Proper Sorting Research Articles

KIF16B delivers for transcytosis

Protein sorting pathways control correct delivery of membrane proteins to specific compartments of the plasma membrane and are required to maintain the physiological functions in all epithelia. Most clathrin‐dependent cargoes require the adaptor protein complexes AP‐1A and AP‐1B for proper sorting to the basolateral plasma membrane. In this issue of The EMBO Journal , Perez Bay et al (2013) shed light on the mechanism of basal‐to‐apical protein transport, or transcytosis, of the transferrin receptor in natively AP‐1B‐deficient epithelia. In AP‐1B‐deficient epithelia, the transferrin receptor transcytoses through the apical recycling endosome, and requires Rab11. Furthermore, they characterize a novel and specific role for the endosomal microtubule motor Kinesin KIF16B in transferrin receptor apical transport. These findings constitute the first characterization of a specific microtubule motor involved in basal‐to‐apical transcytosis in epithelia. Epithelial cells present a compartmentalized plasma membrane, where the composition of each compartment is tightly controlled by a precise protein and lipid sorting machinery (Folsch, 2008). The two most conspicuous compartments are the apical and basolateral domains, which generate and segregate from each other through the formation of apically localized junctional complexes. Protein sorting mechanisms ensure delivery of newly synthesized or recycled, protein components to their proper localization in either the apical or basolateral plasma membrane domains. Vectorial transport of proteins requires sorting determinants that are present in the cytoplasmic, transmembrane or …

Regulation of protein glycosylation and sorting by the Golgi matrix proteins GRASP55/65

The Golgi receives the entire output of newly synthesized cargo from the endoplasmic reticulum (ER), processes it in the stack largely through modification of bound oligosaccharides, and sorts it in the trans-Golgi network (TGN). GRASP65 and GRASP55, two proteins localized to the Golgi stack and early secretory pathway, mediate processes including Golgi stacking, Golgi ribbon linking, and unconventional secretion. Previously we have shown that GRASP depletion in cells disrupts Golgi stack formation. Here we report that knockdown of the GRASP proteins, alone or combined, accelerates protein trafficking through the Golgi membranes but also has striking negative effects on protein glycosylation and sorting. These effects are not caused by Golgi ribbon unlinking, unconventional secretion, or ER stress. We propose that GRASP55/65 are negative regulators of exocytic transport and that this slowdown helps to ensure more complete protein glycosylation in the Golgi stack and proper sorting at the TGN.

Hidden keys in stroma: Unlocking the tumor progression

Malignancy is considered as a pathological imbalance of tissue-cell societies, a state that emerges from tumor-host microenvironment in which host participates in induction, selection and expansion of the neoplastic cells. Invasion of these malignancies can be viewed as a derangement in the proper sorting of cell populations, causing a violation of normal tissue boundaries. This violation is carried out by certain stromal cells like carcinoma associated fibroblasts (CAFs), tumor associated macrophage (TAMs), endothelial cells (ECs) leucocytes, bone marrow derived cells, etc. Tumor cells may alter the surrounding stroma and in turn, stromal cells may promote cancer progression and invasion. Thus, this review compares the role of CAFs, TAMs and ECs in tumor microenvironment towards tumor progression. This compilation aims to collate existing literature on stromal cell with particular emphasis on their role in tumor invasiveness and summarize experimental studies, trials and literature of last 10 years collected from pubmed central indexed journals.

The lipid kinase PI4KIIIβ preserves lysosomal identity

Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIβ with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIβ leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIβ is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.

Economic Risks of Aflatoxin Contamination in Marketing of Peanut in Benin

Aflatoxin (AF) is a human health, nutrition, and financial risk to many people in the developing world. AF contamination in peanut is caused by the fungi:Aspergillus flavusandAspergillus parasiticus. AF is a potent carcinogenic toxin that also causes millions of dollars of financial losses to people in Africa. The fungus producing the AF can be reduced to an acceptable level by proper drying, sorting, storage, and cleaning of peanut. Government intervention and regulation can also encourage market participants to reduce AF contamination. In this paper, we examine the financial risk associated with sorting, and storing of peanut and peanut products along the marketing chain. Study results show that the prices paid for peanut, prices received, the costs of sorting and storage are dominant factors in reducing AF levels in peanut. Practices such as drying, sorting, and storing, however, pose financial risks to market traders of peanut. Unless government intervenes by requesting an AF-reduced peanut and provides assistance for market liberalization where market participants consider quality in trading decisions, suppliers of peanut will be reluctant to adopt AF-reducing techniques.

