Equations 1 and 2 were published incorrectly in both the print and online versions of the paper. The corrected equations are shown below. We apologise for any inconvenience caused. Erratum ne*(t)/ns =

Recently, proteins linked to glycosylphosphatidylinositol (GPI) residues have received considerable attention both for their association with lipid microdomains and for their specific transport between cellular membranes. Basic features of trafficking of GPI-anchored proteins or glycolipids may be explored in flagellated protozoan parasites, which offer the advantage that their surface is dominated by these components. In Trypanosoma brucei, the GPI-anchored variant surface glycoprotein (VSG) is efficiently sorted at multiple intracellular levels, leading to a 50-fold higher membrane concentration at the cell surface compared with the endoplasmic reticulum. We have studied the membrane and VSG flow at an invagination of the plasma membrane, the flagellar pocket, the sole region for endoand exocytosis in this organism. VSG enters trypanosomes in large clathrin-coated vesicles (135 nm in diameter), which deliver their cargo to endosomes. In the lumen of cisternal endosomes, VSG is concentrated by default, because a distinct class of small clathrin-coated vesicles (50 -60 nm in diameter) budding from the cisternae is depleted in VSG. TbRAB11-positive cisternal endosomes, containing VSG, fragment by an unknown process giving rise to intensely TbRAB11-as well as VSG-positive, disk-like carriers (154 nm in diameter, 34 nm in thickness), which are shown to fuse with the flagellar pocket membrane, thereby recycling VSG back to the cell surface.

Embryogenesis in the beetle Tribolium is of increasing interest to both molecular and evolutionary biology because it differs from the Drosophila paradigm by its type of segment specification (short- vs. long-germ) and by the extensive epithelial envelopes - amnion and serosa - that are typical of most insects but not of higher dipterans. Using scanning electron microscopy of DAPI staged embryos we document development in Tribolium castaneum from blastoderm to completion of the envelopes, recording many details not otherwise accessible; we also provide a time table of the respective stages at 30 degrees C. The nascent blastoderm cells remain basally confluent with the yolksac until after the 13th (=last synchronous) mitotic cycle. The cells in the prospective serosa - the first domain to segregate visibly from the uniform blastoderm - carry surface protrusions likely to contact the overlying vitelline envelope. The embryonic rudiment, the other (and larger) blastodermal domain, gives rise to amnion and germ anlage. In the latter, visible differentiation begins with a "primitive pit" reminiscent of the posterior midgut rudiment of Drosophila. The subsequent invagination of the mesoderm resembles Drosophila gastrulation, except in the head region where the median groove extends through the entire preoral region. The prospective amnion starts differing visibly from the germ anlage during early gastrulation. It then folds underneath the spreading serosa and, advancing with the latter, closes the amniotic cavity at the ventral face of the germband. The largest (=posterior) amniotic fold covers a crestlike protrusion of the yolksac. Together with marked changes in the shape and arrangement of the amnion cells, this protrusion may contribute to the fold's elevation and early progress.

Proteins modified by glycosylphosphatidylinositol membrane anchors have become popular for investigating the role of membrane lipid microdomains in cellular sorting processes. To this end, trypanosomatids offer the advantage that they express these molecules in high abundance. The parasitic protozoan Trypanosoma brucei is covered by a dense and nearly homogeneous coat composed of a glycosylphosphatidylinositolanchored protein, the variant surface glycoprotein, which is essential for survival of the parasite in the mammalian blood. Therefore, T. brucei must possess mechanisms to selectively and efficiently deliver variant surface glycoprotein to the cell surface. In this study, we have quantified the steady-state distribution of variant surface glycoprotein by differential biotinylation, by fluorescence microscopy and by immunoelectron microscopy on high-pressure frozen and freezesubstituted samples. These three techniques provide very similar estimates of the fraction of variant surface glycoprotein located on the cell surface, on average 89.4%. The intracellular variant surface glycoprotein (10.6%) is predominantly located in the endosomal compartment (75%), while 25% are associated with the endoplasmic reticulum, Golgi apparatus and lysosomes. The density of variant surface glycoprotein in the plasma membrane including the membrane of the flagellar pocket, the only site for endo-and exocytosis in this organism, is 48-52 times higher than the density in endoplasmic reticulum membranes. The relative densities of the Golgi complex and of the endosomes are 2.7 and 10.8, respectively, compared to the endoplasmic reticulum. This data set provides the basis for an analysis of the dynamics of sorting. Depending on 547 the intracellular itinerary of newly formed variant surface glycoprotein, the high surface density is achieved in two (endoplasmic reticulum » Golgi complex » cell surface) or three enrichment steps (endoplasmic reticulum » Golgi complex » endosomes » cell surface), suggesting sorting between several membrane compartments.

During its life cycle, the parasitic protozoon Leishmania mexicana differentiates from a flagellated form, the promastigote, to an amastigote form carrying a rudimentary flagellum. Besides biochemical changes, this process involves a change in overall cell morphology including flagellar shortening. A mitogen-activated protein kinase kinase homologue designated LmxMKK was identified in a homology screening and found to be critically involved in the regulation of flagellar assembly and cell size. LmxMKK is exclusively expressed in the promastigote stage and is likely to be regulated by posttranslational mechanisms such as phosphorylation. A deletion mutant for the single-copy gene revealed motile flagella dramatically reduced in length and lacking the paraflagellar rod, a structure adjacent to the axoneme in kinetoplastid flagella. Moreover, a fraction of the cells showed perturbance of the axonemal structure. Complementation of the deletion mutant with the wild-type gene restored typical promastigote morphology. We propose that LmxMKK influences anterograde intraflagellar transport to maintain flagellar length in Leishmania promastigotes; as such, it is the first protein kinase known to be involved in organellar assembly.Protein kinases are key regulatory molecules in the proliferation, differentiation, motility, and stress response of all eukaryotic cells. Together with their antagonists, the protein phosphatases, they are organized in complex networks to guarantee proper regulation of cellular processes according to environmental changes and intercellular communication. There is a wealth of information present on protein kinases in higher eukaryotes and Saccharomyces cerevisiae (18,45). Information on signal transduction processes for other unicellular organisms such as the sporozoa (Plasmodium, Toxoplasma, and Theileria) (22) and the kinetoplastida (Trypanosoma and Leishmania) (40, 50) as causative agents of infectious disease or the green alga Chlamydomonas (48) and the slime mold Dictyostelium discoideum (2) as model organisms for flagellar assembly and differentiation, respectively, is relatively scarce. It is likely that in Leishmania, as in other organisms, signal transduction pathways culminate in altered gene expression. As transcription factors and RNA polymerase II promoters are absent in these parasites, it is generally thought that regulation occurs posttranscriptionally on the level of mRNA maturation and turnover, efficiency of translation, and protein stability (10, 40). However, the regulatory molecules involved in these processes have not been identified.Leishmania parasites undergo a digenetic life cycle, differentiating from the promastigote form in the insect vector, the phlebotomine sand fly, to the amastigote form in the lysosomal compartment of the macrophages of mammals. Promastigotes are spindle-shaped cells, 11 to 20 m in length and 2 m in diameter, carrying a single flagellum of at least the length of the cell body at their anterior pole, which pulls the cell forward but also mediates...