Vesicular Sorting Research Articles

Toxoplasma gondii Vps11, a subunit of HOPS and CORVET tethering complexes, is essential for the biogenesis of secretory organelles.

Apicomplexan parasites harbour unique secretory organelles (dense granules, rhoptries and micronemes) that play essential functions in host infection. Toxoplasma gondii parasites seem to possess an atypical endosome-like compartment, which contains an assortment of proteins that appear to be involved in vesicular sorting and trafficking towards secretory organelles. Recent studies highlighted the essential roles of many regulators such as Rab5A, Rab5C, sortilin-like receptor and syntaxin-6 in secretory organelle biogenesis. However, little is known about the protein complexes that recruit Rab-GTPases and SNAREs for membrane tethering in Apicomplexa. In mammals and yeast, transport, tethering and fusion of vesicles from early endosomes to lysosomes and the vacuole, respectively, are mediated by CORVET and HOPS complexes, both built on the same Vps-C core that includes Vps11 protein. Here, we show that a T. gondii Vps11 orthologue is essential for the biogenesis or proper subcellular localization of secretory organelle proteins. TgVps11 is a dynamic protein that associates with Golgi endosomal-related compartments, the vacuole and immature apical secretory organelles. Conditional knock-down of TgVps11 disrupts biogenesis of dense granules, rhoptries and micronemes. As a consequence, parasite motility, invasion, egress and intracellular growth are affected. This phenotype was confirmed with additional knock-down mutants of the HOPS complex. In conclusion, we show that apicomplexan parasites use canonical regulators of the endolysosome system to accomplish essential parasite-specific functions in the biogenesis of their unique secretory organelles.

Extracellular vesicles as emerging intercellular communicasomes.

All living cells release extracellular vesicles having pleiotropic functions in intercellular communication. Mammalian extracellular vesicles, also known as exosomes and microvesicles, are spherical bilayered proteolipids composed of various bioactive molecules, including RNAs, DNAs, proteins, and lipids. Extracellular vesicles directly and indirectly control a diverse range of biological processes by transferring membrane proteins, signaling molecules, mRNAs, and miRNAs, and activating receptors of recipient cells. The active interaction of extracellular vesicles with other cells regulates various physiological and pathological conditions, including cancer, infectious diseases, and neurodegenerative disorders. Recent developments in high-throughput proteomics, transcriptomics, and lipidomics tools have provided ample data on the common and specific components of various types of extracellular vesicles. These studies may contribute to the understanding of the molecular mechanism involved in vesicular cargo sorting and the biogenesis of extracellular vesicles, and, further, to the identification of disease-specific biomarkers. This review focuses on the components, functions, and therapeutic and diagnostic potential of extracellular vesicles under various pathophysiological conditions. [BMB Reports 2014; 47(10): 531-539]

Vesicular transport of a ribonucleoprotein to mitochondria.

ABSTRACTIntracellular trafficking of viruses and proteins commonly occurs via the early endosome in a process involving Rab5. The RNA Import Complex (RIC)-RNA complex is taken up by mammalian cells and targeted to mitochondria. Through RNA interference, it was shown that mito-targeting of the ribonucleoprotein (RNP) was dependent on caveolin 1 (Cav1), dynamin 2, Filamin A and NSF. Although a minor fraction of the RNP was transported to endosomes in a Rab5-dependent manner, mito-targeting was independent of Rab5 or other endosomal proteins, suggesting that endosomal uptake and mito-targeting occur independently. Sequential immunoprecipitation of the cytosolic vesicles showed the sorting of the RNP away from Cav1 in a process that was independent of the endosomal effector EEA1 but sensitive to nocodazole. However, the RNP was in two types of vesicle with or without Cav1, with membrane-bound, asymmetrically orientated RIC and entrapped RNA, but no endosomal components, suggesting vesicular sorting rather than escape of free RNP from endosomes. In vitro, RNP was directly transferred from the Type 2 vesicles to mitochondria. Live-cell imaging captured spherical Cav1− RNP vesicles emerging from the fission of large Cav+ particles. Thus, RNP appears to traffic by a different route than the classical Rab5-dependent pathway of viral transport.

Parkinson's disease-linked mutations in VPS35 induce dopaminergic neurodegeneration.

