Successful completion of cytokinesis relies on addition of new membrane, and requires the recycling endosome regulator Rab11, which localizes to the midzone. Despite the critical role of Rab11 in ...this process, little is known about the formation and composition of Rab11-containing organelles. Here, we identify the phosphatidylinositol (PI) 4-kinase III β Four wheel drive (Fwd) as a key regulator of Rab11 during cytokinesis in Drosophila melanogaster spermatocytes. We show Fwd is required for synthesis of PI 4-phosphate (PI4P) on Golgi membranes and for formation of PI4P-containing secretory organelles that localize to the midzone. Fwd binds and colocalizes with Rab11 on Golgi membranes, and is required for localization of Rab11 in dividing cells. A kinase-dead version of Fwd also binds Rab11 and partially restores cytokinesis to fwd mutant flies. Moreover, activated Rab11 partially suppresses loss of fwd. Our data suggest Fwd plays catalytic and noncatalytic roles in regulating Rab11 during cytokinesis.
Phosphoinositides (PIPs) are a ubiquitous group of seven low-abundance phospholipids that play a crucial role in defining localized membrane properties and that regulate myriad cellular processes, ...including cytoskeletal remodeling, cell signaling cascades, ion channel activity and membrane traffic. PIP homeostasis is tightly regulated by numerous inositol kinases and phosphatases, which phosphorylate and dephosphorylate distinct PIP species. The importance of these phospholipids, and of the enzymes that regulate them, is increasingly being recognized, with the identification of human neurological disorders that are caused by mutations in PIP-modulating enzymes. Genetic disorders of PIP metabolism include forms of epilepsy, neurodegenerative disease, brain malformation syndromes, peripheral neuropathy and congenital myopathy. In this Review, we provide an overview of PIP function and regulation, delineate the disorders associated with mutations in genes that modulate or utilize PIPs, and discuss what is understood about gene function and disease pathogenesis as established through animal models of these diseases.
Gaucher disease is a lysosomal storage disorder caused by a defect in the degradation of glucosylceramide catalyzed by the lysosomal enzyme β-glucocerebrosidase (GBA). GBA reaches lysosomes via ...association with its receptor, lysosomal integral membrane protein type 2 (LIMP-2). We found that distinct phosphatidylinositol 4-kinases (PI4Ks) play important roles at multiple steps in the trafficking pathway of the LIMP-2/GBA complex. Acute depletion of phosphatidylinositol 4-phosphate in the Golgi caused accumulation of LIMP-2 in this compartment, and PI4KIIIβ was found to be responsible for controlling the exit of LIMP-2 from the Golgi. In contrast, depletion of PI4KIIα blocked trafficking at a post-Golgi compartment, leading to accumulation of LIMP-2 in enlarged endosomal vesicles. PI4KIIα depletion also caused secretion of missorted GBA into the medium, which was attenuated by limiting LIMP-2/GBA exit from the Golgi by PI4KIIIβ inhibitors. These studies identified PI4KIIIβ and PI4KIIα as important regulators of lysosomal delivery of GBA, revealing a new element of control to sphingolipid homeostasis by phosphoinositides.
Drosophila sperm development is characterized by extensive post-transcriptional regulation whereby thousands of transcripts are preserved for translation during later stages. A key step in ...translation initiation is the binding of eukaryotic initiation factor 4E (eIF4E) to the 5' mRNA cap. In addition to canonical eIF4E-1, Drosophila has multiple eIF4E paralogs, including four (eIF4E-3, -4, -5, and -7) that are highly expressed in the testis. Among these, only eIF4E-3 has been characterized genetically. Here, using CRISPR/Cas9 mutagenesis, we determined that eIF4E-5 is essential for male fertility. eIF4E-5 protein localizes to the distal ends of elongated spermatid cysts, and eIF4E-5 mutants exhibit defects during post-meiotic stages, including a mild defect in spermatid cyst polarization. eIF4E-5 mutants also have a fully penetrant defect in individualization, resulting in failure to produce mature sperm. Indeed, our data indicate that eIF4E-5 regulates non-apoptotic caspase activity during individualization by promoting local accumulation of the E3 ubiquitin ligase inhibitor Soti. Our results further extend the diversity of non-canonical eIF4Es that carry out distinct spatiotemporal roles during spermatogenesis.
Discovering target and off-target effects of specific compounds is critical to drug discovery and development. We generated a compendium of “chemical-genetic interaction” profiles by testing the ...collection of viable yeast haploid deletion mutants for hypersensitivity to 82 compounds and natural product extracts. To cluster compounds with a similar mode-of-action and to reveal insights into the cellular pathways and proteins affected, we applied both a hierarchical clustering and a factorgram method, which allows a gene or compound to be associated with more than one group. In particular, tamoxifen, a breast cancer therapeutic, was found to disrupt calcium homeostasis and phosphatidylserine (PS) was recognized as a target for papuamide B, a cytotoxic lipopeptide with anti-HIV activity. Further, the profile of crude extracts resembled that of its constituent purified natural product, enabling detailed classification of extract activity prior to purification. This compendium should serve as a valuable key for interpreting cellular effects of novel compounds with similar activities.
Phototransduction in
is mediated by phospholipase C-dependent hydrolysis of PIP
, and is an important model for phosphoinositide signalling. Although generally assumed to operate by generic machinery ...conserved from yeast to mammals, some key elements of the phosphoinositide cycle have yet to be identified in
photoreceptors. Here, we used transgenic flies expressing fluorescently tagged probes (P4M and Tb
), which allow
quantitative measurements of PI4P and PIP
dynamics in photoreceptors of intact living flies. Using mutants and RNA interference for candidate genes potentially involved in phosphoinositide turnover, we identified
PI4KIIIα (CG10260) as the PI4-kinase responsible for PI4P synthesis in the photoreceptor membrane. Our results also indicate that PI4KIIIα activity requires
(the
orthologue of Efr3) and CG8325 (orthologue of YPP1), both of which are implicated as scaffolding proteins necessary for PI4KIIIα activity in yeast and mammals. However, our evidence indicates that the recently reported central role of
(CG3682) in PIP
synthesis in the rhabdomeres should be re-evaluated; although PIP
resynthesis was suppressed by RNAi directed against
, little or no defect was detected in a reportedly null mutant (
).
The complex functions of cellular membranes, and thus overall cell physiology, depend on the distribution of crucial lipid species. Sac1 is an essential, conserved, ER-localized phosphatase whose ...substrate, phosphatidylinositol 4-phosphate (PI4P), coordinates secretory trafficking and plasma membrane function. PI4P from multiple pools is delivered to Sac1 by oxysterol-binding protein and related proteins in exchange for other lipids and sterols, which places Sac1 at the intersection of multiple lipid distribution pathways. However, much remains unknown about the roles of Sac1 in subcellular homeostasis and organismal development. Using a temperature-sensitive allele (
), we show that Sac1 is required for structural integrity of the
retinal floor. The β
-integrin Myospheroid, which is necessary for basal cell adhesion, is mislocalized in
retinas. In addition, the adhesion proteins Roughest and Kirre, which coordinate apical retinal cell patterning at an earlier stage, accumulate within
retinal cells due to impaired endo-lysosomal degradation. Moreover, Sac1 is required for ER homeostasis in
retinal cells. Together, our data illustrate the importance of Sac1 in regulating multiple aspects of cellular homeostasis during tissue development.
Threshold Concepts on the Edge explores new directions in threshold concept research and practice and is of relevance to teachers, learners, educational researchers and academic developers.
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