A phosphoinositide conversion mechanism for exit from endosomes.

Phosphoinositides are a minor class of short-lived membrane phospholipids that serve crucial functions in cell physiology ranging from cell signalling and motility to their role as signposts of compartmental membrane identity. Phosphoinositide 4-phosphates such as phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) are concentrated at the plasma membrane, on secretory organelles, and on lysosomes, whereas phosphoinositide 3-phosphates, most notably phosphatidylinositol 3-phosphate (PI(3)P), are a hallmark of the endosomal system. Directional membrane traffic between endosomal and secretory compartments, although inherently complex, therefore requires regulated phosphoinositide conversion. The molecular mechanism underlying this conversion of phosphoinositide identity during cargo exit from endosomes by exocytosis is unknown. Here we report that surface delivery of endosomal cargo requires hydrolysis of PI(3)P by the phosphatidylinositol 3-phosphatase MTM1, an enzyme whose loss of function leads to X-linked centronuclear myopathy (also called myotubular myopathy) in humans. Removal of endosomal PI(3)P by MTM1 is accompanied by phosphatidylinositol 4-kinase-2α (PI4K2α)-dependent generation of PI(4)P and recruitment of the exocyst tethering complex to enable membrane fusion. Our data establish a mechanism for phosphoinositide conversion from PI(3)P to PI(4)P at endosomes en route to the plasma membrane and suggest that defective phosphoinositide conversion at endosomes underlies X-linked centronuclear myopathy caused by mutation of MTM1 in humans.

PI4K2ß/AP-1-based TGN-endosomal sorting regulates Wnt signaling.

Endosomal membrane traffic serves crucial roles in cell physiology, signaling, and development. Sorting between endosomes and the trans-Golgi network (TGN) is regulated among other factors by the adaptor AP-1, an essential component of multicellular organisms. Membrane recruitment of AP-1 requires phosphatidylinositol 4-phosphate [PI(4)P], though the precise mechanisms and PI4 kinase isozyme (or isozymes) involved in generation of this PI(4)P pool remain unclear. The Wnt pathway is a major developmental signaling cascade and depends on endosomal sorting in Wnt-sending cells. Whether TGN/endosomal sorting modulates signaling downstream of Frizzled (Fz) receptors in Wnt-receiving cells is unknown. Here, we identify PI4-kinase type 2ß (PI4K2ß) as a regulator of TGN/endosomal sorting and Wnt signaling. PI4K2ß and AP-1 interact directly and are required for efficient sorting between endosomes and the TGN. Zebrafish embryos depleted of PI4K2ß or AP-1 lack pectoral fins due to defective Wnt signaling. Rescue experiments demonstrate requirements for PI4K2ß-AP-1 complex formation and PI4K2ß-mediated PI(4)P synthesis. Furthermore, PI4K2ß binds to the Fz-associated component Dishevelled (Dvl) and regulates endosomal recycling of Fz receptors and Wnt target gene expression. These data reveal an evolutionarily conserved role for PI4K2ß and AP-1 in coupling phosphoinositide metabolism to AP-1-mediated sorting and Wnt signaling.

Phosphatidylinositol 4-kinase IIα function at endosomes is regulated by the ubiquitin ligase Itch.

Phosphatidylinositol (PI) 4-phosphate (PI(4)P) and its metabolizing enzymes serve important functions in cell signalling and membrane traffic. PI 4-kinase type IIα (PI4KIIα) regulates Wnt signalling, endosomal sorting of signalling receptors, and promotes adaptor protein recruitment to endosomes and the trans-Golgi network. Here we identify the E3 ubiquitin ligase Itch as binding partner and regulator of PI4KIIα function. Itch directly associates with and ubiquitinates PI4KIIα, and both proteins colocalize on endosomes containing Wnt-activated frizzled 4 (Fz4) receptor. Depletion of PI4KIIα or Itch regulates Wnt signalling with corresponding changes in Fz4 internalization and degradative sorting. These findings unravel a new molecular link between phosphoinositide-regulated endosomal membrane traffic, ubiquitin and the modulation of Wnt signalling.

Regulation of phosphoinositide-metabolizing enzymes by clathrin coat proteins.

Clathrin plays key roles in endocytic and endo-lysosomal membrane dynamics by facilitating the formation of coated vesicles at the plasma membrane and at the trans-Golgi network (TGN)/endosomal boundary. Assembly of the clathrin lattice critically depends on adaptor proteins and accessory proteins, which connect the clathrin scaffold to the membrane and to transmembrane cargo including receptors, transporters, channels, and SNARE proteins. The recruitment of adaptor proteins to membrane surfaces is triggered by coincidence-detection mechanisms involving phosphoinositides (PIs), cargo proteins, and in many cases small GTPases. To tightly regulate coat formation, there is extensive cross-talk between PI-metabolizing enzymes and adaptor proteins. One of the best studied examples is the endocytic clathrin adaptor complex AP-2, which binds plasma membrane-enriched PI(4,5)P(2). In neurons, PI(4,5)P(2) is synthesized from PI(4)P primarily by the γ-isoform of the type I phosphatidylinositol 4-phosphate 5-kinase family (PIPKIγ), whose enzymatic activity is regulated by direct binding to, amongst others, the small GTPase Arf6 and AP-2. Cargo-bound AP-2 potently stimulates PIPK1γ activity and thereby drives AP-2-membrane interactions. This feed-forward loop is thought to facilitate membrane translocation of additional AP-2 molecules and concomitantly clathrin, but also of endocytic accessory proteins, many of which directly associate with PI(4,5)P(2). It is likely that similar mechanisms support the formation of coated vesicles at the trans-Golgi network (TGN) and on endosomes, involving PI(4)P and PI(3)P respectively, but detailed knowledge is lacking to date. To explore how coat proteins regulate kinase activity, assays are needed to sensitively detect subtle changes in PI synthesis and to discriminate between the various PI species. Here we describe a sensitive and specific radioactivity-based assay to measure PI kinase activity.