Mutations in the vacuolar protein sorting 35 homolog (VPS35) gene at the PARK17 locus, encoding a key component of the retromer complex, were recently identified as a new cause of late-onset, autosomal dominant Parkinson's disease (PD). Here we explore the pathogenic consequences of PD-associated mutations in VPS35 using a number of model systems. VPS35 exhibits a broad neuronal distribution throughout the rodent brain, including within the nigrostriatal dopaminergic pathway. In the human brain, VPS35 protein levels and distribution are similar in tissues from control and PD subjects, and VPS35 is not associated with Lewy body pathology. The common D620N missense mutation in VPS35 does not compromise its protein stability or localization to endosomal and lysosomal vesicles, or the vesicular sorting of the retromer cargo, sortilin, SorLA and cation-independent mannose 6-phosphate receptor, in rodent primary neurons or patient-derived human fibroblasts. In yeast we show that PD-linked VPS35 mutations are functional and can normally complement VPS35 null phenotypes suggesting that they do not result in a loss-of-function. In rat primary cortical cultures the overexpression of human VPS35 induces neuronal cell death and increases neuronal vulnerability to PD-relevant cellular stress. In a novel viral-mediated gene transfer rat model, the expression of D620N VPS35 induces the marked degeneration of substantia nigra dopaminergic neurons and axonal pathology, a cardinal pathological hallmark of PD. Collectively, these studies establish that dominant VPS35 mutations lead to neurodegeneration in PD consistent with a gain-of-function mechanism, and support a key role for VPS35 in the development of PD.

Analysis of three μ1‐AP1 subunits during zebrafish development

The family of AP-1 complexes mediates protein sorting in the late secretory pathway and it is essential for the development of mammals. The ubiquitously expressed AP-1A complex consists of four adaptins γ1, β1, μ1A, and σ1A. AP-1A mediates protein transport between the trans-Golgi network and early endosomes. The polarized epithelia AP-1B complex contains the μ1B-adaptin. AP-1B mediates specific transport of proteins from basolateral recycling endosomes to the basolateral plasma membrane of polarized epithelial cells. Analysis of the zebrafish genome revealed the existence of three μ1-adaptin genes, encoding μ1A, μ1B, and the novel isoform μ1C, which is not found in mammals. μ1C shows 80% sequence identity with μ1A and μ1B. The μ1C expression pattern largely overlaps with that of μ1A, while μ1B is expressed in epithelial cells. By knocking-down the synthesis of μ1A, μ1B and μ1C with antisense morpholino techniques we demonstrate that each of these μ1 adaptins is essential for zebrafish development, with μ1A and μ1C being involved in central nervous system development and μ1B in kidney, gut and liver formation. Zebrafish is unique in expressing three AP-1 complexes: AP-1A, AP-1B, and AP-1C. Our results demonstrate that they are not redundant and that each of them has specific functions, which cannot be fulfilled by one of the other isoforms. Each of the μ1 adaptins appears to mediate specific molecular mechanisms essential for early developmental processes, which depends on specific intracellular vesicular protein sorting pathways.

Hepatocyte growth factor regulated tyrosine kinase substrate in the peripheral development and function of B-cells

Hepatocyte growth factor (HGF)-regulated tyrosine kinase substrate (Hrs) is a vesicular sorting protein that functions as one of the endosomal-sorting proteins required for transport (ESCRT). Hrs, which binds to ubiquitinated proteins through its ubiquitin-interacting motif (UIM), contributes to the lysosomal transport and degradation of ubiquitinated membrane proteins. However, little is known about the relationship between B-cell functions and ESCRT proteins in vivo. Here we examined the immunological roles of Hrs in B-cell development and functions using B-cell-specific Hrs-deficient (Hrsflox/flox;mb1cre/+:Hrs-cKO) mice, which were generated using a cre-LoxP recombination system. Hrs deficiency in B-cells significantly reduced T-cell-dependent antibody production in vivo and impaired the proliferation of B-cells treated in vitro with an anti-IgM monoclonal antibody but not with LPS. Although early development of B-cells in the bone marrow was normal in Hrs-cKO mice, there was a significant decrease in the number of the peripheral transitional B-cells and marginal zone B-cells in the spleen of Hrs-cKO mice. These results indicate that Hrs plays important roles during peripheral development and physiological functions of B lymphocytes.

