Two endosome populations involved in recycling of membranes and receptors to the plasma membrane have been described, the early and the recycling endosome. However, this distinction is mainly based on the flow of cargo molecules and the spatial distribution of these membranes within the cell. To get insights into the membrane organization of the recycling pathway, we have studied Rab4, Rab5, and Rab11, three regulatory components of the transport machinery. Following transferrin as cargo molecule and GFP-tagged Rab proteins we could show that cargo moves through distinct domains on endosomes. These domains are occupied by different Rab proteins, revealing compartmentalization within the same continuous membrane. Endosomes are comprised of multiple combinations of Rab4, Rab5, and Rab11 domains that are dynamic but do not significantly intermix over time. Three major populations were observed: one that contains only Rab5, a second with Rab4 and Rab5, and a third containing Rab4 and Rab11. These membrane domains display differential pharmacological sensitivity, reflecting their biochemical and functional diversity. We propose that endosomes are organized as a mosaic of different Rab domains created through the recruitment of specific effector proteins, which cooperatively act to generate a restricted environment on the membrane.

During the maternal-to-zygotic transition, a developing embryo integrates post-transcriptional regulation of maternal mRNAs with transcriptional activation of its own genome. By combining chromosomal ablation in Drosophila with microarray analysis, we characterized the basis of this integration. We show that the expression profile for at least one third of zygotically active genes is coupled to the concomitant degradation of the corresponding maternal mRNAs. The embryo uses transcription and degradation to generate localized patterns of expression, and zygotic transcription to degrade distinct classes of maternal transcripts. Although degradation does not appear to involve a simple regulatory code, the activation of the zygotic genome starts from intronless genes sharing a common cis-element. This cis-element interacts with a single protein, the Bicoid stability factor, and acts as a potent enhancer capable of timing the activity of an exogenous transactivator. We propose that this regulatory mode links morphogen gradients with temporal regulation during the maternal-to-zygotic transition.

The three GTPases Rab5, Rab4 and Rab11 regulate sequential transport steps along the endocytic/recycling pathway, and occupy distinct membrane domains on early and recycling endosomes. To address the mechanisms that regulate communication between such domains, we searched for proteins that interact with both Rab5 and Rab4. Here, we report that Rabenosyn-5, a previously identified Rab5 effector, also binds to Rab4. Rabenosyn-5 overexpression increased the association between Rab5 and Rab4 endosomal domains and decreased the fraction of Rab4- and Rab11-positive structures. This redistribution was accompanied by a faster rate of transferrin recycling from early endosomes to the cell surface and reduced transport to Rab11-containing perinuclear recycling endosomes. These effects depend on the ability of Rabenosyn-5 to interact with Rab4. We propose that divalent Rab effectors regulate protein sorting and recycling by connecting Rab domains on early endosomes.

The synchronous cleavage divisions of early embryogenesis require coordination of the cell-cycle oscillator, the dynamics of the cytoskeleton, and the cytoplasm. Yet, it remains unclear how spatially restricted biochemical signals are integrated with physical properties of the embryo to generate collective dynamics. Here, we show that synchronization of the cell cycle in Drosophila embryos requires accurate nuclear positioning, which is regulated by the cell-cycle oscillator through cortical contractility and cytoplasmic flows. We demonstrate that biochemical oscillations are initiated by local Cdk1 inactivation and spread through the activity of phosphatase PP1 to generate cortical myosin II gradients. These gradients cause cortical and cytoplasmic flows that control proper nuclear positioning. Perturbations of PP1 activity and optogenetic manipulations of cortical actomyosin disrupt nuclear spreading, resulting in loss of cell-cycle synchrony. We conclude that mitotic synchrony is established by a self-organized mechanism that integrates the cell-cycle oscillator and embryo mechanics.

SummaryMorphogenesis of multicellular organisms is driven by localized cell shape changes. How, and to what extent, changes in behavior in single cells or groups of cells influence neighboring cells and large-scale tissue remodeling remains an open question. Indeed, our understanding of multicellular dynamics is limited by the lack of methods allowing the modulation of cell behavior with high spatiotemporal precision. Here, we developed an optogenetic approach to achieve local modulation of cell contractility and used it to control morphogenetic movements during Drosophila embryogenesis. We show that local inhibition of apical constriction is sufficient to cause a global arrest of mesoderm invagination. By varying the spatial pattern of inhibition during invagination, we further demonstrate that coordinated contractile behavior responds to local tissue geometrical constraints. Together, these results show the efficacy of this optogenetic approach to dissect the interplay between cell-cell interaction, force transmission, and tissue geometry during complex morphogenetic processes.

