Branched-chain actin dynamics polarizes vesicle trajectories and partitions apicobasal epithelial membrane domains.

In prevailing epithelial polarity models, membrane- and junction-based polarity cues such as the partitioning-defective PARs specify the positions of apicobasal membrane domains. Recent findings indicate, however, that intracellular vesicular trafficking can determine the position of the apical domain, upstream of membrane-based polarity cues. These findings raise the question of how vesicular trafficking becomes polarized independent of apicobasal target membrane domains.

The biosynthetic-secretory pathway, supplemented by recycling routes, determines epithelial membrane polarity.

In prevailing epithelial polarity models, membrane-based polarity cues (e.g., the partitioning-defective PARs) position apicobasal cellular membrane domains.

Uncovering the 'sphinx' of sphingosine 1-phosphate signalling: from cellular events to organ morphogenesis.

Sphingosine 1-phosphate (S1P) is a bioactive sphingolipid metabolite, functioning as a signalling molecule in diverse cellular processes. Over the past few decades, studies of S1P signalling have revealed that the physiological activity of S1P largely depends on S1P metabolizing enzymes, transporters and receptors on the plasma membrane, as well as on the intracellular proteins that S1P binds directly to. In addition to its roles in cancer signalling, immunity and inflammation, a large body of evidence has identified a close link of S1P signalling with organ morphogenesis.

A tensile trilayered cytoskeletal endotube drives capillary-like lumenogenesis.

Unicellular tubes are components of internal organs and capillaries. It is unclear how they meet the architectural challenge to extend a centered intracellular lumen of uniform diameter. In an RNAi-based screen, we identified three intermediate filaments (IFs)-IFA-4, IFB-1, and IFC-2-as interactors of the lumenal membrane-actin linker ERM-1 in excretory-canal tubulogenesis.

A Model of Hereditary Sensory and Autonomic Neuropathy Type 1 Reveals a Role of Glycosphingolipids in Neuronal Polarity.

Hereditary sensory and autonomic neuropathy Type 1 (HSAN1) is a rare autosomal dominantly inherited neuropathy, clinically characterized by a loss of distal peripheral sensory and motoneuronal function. Mutations in subunits of serine palmitoyltransferase (SPT) have been linked to the majority of HSAN1 cases. SPTs catalyze the condensation of l-serine with palmitoyl-CoA, the first committed and rate-limiting step in sphingolipid biosynthesis.

The galectin LEC-5 is a novel binding partner for RAB-11.

RAB-11/Rab11 is an endosomal GTPase with conserved roles in directional trafficking and apical domain formation in polarized epithelial cells. From a yeast two-hybrid screen using full-length C. elegans RAB-11 as bait, we identified LEC-5 as a novel binding protein for RAB-11.

Degradation for better survival? Role of ubiquitination in epithelial morphogenesis.

As a prevalent post-translational modification, ubiquitination is essential for many developmental processes. Once covalently attached to the small and conserved polypeptide ubiquitin (Ub), a substrate protein can be directed to perform specific biological functions via its Ub-modified form. Three sequential catalytic reactions contribute to this process, among which E3 ligases serve to identify target substrates and promote the activated Ub to conjugate to substrate proteins.

The C. elegans Excretory Canal as a Model for Intracellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis in a Single Cell: labeling by GFP-fusions, RNAi Interaction Screen and Imaging.

The four C. elegans excretory canals are narrow tubes extended through the length of the animal from a single cell, with almost equally far extended intracellular endotubes that build and stabilize the lumen with a membrane and submembraneous cytoskeleton of apical character. The excretory cell expands its length approximately 2,000 times to generate these canals, making this model unique for the in vivo assessment of de novo polarized membrane biogenesis, intracellular lumen morphogenesis and unicellular tubulogenesis.

The C. elegans Intestine As a Model for Intercellular Lumen Morphogenesis and In Vivo Polarized Membrane Biogenesis at the Single-cell Level: Labeling by Antibody Staining, RNAi Loss-of-function Analysis and Imaging.

Multicellular tubes, fundamental units of all internal organs, are composed of polarized epithelial or endothelial cells, with apical membranes lining the lumen and basolateral membranes contacting each other and/or the extracellular matrix. How this distinctive membrane asymmetry is established and maintained during organ morphogenesis is still an unresolved question of cell biology. This protocol describes the C.