Possible role for the adaptor protein complexes in sorting of human papillomavirus

Human papillomaviruses (HPVs) infect genital and mucosal epithelium and persistent infection with high‐risk strains like HPV16 are associated with cervical, anogenital, and nasopharyngeal cancers. HPV16 particles consist of two structural proteins: the major capsid protein L1 and the minor capsid protein L2. In a previous yeast two hybrid screen using L2 as bait, we identified a potential interaction with a truncated μ chain (μ2) of the adaptor protein complex‐2 (AP2). The AP complex family directs vesicular sorting of cargo via interactions between the AP complex μ subunits and YxxΦ motifs within the cytoplasmic tails of transmembrane receptors. Interestingly L2 contains a number of YxxΦ motifs with variable conservation among different HPV types. The goal of this project is to determine which AP complexes L2 interacts with, how these interactions occur, and the functions of these interactions during infection. Yeast two‐hybrid screens indicate that full‐length L2 interacts with all AP complex μ chains but smaller L2‐YxxΦ peptides have preference for specific μ chains. Infections with YxxΦ‐mutant viruses show decreased infectivity, strongly supporting a role for AP complexes in infection. siRNA single knockdowns of AP complex μ chains show varying effects on infectivity. Current work seeks to understand the nature of these defects and to assess specific roles for L2‐AP complex interactions during infection.Support for this research comes from the Undergraduate Biology Research Program (U. of Arizona UBRP) and the American Cancer Society Research Scholar Grant RSG‐117469.

COG complexes form spatial landmarks for distinct SNARE complexes

Vesicular tethers and SNAREs are two key protein components of the intracellular membrane trafficking machinery. The COG (conserved oligomeric Golgi) complex has been implicated in the tethering of retrograde intra-Golgi vesicles. Here, using yeast two hybrid and co-immunoprecipitation approaches, we show that three COG subunits, namely COG4, 6, and 8, are capable of interacting with defined Golgi SNAREs, namely STX5, STX6, STX16, GS27, and SNAP29. Comparative analysis of COG8-STX16 and COG4-STX5 interactions by a COG-based mitochondrial re-localization assay reveals that the COG8 and COG4 proteins initiate the formation of two different tethering platforms that can facilitate the redirection of two populations of Golgi transport intermediates to the mitochondrial vicinity. Our results uncover a role for COG subcomplexes in defining the specificity of vesicular sorting within the Golgi.

Particle Tracking Analysis for the Intracellular Trafficking of Nanoparticles Modified with African Swine Fever Virus Protein p54-derived Peptide

Previous studies showed that the cytoplasmic transport of nanoparticles to the nucleus is driven by a vesicular sorting system. Artificial approaches for targeting a microtubule-associating motor complex is also a challenge. We describe herein the development of a liposomal nanoparticle, the surface of which is modified with stearylated octa-arginine (STR-R8), and a dynein light chain (LC8)-associated peptide derived from an African swine fever virus protein p54 (p54(149-161)) with polyethyleneglycol (PEG) as a spacer (p54(149-161)-PEG/R8-liposomal nanoparticles (LNPs)). The p54(149-161)-PEG/R8-LNPs preferentially gain access to the nucleus, resulting in a one- to two-order of magnitude higher transfection activity in comparison with p54(149-161)-free nanoparticles (PEG/R8-LNPs). Further studies of particle tracking in HeLa cells stably expressing green fluorescent protein (GFP)-tagged tubulin (GFP/Tub-HeLa) indicate that p54(149-161) stimulated the transport of nanoparticles along fibrous tubulin structures. Moreover, a part of the p54(149-161)-PEG/R8-LNPs appeared to undergo quasi-straight transport without sharing the tracks corresponding to PKH67, the plasma membrane of which had been prestained with a marker just before transfection, while corresponding movement was never observed in the case of PEG/R8-LNPs. These findings suggest that a portion of the p54(149-161)-modified nanoparticles can use microtubule-dependent transport without the need for an assist by a vesicular sorting system.