Methods for analysis of rab27a/Munc13-4 in secretory lysosome release in hematopoietic cells.

Secretory lysosomes constitute a heterogeneous organelle of hematopoietic cells that combines the properties of regular lysosomes with those of secretory granules. Although secretory lysosomes serve essential functions, such as in the immune system and blood clotting, the mechanisms underlying the release of contents are incompletely understood. It is clear, however, that rab27a and the C2 domain protein munc13-4 serve essential functions. Mutations in these genes lead to immune disorders where the lytic granule function of cytotoxic T cells is jeopardized in humans. We identified munc13-4 as a rab27a binding protein from spleen. Munc13-4 is highly expressed in several hematopoietic cells including cytotoxic T cells and mast cells. We describe the molecular features of the interaction and requirements for localization, and show that munc13-4 is a positive regulator of secretory lysosome exocytosis.

Expression clustering reveals detailed co-expression patterns of functionally related proteins during B cell differentiation: a proteomic study using a combination of one-dimensional gel electrophoresis, LC-MS/MS, and stable isotope labeling by amino acids in cell culture (SILAC).

B cells play an essential role in the immune response. Upon activation they may differentiate into plasma cells that secrete specific antibodies against potentially pathogenic non-self antigens. To identify the cellular proteins that are important for efficient production of these antibodies we set out to study the B cell differentiation process at the proteome level. We performed an in-depth proteomic study to quantify dynamic relative protein expression patterns of several hundreds of proteins at five consecutive time points after lipopolysaccharide-induced activation of B lymphocytes. The proteome analysis was performed using a combination of stable isotope labeling using [13C6]leucine added to the murine B cell cultures, one-dimensional gel electrophoresis, and LC-MS/MS. In this study we identified 1,001 B cell proteins. We were able to quantify the expression levels of a quarter of all identified proteins (i.e. 234) at each of the five different time points. Nearly all proteins revealed changes in expression patterns. The quantitative dataset was further analyzed using an unbiased clustering method. Based on their expression profiles, we grouped the entire set of 234 quantified proteins into a limited number of 12 distinct clusters. Functionally related proteins showed a strong correlation in their temporal expression profiles. The quality of the quantitative data allowed us to even identify subclusters within functionally related classes of proteins such as in the endoplasmic reticulum proteins that are involved in antibody production.

Munc13-4 is an effector of rab27a and controls secretion of lysosomes in hematopoietic cells.

Griscelli syndrome type 2 (GS2) is a genetic disorder in which patients exhibit life-threatening defects of cytotoxic T lymphocytes (CTLs) whose lytic granules fail to dock on the plasma membrane and therefore do not release their contents. The disease is caused by the absence of functional rab27a, but how rab27a controls secretion of lytic granule contents remains elusive. Mutations in Munc13-4 cause familial hemophagocytic lymphohistiocytosis subtype 3 (FHL3), a disease phenotypically related to GS2. We show that Munc13-4 is a direct partner of rab27a. The two proteins are highly expressed in CTLs and mast cells where they colocalize on secretory lysosomes. The region comprising the Munc13 homology domains is essential for the localization of Munc13-4 to secretory lysosomes. The GS2 mutant rab27aW73G strongly reduced binding to Munc13-4, whereas the FHL3 mutant Munc13-4Delta608-611 failed to bind rab27a. Overexpression of Munc13-4 enhanced degranulation of secretory lysosomes in mast cells, showing that it has a positive regulatory role in secretory lysosome fusion. We suggest that the secretion defects seen in GS2 and FHL3 have a common origin, and we propose that the rab27a/Munc13-4 complex is an essential regulator of secretory granule fusion with the plasma membrane in hematopoietic cells. Mutations in either of the two genes prevent formation of this complex and abolish secretion.

Fluorescent protein tagging in Toxoplasma gondii: identification of a novel inner membrane complex component conserved among Apicomplexa.

Toxoplasma gondii is an obligate intracellular parasite, and its sub-cellular organization shows clear adaptations to this life-style. In addition to organelles shared among all eukaryotes, the organism possesses a number of specialized compartments with important roles in host cell invasion and intra-cellular survival. These unique aspects of the parasite's biology are also reflected in its genome. The ongoing genome sequencing efforts for T. gondii and related apicomplexans predict a high proportion of genes unique to the phylum, which lack homologs in other model organisms. Knowing the sub-cellular localization of these gene products will be an important first step towards their functional characterization. We used a library approach wherein parasite genomic DNA was fused to the yellow fluorescent protein (YFP) gene. Parasites transformed with this library were screened by flow cytometry and fluorescence microscopy. Clones tagged in a wide variety of sub-cellular compartments (nucleus, mitochondria, ER, dense granules (secreted), spliceosome, plasma membrane, apicoplast, inner membrane complex) were isolated and confirmed using compartment specific markers. Clones with tags in parasite-specific localizations were subjected to insert rescue and phenotypic verification using an in vitro recombination system. Among the genes identified is a novel inner membrane complex gene (IMC3) conserved among Apicomplexa.