Facilitation of Endosomal Recycling by an IRG Protein Homolog Maintains Apical Tubule Structure in Caenorhabditis elegans.

Determination of luminal diameter is critical to the function of small single-celled tubes. A series of EXC proteins, including EXC-1, prevent swelling of the tubular excretory canals in Caenorhabditis elegans In this study, cloning of exc-1 reveals it to encode a homolog of mammalian IRG proteins, which play roles in immune response and autophagy and are associated with Crohn's disease. Mutants in exc-1 accumulate early endosomes, lack recycling endosomes, and exhibit abnormal apical cytoskeletal structure in regions of enlarged tubules.

RNAi-based biosynthetic pathway screens to identify in vivo functions of non-nucleic acid-based metabolites such as lipids.

The field of metabolomics continues to catalog new compounds, but their functional analysis remains technically challenging, and roles beyond metabolism are largely unknown. Unbiased genetic/RNAi screens are powerful tools to identify the in vivo functions of protein-encoding genes, but not of nonproteinaceous compounds such as lipids. They can, however, identify the biosynthetic enzymes of these compounds-findings that are usually dismissed, as these typically synthesize multiple products.

Vesicular sorting controls the polarity of expanding membranes in the C. elegans intestine.

Biological tubes consist of polarized epithelial cells with apical membranes building the central lumen and basolateral membranes contacting adjacent cells or the extracellular matrix. Cellular polarity requires distinct inputs from outside the cell, e.g.

Intracellular lumen extension requires ERM-1-dependent apical membrane expansion and AQP-8-mediated flux.

Many unicellular tubes such as capillaries form lumens intracellularly, a process that is not well understood. Here we show that the cortical membrane organizer ERM-1 is required to expand the intracellular apical/lumenal membrane and its actin undercoat during single-cell Caenorhabditis elegans excretory canal morphogenesis. We characterize AQP-8, identified in an ERM-1-overexpression (ERM-1[++]) suppressor screen, as a canalicular aquaporin that interacts with ERM-1 in lumen extension in a mercury-sensitive manner, implicating water-channel activity.

Clathrin and AP-1 regulate apical polarity and lumen formation during C. elegans tubulogenesis.

Clathrin coats vesicles in all eukaryotic cells and has a well-defined role in endocytosis, moving molecules away from the plasma membrane. Its function on routes towards the plasma membrane was only recently appreciated and is thought to be limited to basolateral transport. Here, an unbiased RNAi-based tubulogenesis screen identifies a role of clathrin (CHC-1) and its AP-1 adaptor in apical polarity during de novo lumenal membrane biogenesis in the C.

Apicobasal domain identities of expanding tubular membranes depend on glycosphingolipid biosynthesis.

Metazoan internal organs are assembled from polarized tubular epithelia that must set aside an apical membrane domain as a lumenal surface. In a global Caenorhabditis elegans tubulogenesis screen, interference with several distinct fatty-acid-biosynthetic enzymes transformed a contiguous central intestinal lumen into multiple ectopic lumens. We show that multiple-lumen formation is caused by apicobasal polarity conversion, and demonstrate that in situ modulation of lipid biosynthesis is sufficient to reversibly switch apical domain identities on growing membranes of single post-mitotic cells, shifting lumen positions.

Targeted gene alteration in Caenorhabditis elegans by gene conversion.

Now that some genomes have been completely sequenced, the ability to direct specific mutations into genomes is particularly desirable. Here we present a method to create mutations in the Caenorhabditis elegans genome efficiently through transgene-directed, transposon-mediated gene conversion. Engineered deletions targeted into two genes show that the frequency of obtaining the desired mutation was higher using this approach than using standard transposon insertion-deletion approaches.

Lumen morphogenesis in C. elegans requires the membrane-cytoskeleton linker erm-1.

Epithelial tubes are basic building blocks of complex organs, but their architectural requirements are not well understood. Here we show that erm-1 is a unique C. elegans ortholog of the ERM family of cytoskeleton-membrane linkers, with an essential role in lumen morphogenesis.