Vesicular sorting controls the polarity of expanding membranes in theC. elegansintestine

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., the matrix, inside the cell, e.g., vesicular trafficking and the plasma membrane and its junctions.1 Many highly conserved polarity cues have been identified, but their integration during the complex process of polarized tissue and organ morphogenesis is not well understood. It is assumed that plasma-membrane-associated polarity determinants, such as the partitioning-defective (PAR) complex, define plasma membrane domain identities, whereas vesicular trafficking delivers membrane components to these domains, but lacks the ability to define them. In vitro studies on lumenal membrane biogenesis in mammalian cell lines now indicate that trafficking could contribute to defining membrane domains by targeting the polarity determinants, e.g., the PARs, themselves.2 This possibility suggests a mechanism for PARs’ asymmetric distribution on membranes and places vesicle-associated polarity cues upstream of membrane-associated polarity determinants. In such an upstream position, trafficking might even direct multiple membrane components, not only polarity determinants, an original concept of polarized plasma membrane biogenesis3,4that was largely abandoned due to the failure to identify a molecularly defined intrinsic vesicular sorting mechanism. Our two recent studies on C. elegans intestinal tubulogenesis reveal that glycosphingolipids (GSLs) and the well-recognized vesicle components clathrin and its AP-1 adaptor are required for targeting multiple apical molecules, including polarity regulators, to the expanding apical/lumenal membrane.5,6 These findings support GSLs’ long-proposed role in in vivo polarized epithelial membrane biogenesis and development and identify a novel function in apical polarity for classical post-Golgi vesicle components. They are also compatible with a vesicle-intrinsic sorting mechanism during membrane biogenesis and suggest a model for how vesicles could acquire apical directionality during the assembly of the functionally critical polarized lumenal surfaces of epithelial tubes.

Abnormal expression of PI3K isoforms in patients with tobacco-related oral squamous cell carcinoma

BackgroundThe phosphatidylinositol 3-kinase (PI3K) signaling regulates several cellular functions such as motility, proliferation, angiogenesis and survival. MethodsSince there is no information on expression of PI3K isoforms in oral cancer, we studied the expression of different isoforms of PI3K (p110α, p110γ, PI3K-C2, Vps34p and p85α) in tumor samples and PBMC by RT and q-RTPCR and serum levels of PI3K p110α by SPR and ELISA techniques in 108 patients with tobacco-related oral squamous cell carcinoma (OSCC) and 46 normal subjects. ResultsWe observed significantly higher PI3K p110α (p<0.0001) and lower (p<0.0001) vesicular sorting protein 34p (Vps34p) mRNA both in PBMC and tissue samples of oral cancer patients as compared to the normal controls. Other PI3K isoforms did not show such change. Circulating PI3K p110α levels were higher in patients (p<0.0001) as compared to healthy subjects, the SPR data showed direct correlation with advancing stage of the disease. PI3K p110α was overexpressed in tumor samples but not in the normal buccal mucosa. ConclusionsUpregulation of circulating PI3K p110α isoform and its direct correlation with increasing tumor load in OSCC patients indicates that it may be a significant prognostic indicator and a suitable target for therapeutic/chemo-preventive strategies for tobacco-related OSCC.

Birt–Hogg–Dubé: tumour suppressor function and signalling dynamics central to folliculin

The cellular function of folliculin (FLCN) is a mystery that still needs to be solved. It is known that mutation of FLCN can predispose Birt-Hogg-Dubé (BHD) patient's to renal cell carcinoma , renal and lung cysts, as well as skin fibrofolliculomas. FLCN has been classed as a tumour suppressor, but it is probable that cystic and the skin manifestations do not occur as a consequence of FLCN loss of heterozygosity. Discovery that FLCN is a direct substrate of AMP dependent protein kinase (AMPK) placed FLCN on the cell signalling map, downstream of AMPK. This breakthrough suggested that FLCN might be involved in cell energy homeostasis. Over these more recent years, BHD research has become much more complicated and interesting from a cell signalling perspective. Folliculin has been linked to numerous cell pathways that are known to cause cancer, involving cell growth, metabolism, cell adhesion, cell motility, cytokinesis, and cell survival. The collective evidence implies that FLCN may have a broader housekeeping role in the cell. Of particular importance, FLCN was recently been reported to have guanine exchange factor activity towards the small G protein Rab35 and implicates FLCN in vesicular trafficking and/or membrane sorting. This newer discovery will undoubtedly help in the continued challenge of solving the signalling puzzle that shrouds FLCN function.

An SNX10 mutation causes malignant osteopetrosis of infancy

BackgroundOsteopetrosis is a life-threatening, rare disorder typically resulting from osteoclast dysfunction and infrequently from failure to commitment to osteoclast lineage. Patients commonly present in infancy with macrocephaly, feeding difficulties, evolving...

