, and allospecificity to downregulate posttransplant alloantibody production. In vivo CD8-mediated clearance of alloprimed B cells was also FasL- and perforin-dependent. In vitro data demonstrated that recipient CD8+ T cells directly induce apoptosis of alloprimed IgG1+ B cells in co-culture in an allospecific and MHC class I-dependent fashion. Altogether these data are consistent with the interpretation that CD8+ T cells downregulate posttransplant alloantibody production by FasL- and perforin-dependent direct elimination of alloprimed IgG1+ B cells.

In order for eukaryotic cells to function properly, they must establish polarity. The Drosophila oocyte uses mRNA localization to establish polarity and hence provides a genetically tractable model in which to study this process. The spatial restriction of oskar mRNA and its subsequent protein product is necessary for embryonic patterning. The localization of oskar mRNA requires microtubules and microtubule-based motor proteins. Null mutants in Kinesin heavy chain (Khc), the motor subunit of the plus end-directed Kinesin-1, result in oskar mRNA delocalization. Although the majority of oskar particles are non-motile in khc nulls, a small fraction of particles display active motility. Thus, a motor other than Kinesin-1 could conceivably also participate in oskar mRNA localization. Here we show that Dynein heavy chain (Dhc), the motor subunit of the minus end-directed Dynein complex, extensively co-localizes with Khc and oskar mRNA. In addition, immunoprecipitation of the Dynein complex specifically co-precipitated oskar mRNA and Khc. Lastly, germline-specific depletion of Dhc resulted in oskar mRNA and Khc delocalization. Our results therefore suggest that efficient posterior localization of oskar mRNA requires the concerted activities of both Dynein and Kinesin-1.

SummaryHow the opposing activity of kinesin and dynein motors generates polarized distribution of organelles inside cells is poorly understood and hotly debated [1, 2]. Possible explanations include stochastic mechanical competition [3, 4], coordinated regulation by motor-associated proteins [5, 6, 7], mechanical activation of motors [8], and lipid-induced organization [9]. Here, we address this question by using phagocytosed latex beads to generate early phagosomes (EPs) that move bidirectionally along microtubules (MTs) in an in vitro assay [9]. Dynein/kinesin activity on individual EPs is recorded as real-time force generation of the motors against an optical trap. Activity of one class of motors frequently coincides with, or is rapidly followed by opposite motors. This leads to frequent and rapid reversals of EPs in the trap. Remarkably, the choice between dynein and kinesin can be explained by the tossing of a coin. Opposing motors therefore appear to function stochastically and independently of each other, as also confirmed by observing no effect on kinesin function when dynein is inhibited on the EPs. A simple binomial probability calculation based on the geometry of EP-microtubule contact explains the observed activity of dynein and kinesin on phagosomes. This understanding of intracellular transport in terms of a hypothetical coin, if it holds true for other cargoes, provides a conceptual framework to explain the polarized localization of organelles inside cells.