Grégoire Michaux

An AP-1/clathrin coat plays a novel and essential role in forming the Weibel-Palade bodies of endothelial cells

Clathrin provides an external scaffold to form small 50–100-nm transport vesicles. In contrast, formation of much larger dense-cored secretory granules is driven by selective aggregation of internal cargo at the trans-Golgi network; the only known role of clathrin in dense-cored secretory granules formation is to remove missorted proteins by small, coated vesicles during maturation of these spherical organelles. The formation of Weibel-Palade bodies (WPBs) is also cargo driven, but these are cigar-shaped organelles up to 5 μm long. We hypothesized that a cytoplasmic coat might be required to make these very different structures, and we found that new and forming WPBs are extensively, sometimes completely, coated. Overexpression of an AP-180 truncation mutant that prevents clathrin coat formation or reduced AP-1 expression by small interfering RNA both block WPB formation. We propose that, in contrast to other secretory granules, cargo aggregation alone is not sufficient to form immature WPBs and that an external scaffold that contains AP-1 and clathrin is essential.

How to roll an endothelial cigar: The biogenesis of Weibel-Palade bodies

Weibel‐Palade bodies (WPB) are the regulated secretory organelles of endothelial cells. These cigar‐shaped membrane‐bound structures function in both hemostasis and inflammation but their biogenesis is poorly understood. Here, we review what is currently known about their formation. The content of WPBs is dominated by the hemostatic factor von Willebrand factor (VWF), whose complex biogenesis ends in the formation of high molecular weight multimers. VWF is also organized into proteinaceous tubules which underlie the striated interior of WPBs as seen in the EM. VWF expression is necessary for formation of WPBs, and its heterologous expression can even lead to the specific recruitment of WPB membrane proteins, including the leukocyte receptor P‐selectin, the tetraspanin CD63, and Rab27a. Unusually, the VWF propeptide is implicated in the biogenesis of WPBs, being essential for formation of the storage compartment. The elongation of the cigars and the formation of the tubules are determined by non‐covalent interactions between pro‐ and mature VWF proteins. Surprisingly, high molecular weight multimers seem neither necessary nor sufficient to trigger formation of a storage compartment, and do not seem to have any role in WPB biogenesis. Von Willebrands disease, usually caused by mutations within VWF, has provided many of the insights into the way in which VWF drives the formation of these organelles.

CHE-14, a protein with a sterol-sensing domain, is required for apical sorting in C. elegans ectodermal epithelial cells.

BACKGROUND Polarised trafficking of proteins is critical for normal expression of the epithelial phenotype, but its genetic control is not understood. The regulatory gene lin-26 is essential for normal epithelial differentiation in the nematode Caenorhabditis elegans. To identify potential effectors of lin-26, we characterised mutations that result in lin-26-like phenotypes. Here, we report the phenotypic and molecular analysis of one such mutant line, che-14. RESULTS Mutations in che-14 resulted in several partially penetrant phenotypes affecting the function of most epithelial or epithelial-like cells of the ectoderm, including the hypodermis, excretory canal, vulva, rectum and several support cells. The defects were generally linked to the accumulation of vesicles or amorphous material near the apical surface, suggesting that secretion was defective. The CHE-14 protein showed similarity to proteins containing sterol-sensing domains, including Dispatched, Patched and NPC1. A fusion protein between full-length CHE-14 and the green fluorescent protein became localised to the apical surface of epithelial cells that require che-14 function. Deletions that removed the predicted transmembrane domains or extracellular loops of CHE-14 abolished apical localisation and function of the protein. CONCLUSIONS We propose that CHE-14 is involved in a novel secretory pathway dedicated to the exocytosis of lipid-modified proteins at the apical surface of certain epithelial cells. Our data raise the possibility that the primordial function of proteins containing a sterol-sensing domain is to control vesicle trafficking: CHE-14 and Dispatched in exocytosis, Patched and NPC1 in endocytosis.

High-pressure freezing provides insights into Weibel-Palade body biogenesis

The Weibel-Palade bodies (WPBs) of endothelial cells play an important role in haemostasis and the initiation of inflammation, yet their biogenesis is poorly understood. Tubulation of their major content protein, von Willebrand factor (VWF), is crucial to WPB function, and so we investigated further the relationship between VWF tubule formation and WPB formation in human umbilical vein endothelial cells (HUVECs). By using high-pressure freezing and freeze substitution before electron microscopy, we visualised VWF tubules in the trans-Golgi network (TGN), as well as VWF subunits in vesicular structures. Tubules were also seen in WPBs that were connected to the TGN by membranous stalks. Tubules are disorganised in the immature WPBs but during maturation we found a dramatic increase in the spatial organisation of the tubules and in organelle electron density. We also found coated budding profiles suggestive of the removal of missorted material after initial formation of these granules. Finally, we discovered that these large, seemingly rigid, organelles flex at hinge points and that the VWF tubules are interrupted at these hinges, facilitating organelle movement around the cell. The use of high-pressure freezing was vital in this study and it suggests that this technique might prove essential to any detailed characterisation of organelle biogenesis.