Split decision: a thaumarchaeon encoding both FtsZ and Cdv cell division proteins chooses Cdv for cytokinesis

Cytoskeletal proteins play a pivotal role in cytokinesis in prokaryotes and eukaryotes. Most bacteria and a major branch of the archaea called the Euryarchaeota harbour a tubulin homologue, FtsZ, which assembles into a dynamic polymeric ring structure required for cytokinesis. However, Crenarchaeota, another branch of the archaea, lack FtsZ and instead use Cdv proteins, which are homologues of the ESCRT-III-like system involved in vesicular sorting and cytokinesis in eukaryotes, for cell division. Recently, a group of Crenarchaeota that grow in non-extreme environments was found to be sufficiently divergent to warrant its own branch of the archaea called the Thaumarchaeota. Notably, Thaumarchaeota have both Cdv and FtsZ homologues, which begs the question of which system is used for cell division. In this issue of Molecular Microbiology,Pelve et al. (2011) Pelve and colleagues tackle this question. They found that cells of the thaumarchaeon Nitrosopumilus maritimus likely divide using the Cdv system and not FtsZ, based on localization of Cdv proteins but not FtsZ to division sites. The authors also provide evidence that the cell cycle during growth of N. maritimus differs significantly from those of other archaea.

Translation Initiator EIF4G1 Mutations in Familial Parkinson Disease

Genome-wide analysis of a multi-incident family with autosomal-dominant parkinsonism has implicated a locus on chromosomal region 3q26-q28. Linkage and disease segregation is explained by a missense mutation c.3614G>A (p.Arg1205His) in eukaryotic translation initiation factor 4-gamma (EIF4G1). Subsequent sequence and genotype analysis identified EIF4G1 c.1505C>T (p.Ala502Val), c.2056G>T (p.Gly686Cys), c.3490A>C (p.Ser1164Arg), c.3589C>T (p.Arg1197Trp) and c.3614G>A (p.Arg1205His) substitutions in affected subjects with familial parkinsonism and idiopathic Lewy body disease but not in control subjects. Despite different countries of origin, persons with EIF4G1 c.1505C>T (p.Ala502Val) or c.3614G>A (p.Arg1205His) mutations appear to share haplotypes consistent with ancestral founders. eIF4G1 p.Ala502Val and p.Arg1205His disrupt eIF4E or eIF3e binding, although the wild-type protein does not, and render mutant cells more vulnerable to reactive oxidative species. EIF4G1 mutations implicate mRNA translation initiation in familial parkinsonism and highlight a convergent pathway for monogenic, toxin and perhaps virally-induced Parkinson disease.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Role of c-Cbl Carboxyl Terminus in Serotonin 5-HT2A Receptor Recycling and Resensitization

The 5-hydroxytryptamine 2A receptor (5-HT(2A)R) undergoes constitutive and agonist-dependent internalization. Despite many advances in our understanding of G protein-coupled receptor trafficking, the exact mechanism of endocytic sorting of G protein-coupled receptors remains obscure. Recently, we have reported a novel finding documenting a global role for the ubiquitin ligase c-Cbl in regulating vesicular sorting of epidermal growth factor receptor (Baldys, A., Göoz, M., Morinelli, T. A., Lee, M. H., Raymond, J. R., Jr., Luttrell, L. M., and Raymond, J. R., Sr. (2009) Biochemistry 48, 1462-1473). Thus, we tested the hypothesis that c-Cbl might play a role in 5-HT(2A)R recycling. In this study, we demonstrated an association of 5-HT(2A)R with c-Cbl. Furthermore, down-regulation of c-Cbl by RNA interference blocked efficient recycling of 5-HT(2A)R to the plasma membrane. Immunofluorescence microscopy revealed that 5-HT(2A) receptors were trapped in early endosome antigen 1- and Rab11-positive sorting endosomes in cells overexpressing c-Cbl mutants lacking carboxyl termini. This inhibitory effect was associated with a relative decrease in association of c-Cbl truncation proteins with the 5-HT(2A)R, compared with that observed for the full-length c-Cbl fusion protein. Consistent with the delayed recycling, 5-HT(2A)R resensitization was greatly attenuated in the presence of c-Cbl mutants lacking carboxyl termini, as detected by changes in the cytosolic calcium. Taken together, these studies have led to the discovery that the C-terminal region of c-Cbl plays a crucial role in the temporal and spatial control of 5-HT(2A)R recycling.

