Optogenetic inhibition of apical constriction during Drosophila embryonic development.

Morphogenesis of multicellular organisms is driven by changes in cell behavior, which happen at precise locations and defined developmental stages. Therefore, the studying of morphogenetic events would greatly benefit from tools that allow the perturbation of cell activity with spatial and temporal precision. We recently developed an optogenetic approach to modulate cell contractility with cellular precision and on fast (seconds) timescales during Drosophila embryogenesis. We present here a protocol to handle genetically engineered photosensitive Drosophila embryos and achieve light-mediated inhibition of apical constriction during tissue invagination. The possibility to modulate the levels of optogenetic activation at different laser powers makes this method suited also for studying how mechanical stresses are sensed and interpreted in vivo. Given the conserved function of cell contractility during animal development, the application of this method to other morphogenetic processes will facilitate our understanding of tissue mechanics and cell-cell interaction during morphogenesis.

Distinct membrane domains on endosomes in the recycling pathway visualized by multicolor imaging of Rab4, Rab5, and Rab11.

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.

Fe65 and the protein network centered around the cytosolic domain of the Alzheimer's beta-amyloid precursor protein.

A distinctive tract of all the forms of Alzheimer's disease is the extracellular deposition of a 40-42/43 amino acid-long peptide derived from the so-called beta-amyloid precursor protein (APP). This is a membrane protein of unknown function, whose short cytosolic domain has been recently demonstrated to interact with several proteins. One of these proteins, named Fe65, has the characteristics of an adaptor protein; in fact, it possesses three protein-protein interaction domains: a WW domain and two PID/PTB domains. The interaction with APP requires the most C-terminal PID/PTB domain, whereas the WW domain is responsible for the interaction with various proteins, one of which was demonstrated to be the mammalian homolog of the Drosophila enabled protein (Mena), which in turn interacts with the cytoskeleton. The second PID/PTB domain of Fe65 binds to the CP2/LSF/LBP1 protein, which is an already known transcription factor. The other proteins interacting with the cytosolic domain of APP are the G(o) heterotrimeric protein, APP-BP1 and X11. The latter interacts with APP through a PID/PTB domain and possesses two other protein-protein interaction domains. The small size of the APP cytodomain and the overlapping of its regions involved in the binding of Fe65 and X11 suggest the existence of competitive mechanisms regulating the binding of the various ligands to this cytosolic domain. In this short review the possible functional roles of this complex protein network and its involvement in the generation of Alzheimer's phenotype are discussed.

Interaction of the phosphotyrosine interaction/phosphotyrosine binding-related domains of Fe65 with wild-type and mutant Alzheimer's beta-amyloid precursor proteins.

The two tandem phosphotyrosine interaction/phosphotyrosine binding (PID/PTB) domains of the Fe65 protein interact with the intracellular region of the Alzheimer's beta-amyloid precursor protein (APP). This interaction, previously demonstrated in vitro and in the yeast two hybrid system, also takes place in vivo in mammalian cells, as demonstrated here by anti-Fe65 co-immunoprecipitation experiments. This interaction differs from that occurring between other PID/PTB domain-containing proteins, such as Shc and insulin receptor substrate 1, and activated growth factor receptors as follows: (i) the Fe65-APP interaction is phosphorylation-independent; (ii) the region of the APP intracellular domain involved in the binding is larger than that of the growth factor receptor necessary for the formation of the complex with Shc; and (iii) despite a significant similarity the carboxyl-terminal regions of PID/PTB of Fe65 and of Shc are not functionally interchangeable in terms of binding cognate ligands. A role for Fe65 in the pathogenesis of familial Alzheimer's disease is suggested by the finding that mutant APP, responsible for some cases of familial Alzheimer's disease, shows an altered in vivo interaction with Fe65.

DNA-binding protein Pur alpha and transcription factor YY1 function as transcription activators of the neuron-specific FE65 gene promoter.

Fe65 is an adaptor protein that interacts with the Alzheimer beta-amyloid precursor protein and is expressed mainly in the neurons of several regions of the nervous system. The FE65 gene has a TATA-less promoter that drives an efficient transcription in cells showing a neuronal phenotype, whereas its efficiency is poor in non-neuronal cells. A short sequence encompassing the transcription start site contains sufficient information to drive the transcription in neuronal cells but not in non-neural cells. Electrophoretic mobility-shift assays performed with rat brain nuclear extracts showed that three major DNA-protein complexes, named BI, BII and BIII, are formed by the FE65 minimal promoter. The proteins present in complexes BI and BII were purified from bovine brain; internal microsequencing of the purified proteins demonstrated that they corresponded to the previously isolated single-stranded-DNA-binding protein Pur alpha, abundantly expressed in the brain. In Chinese hamster ovary (CHO) cells, where the efficiency of FE65 promoter is very low, transient expression of Pur alpha increased the transcription efficiency of the FE65 minimal promoter. By using oligonucleotide competition and a specific antibody we demonstrated that the transcription factor YY1 is responsible for the formation of complex BIII. Also in this case, the transient expression of the YY1 cDNA in CHO cells resulted in an increased transcription from the FE65 minimal promoter. The absence of any co-operative effect when CHO cells were co-transfected with both YY1 and Pur alpha cDNA species suggests that two different transcription regulatory mechanisms could have a role in the regulation of the FE65 gene.