Genes Required for Osmoregulation and Apical Secretion in Caenorhabditis elegans

Few studies have investigated whether or not there is an interdependence between osmoregulation and vesicular trafficking. We previously showed that in Caenorhabditis elegans che-14 mutations affect osmoregulation, cuticle secretion, and sensory organ development. We report the identification of seven lethal mutations displaying che-14-like phenotypes, which define four new genes, rdy-1–rdy-4 (rod-like larval lethality and dye-filling defective). rdy-1, rdy-2, and rdy-4 mutations affect excretory canal function and cuticle formation. Moreover, rdy-1 and rdy-2 mutations reduce the amount of matrix material normally secreted by sheath cells in the amphid channel. In contrast, rdy-3 mutants have short cystic excretory canals, suggesting that it acts in a different process. rdy-1 encodes the vacuolar H+-ATPase a-subunit VHA-5, whereas rdy-2 encodes a new tetraspan protein. We suggest that RDY-1/VHA-5 acts upstream of RDY-2 and CHE-14 in some tissues, since it is required for their delivery to the epidermal, but not the amphid sheath, apical plasma membrane. Hence, the RDY-1/VHA-5 trafficking function appears essential in some cells and its proton pump function essential in others. Finally, we show that RDY-1/VHA-5 distribution changes prior to molting in parallel with that of actin microfilaments and propose a model for molting whereby actin provides a spatial cue for secretion.

P-selectin and CD63 use different mechanisms for delivery to Weibel-Palade bodies.

The biogenesis of endothelial‐specific Weibel–Palade bodies (WPB) is poorly understood, despite their key role in both haemostasis and inflammation. Biogenesis of specialized organelles of haemopoietic cells is often adaptor protein complex 3‐dependent (AP‐3‐dependent), and AP‐3 has previously been shown to play a role in the trafficking of both WPB membrane proteins, P‐selectin and CD63. However, WPB are thought to form at the trans Golgi network (TGN), which is inconsistent with a role for AP‐3, which operates in post‐Golgi trafficking. We have therefore investigated in detail the mechanisms of delivery of these two membrane proteins to WPB. We find that P‐selectin is recruited to forming WPB in the trans‐Golgi by AP‐3‐independent mechanisms that use sorting information within both the cytoplasmic tail and the lumenal domain of the receptor. In contrast, CD63 is recruited to already‐budded WPB by an AP‐3‐dependent route. These different mechanisms of recruitment lead to the presence of distinct immature and mature populations of WPB in human umbilical vein endothelial cells (HUVEC).

Epithelial biology: lessons from Caenorhabditis elegans

Epithelial cells are essential and abundant in all multicellular animals where their dynamic cell shape changes orchestrate morphogenesis of the embryo and individual organs. Genetic analysis in the simple nematode Caenorhabditis elegans provides some clues to the mechanisms that are involved in specifying epithelial cell fates and in controlling specific epithelial processes such as junction assembly, trafficking or cell fusion and cell adhesion. Here we review recent findings concerning C. elegans epithelial cells, focusing in particular on epithelial polarity, and transcriptional control.

AP-1 is required for the maintenance of apico-basal polarity in the C. elegans intestine

Epithelial tubes perform functions that are essential for the survival of multicellular organisms. Understanding how their polarised features are maintained is therefore crucial. By analysing the function of the clathrin adaptor AP-1 in the C. elegans intestine, we found that AP-1 is required for epithelial polarity maintenance. Depletion of AP-1 subunits does not affect epithelial polarity establishment or the formation of the intestinal lumen. However, the loss of AP-1 affects the polarised distribution of both apical and basolateral transmembrane proteins. Moreover, it triggers de novo formation of ectopic apical lumens between intestinal cells along the lateral membranes later during embryogenesis. We also found that AP-1 is specifically required for the apical localisation of the small GTPase CDC-42 and the polarity determinant PAR-6. Our results demonstrate that AP-1 controls an apical trafficking pathway required for the maintenance of epithelial polarity in vivo in a tubular epithelium.

PAR-4 and anillin regulate myosin to coordinate spindle and furrow position during asymmetric division.

PAR-4/LKB-1, PIG-1/MELK, and the anillin ANI-1 inhibit the accumulation of myosin at the anterior cortex of asymmetrically dividing one-cell C. elegans embryos, thereby preventing myosin from uncoupling cytokinetic furrow and mitotic spindle positions.

Contractile forces at tricellular contacts modulate epithelial organization and monolayer integrity

Monolayered epithelia are composed of tight cell assemblies that ensure polarized exchanges. EpCAM, an unconventional epithelial-specific cell adhesion molecule, is assumed to modulate epithelial morphogenesis in animal models, but little is known regarding its cellular functions. Inspired by the characterization of cellular defects in a rare EpCAM-related human intestinal disease, we find that the absence of EpCAM in enterocytes results in an aberrant apical domain. In the course of this pathological state, apical translocation towards tricellular contacts (TCs) occurs with striking tight junction belt displacement. These unusual cell organization and intestinal tissue defects are driven by the loss of actomyosin network homoeostasis and contractile activity clustering at TCs, yet is reversed by myosin-II inhibitor treatment. This study reveals that adequate distribution of cortical tension is crucial for individual cell organization, but also for epithelial monolayer maintenance. Our data suggest that EpCAM modulation protects against epithelial dysplasia and stabilizes human tissue architecture.