During organismal development, cells undergo complex changes in shape whose causal relationship to individual morphogenetic processes remains unclear. The modular nature of such processes suggests that it should be possible to isolate individual modules, determine the minimum set of requirements sufficient to drive tissue remodeling, and re-construct morphogenesis. Here we use optogenetics to reconstitute epithelial folding in embryonic Drosophila tissues that otherwise would not undergo invagination. We show that precise spatial and temporal activation of Rho signaling is sufficient to trigger apical constriction and tissue folding. Induced furrows can occur at any position along the dorsal–ventral or anterior–posterior embryo axis in response to the spatial pattern and level of optogenetic activation. Thus, epithelial folding is a direct function of the spatio-temporal organization and strength of Rho signaling that on its own is sufficient to drive tissue internalization independently of any pre-determined condition or differentiation program associated with endogenous invagination processes.

The ␤-amyloid precursor protein (APP) 1 is an integral membrane protein from which the ␤-amyloid peptide is generated. The ␤-amyloid peptide forms the extracellular insoluble aggregates characteristic of Alzheimer's disease. The function of APP and the regulation of the proteolytic events generating the ␤-amyloid peptide are still unknown. APP was expected to be involved in signal transduction processes, because of its transmembrane topology. Three main isoforms of APP exist, generated by alternative splicing (APP 770 , APP 751 , and APP 695 ) and all possessing the same intracellular domain (reviewed in Ref.1). Although little is known about the putative extracellular ligand(s) for APP, several results describe the interaction of its intracellular domain with other proteins. These include the interaction with the heterotrimeric G protein Go (2), a 59-kDa ubiquitously expressed protein named APP-BP1 (3), the X11 protein (4), the neuron-abundant Fe65 protein, and an Fe65-like protein (4 -6). It was shown that intact APP binds to oligomeric Go protein and that the intracellular region of APP spanning residues 657-676 activates Go (2, 7). Furthermore, the interaction of APP with a monoclonal antibody directed against its extracellular domain mimics a ligand-receptor binding that triggers Go activation (7). APP-BP1 interacts both in vitro and in vivo with the carboxyl-terminal region of APP, which represents its intracellular domain. This protein is homologous to the product of the Arabidopsis auxin resistance gene AXR1 and to a Caenorabditis elegans protein of unknown function (3).The Fe65 gene is mainly expressed in the neurons of specific regions of the mammalian nervous system (8, 9) and encodes a protein containing two different types of protein-protein interaction domains: the WW domain (reviewed in Ref. 10) and the phosphotyrosine interaction/phosphotyrosine binding (PID/ PTB) domain (reviewed in Ref. 11). The latter was found in the oncoprotein Shc (12, 13), in its relatives , in other apparently unrelated proteins, such as Numb, X11, and Dab (15), and in insulin receptor substrate 1 (IRS-1) and 17). The PID/PTB domains interact with phosphotyrosine residues located in the intracellular domains of growth factor receptors, such as EGF-R, trkA, and plateletderived growth factor receptor in the case of Shc (13) and insulin receptor and interleukin 4 receptor in the case of IRS-1 (16). In contrast, the Fe65 region containing the two PID/PTB domains was demonstrated to interact with the intracellular domain of APP (5).All the PID/PTB domains present in the Shc family, IRS-1, and Fe65 interact with intracellular regions of membrane proteins containing the consensus motif ⌽XNPXY (where ⌽ is hydrophobic and X is any amino acid). However, Fe65 possesses at least two unique characteristics: (i) although all the known members of the PID/PTB family contain only one PID/ PTB element (13), Fe65 is an exception, because its sequence interacting with APP shows two consecutive PID/PTB domains; and (ii) although the Tyr prese...

The intracellular trafficking of the Notch ligand Delta plays an important role in the activation of the Notch pathway. We have addressed the snail-dependent regulation of Delta trafficking during the plasma membrane growth of the mesoderm in the Drosophila embryo. We show that Delta is retained in endocytic vesicles in the mesoderm but expressed on the surface of the adjacent ectoderm. This trafficking pattern requires Neuralized. We developed a protocol based on chromosomal deletion and microarray analysis that led to the identification of tom as the target of snail regulating Delta trafficking. Snail represses Tom expression in the mesoderm and thereby activates Delta trafficking. Overexpression of Tom abolishes Delta trafficking and signaling to the adjacent mesoectoderm. Loss of Tom produces mesoderm-type Delta trafficking in the entire blastoderm epithelium and an expansion of mesoectoderm gene expression. We propose that Tom antagonizes the activity of Neuralized and thus establishes a sharp mesoderm-mesoectoderm boundary of Notch signaling.