Analysis of Ca2+-dependent Weibel-Palade body tethering by live cell TIRF microscopy: involvement of a Munc13-4/S100A10/annexin A2 complex

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated, Ca2+-dependent exocytosis of WPBs critically depends on their proper targeting to the plasma membrane, but the mechanism of WPB-plasma membrane tethering prior to fusion is not well characterized. Here we describe a method to visualize and analyze WPB tethering and fusion in living human umbilical vein endothelial cells (HUVEC) by total internal reflection fluorescence (TIRF) microscopy. This method is based on automated object detection and allowed us to identify components of the tethering complex of WPBs and to monitor their dynamics in space and time. An important tethering factor identified by this means was Munc13-4 that was shown to interact with S100A10 residing in a complex with plasma membrane-bound annexin A2

Tertiary structure modeling of the mutant HGD protein.

<p>(<b>A</b>) Crystal structure of the normal and (<b>B</b>) tertiary structure model of the mutant HGD protein α-helical coils, β-pleated sheets and interconnecting loops are colored in turquoise, magenta and pink, respectively. In (A), exon 2 is represented by the two yellow anti-parallel β-pleated sheets and green interconnecting loops. The adjoining four amino acids from exons 1 and 3 are shown in blue. Note in (B), the absence of exon 2 β-pleated sheets and loops and the presence of a novel loop (shown in blue) that represents 4 amino acids from each of exons 1 and 3. Despite the similarities of the β-sheets between the two structures, most of the amino acids in the α-helical coils are different from the native structure. This figure does not contain any copyrighted image.</p

Regions of homozygosity (ROH) derived from the proband in the SNP array.

<p>(<b>A</b>) The cytogenetic location, genomic coordinates (hg19) and sizes of each ROH. (<b>B</b>) Location and size (in Mb) of each ROH on chromosomes 3, 4, 9 and 11.</p

Identification of the 649 bp deletion in the proband.

<p>(<b>A</b>) PCR genotyping of the 649 bp deletion in a normal control (NC), mother (M), father (F) and proband (P). Size markers (M) consist of a 500 bp ladder. The sizes of the amplicons representing the normal (1073 bp) and mutant (424 bp) alleles are indicated next to the gel. (<b>B</b>) Sanger DNA sequencing electropherogram of the mutant amplicon, showing the deletion breakpoint (indicated by the arrow). (<b>C</b>) Representative DNA sequence of the 1073 bp amplicon extending from the forward and reverse primer (shown in red color). The strikethrough bases indicate the deleted DNA sequences and those of exon 2 are highlighted.</p

Amino acid sequences of α-helical coils and β-pleated sheets in the normal and mutant HGD proteins are listed from their amino to carboxy termini.

<p>Note that all the coils in the predicted protein are different whereas only some β-pleated sheets are missing. The two pleated sheets spanned by exon 2 are denoted as deleted from the mutant protein.</p><p>Amino acid sequences of α-helical coils and β-pleated sheets in the normal and mutant HGD proteins are listed from their amino to carboxy termini.</p

CC chemokine receptor 10 cell surface presentation in melanocytes is regulated by the novel interaction partner S100A10

The superfamily of G-protein-coupled receptors (GPCR) conveys signals in response to various endogenous and exogenous stimuli. Consequently, GPCRs are the most important drug targets. CCR10, the receptor for the chemokines CCL27/CTACK and CCL28/MEC, belongs to the chemokine receptor subfamily of GPCRs and is thought to function in immune responses and tumour progression. However, there is only limited information on the intracellular regulation of CCR10. We find that S100A10, a member of the S100 family of Ca2+ binding proteins, binds directly to the C-terminal cytoplasmic tail of CCR10 and that this interaction regulates the CCR10 cell surface presentation. This identifies S100A10 as a novel interaction partner and regulator of CCR10 that might serve as a target for therapeutic intervention

First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5-16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband's phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin

Saudi guidelines on the diagnosis and treatment of pulmonary hypertension: 2014 updates

The Saudi Association for Pulmonary Hypertension (previously called Saudi Advisory Group for Pulmonary Hypertension) has published the first Saudi Guidelines on Diagnosis and Treatment of Pulmonary Arterial Hypertension back in 2008. [1] That guideline was very detailed and extensive and reviewed most aspects of pulmonary hypertension (PH). One of the disadvantages of such detailed guidelines is the difficulty that some of the readers who just want to get a quick guidance or looking for a specific piece of information might face. All efforts were made to develop this guideline in an easy-to-read form, making it very handy and helpful to clinicians dealing with PH patients to select the best management strategies for the typical patient suffering from a specific condition. This Guideline was designed to provide recommendations for problems frequently encountered by practicing clinicians involved in management of PH. This publication targets mainly adult and pediatric PH-treating physicians, but can also be used by other physicians interested in PH

A novel Munc13-4/S100A10/annexin A2 complex promotes Weibel–Palade body exocytosis in endothelial cells

Endothelial cells respond to blood vessel injury by the acute release of the procoagulant von Willebrand factor, which is stored in unique secretory granules called Weibel-Palade bodies (WPBs). Stimulated WPB exocytosis critically depends on their proper recruitment to the plasma membrane, but factors involved in WPB-plasma membrane tethering are not known. Here we identify Munc13-4, a protein mutated in familial hemophagocytic lymphohistiocytosis 3, as a WPB-tethering factor. Munc13-4 promotes histamine-evoked WPB exocytosis and is present on WPBs, and secretagogue stimulation triggers an increased recruitment of Munc13-4 to WPBs and a clustering of Munc13-4 at sites of WPB-plasma membrane contact. We also identify the S100A10 subunit of the annexin A2 (AnxA2)-S100A10 protein complex as a novel Munc13-4 interactor and show that AnxA2-S100A10 participates in recruiting Munc13-4 to WPB fusion sites. These findings indicate that Munc13-4 supports acute WPB exocytosis by tethering WPBs to the plasma membrane via AnxA2-S100A10