Failure of acrosome formation and globozoospermia in the wobbler mouse, a Vps54 spontaneous recessive mutant

The acrosome is a unique organelle that plays an important role at fertilization and during sperm morphogenesis and that is absent in globozoospermia, an inherited infertility syndrome in humans. At the light of recent experimental evidence, the acrosome is considered a lysosome-related organelle to whose biogenesis both the endocytic and biosynthetic pathways contribute. Vps54 is a vesicular sorting protein involved in the retrograde traffic; the recessive Vps54(L967Q) mutation in the mouse results in the wobbler phenotype, characterized by motor-neuron degeneration and male infertility. Here we have investigated the spatio-temporal occurrence/progression of the wobbler fertility disorder starting from mice at post-natal day 35, the day of the first event of spermiation. We show that the pathogenesis of wobbler infertility originates at the first spermiogenetic wave, affecting acrosome formation and sperm head elongation. Vps54(L967Q)-labeled vesicles, on the contrary of the wild-type Vps54-labeled ones, are not able to coalesce into a larger vesicle that develops, flattens and shapes to give rise to the acrosome. Evidence that it is the malfunctioning of the endocytic traffic to hamper the development of the acrosome comes out from the study on UBPy. UBPy, a deubiquitinating enzyme, is a marker of acrosome biogenesis from the endocytic pathway. In wobbler spermatids UBPy-positive endosomes remain single, scattered vesicles that do not contribute to acrosome formation. As secondary defect of wobbler spermiogenesis, spermatid mitochondria are misorted; moreover, with the progression of the age/disease also Sertoli-germ cell adhesions are compromised suggesting a derailment in the endocytic route that underlies their restructuring.

Role of Sphingomyelin Synthase in Controlling the Antimicrobial Activity of Neutrophils against Cryptococcus neoformans

The key host cellular pathway(s) necessary to control the infection caused by inhalation of the environmental fungal pathogen Cryptococcus neoformans are still largely unknown. Here we have identified that the sphingolipid pathway in neutrophils is required for them to exert their killing activity on the fungus. In particular, using both pharmacological and genetic approaches, we show that inhibition of sphingomyelin synthase (SMS) activity profoundly impairs the killing ability of neutrophils by preventing the extracellular release of an antifungal factor(s). We next found that inhibition of protein kinase D (PKD), which controls vesicular sorting and secretion and is regulated by diacylglycerol (DAG) produced by SMS, totally blocks the extracellular killing activity of neutrophils against C. neoformans. The expression of SMS genes, SMS activity and the levels of the lipids regulated by SMS (namely sphingomyelin (SM) and DAG) are up-regulated during neutrophil differentiation. Finally, tissue imaging of lungs infected with C. neoformans using matrix-assisted laser desorption-ionization mass spectrometry (MALDI-MS), revealed that specific SM species are associated with neutrophil infiltration at the site of the infection. This study establishes a key role for SMS in the regulation of the killing activity of neutrophils against C. neoformans through a DAG-PKD dependent mechanism, and provides, for the first time, new insights into the protective role of host sphingolipids against a fungal infection.

Proteolytic processing of protocadherin proteins requires endocytosis

The α-, β-, and γ-protocadherins (Pcdhα, Pcdhβ, and Pcdhγ) comprise a large family of single-pass transmembrane proteins predominantly expressed in the nervous system. These proteins contain six cadherin-like extracellular domains, and proteolysis of Pcdhα and Pcdhγ by the γ-secretase complex releases their intracellular domains into the cytoplasm where they may function locally and/or enter the nucleus and affect gene expression. Thus, cleavage of Pcdhs may function to link intercellular contacts and intracellular signaling. Here we report that shedding of the Pcdhα extracellular domain and subsequent processing by γ-secretase require endocytosis and that Pcdhs interact with the regulator of vesicular sorting ESCRT-0 in undifferentiated cells. We also find that the accumulation of Pcdh cleavage products is regulated during development. Differentiation leads to an increase in the interactions between Pcdh proteins and a decrease in the accumulation of cleavage products. We conclude that Pcdh processing requires endocytosis and that the level of cleavage products is regulated during neuronal differentiation.