Knowing the Boundaries: Extending the Differential Adhesion Hypothesis in Embryonic Cell Sorting

Successful embryogenesis requires proper sorting and compartmentalization of different cell types. Mechanical interactions between cells help govern these processes. In the past, physics-based theories have guided in vitro studies of cell sorting and tissue surface tension. Recent experiments have challenged this approach, indicating that adhesive molecules also act as signaling molecules that initiate local reorganization of actomyosin and demonstrating that cells at the boundary of a colony of initially identical cells become "mechanically polarized." Extending physical models to account for mechanical polarization helps solve a long-standing paradox about magnitudes of tissue surface tensions and potentially explains discrepancies between recent in vivo and in vitro cell-sorting experiments. New experiments are needed to further explore the connection between mechanical polarization and tissue boundary formation in vivo.

RNA secondary structure, an important bioinformatics tool to enhance multiple sequence alignment: a case study (Sordariomycetes, Fungi)

In a case study of fungi of the class Sordariomycetes, we evaluated the effect of multiple sequence alignment (MSA) on the reliability of the phylogenetic trees, topology and confidence of major phylogenetic clades. We compared two main approaches for constructing MSA based on (1) the knowledge of the secondary (2D) structure of ribosomal RNA (rRNA) genes, and (2) automatic construction of MSA by four alignment programs characterized by different algorithms and evaluation methods, CLUSTAL, MAFFT, MUSCLE, and SAM. In the primary fungal sequences of the two functional rRNA genes, the nuclear small and large ribosomal subunits (18 S and 28 S), we identified four and six, respectively, highly variable regions, which correspond mainly to hairpin loops in the 2D structure. These loops are often positioned in expansion segments, which are missing or are not completely developed in the Archaeal and Eubacterial kingdoms. Proper sorting of these sites was a key for constructing an accurate MSA. We utilized DNA sequences from 28 S as an example for one-gene analysis. Five different MSAs were created and analyzed with maximum parsimony and maximum likelihood methods. The phylogenies inferred from the alignments improved with 2D structure with identified homologous segments, and those constructed using the MAFFT alignment program, with all highly variable regions included, provided the most reliable phylograms with higher bootstrap support for the majority of clades. We illustrate and provide examples demonstrating that re-evaluating ambiguous positions in the consensus sequences using 2D structure and covariance is a promising means in order to improve the quality and reliability of sequence alignments.

Identification of a New Rhoptry Neck Complex RON9/RON10 in the Apicomplexa Parasite Toxoplasma gondii

Apicomplexan parasites secrete and inject into the host cell the content of specialized secretory organelles called rhoptries, which take part into critical processes such as host cell invasion and modulation of the host cell immune response. The rhoptries are structurally and functionally divided into two compartments. The apical duct contains rhoptry neck (RON) proteins that are conserved in Apicomplexa and are involved in formation of the moving junction (MJ) driving parasite invasion. The posterior bulb contains rhoptry proteins (ROPs) unique to an individual genus and, once injected in the host cell act as effector proteins to co-opt host processes and modulate parasite growth and virulence. We describe here two new RON proteins of Toxoplasma gondii, RON9 and RON10, which form a high molecular mass complex. In contrast to the other RONs described to date, this complex was not detected at the MJ during invasion and therefore was not associated to the MJ complex RON2/4/5/8. Disruptions of either RON9 or RON10 gene leads to the retention of the partner in the ER followed by subsequent degradation, suggesting that the RON9/RON10 complex formation is required for proper sorting to the rhoptries. Finally, we show that the absence of RON9/RON10 has no significant impact on the morphology of rhoptry, on the invasion and growth in fibroblasts in vitro or on virulence in vivo. The conservation of RON9 and RON10 in Coccidia and Cryptosporidia suggests a specific relation with development in intestinal epithelial cells.

Growth and metabolic control of lipid signalling at the Golgi

PtdIns4P is a key regulator of the secretory pathway and plays an essential role in trafficking from the Golgi. Our recent work demonstrated that spatial control of PtdIns4P at the ER (endoplasmic reticulum) and Golgi co-ordinates secretion with cell growth. The central elements of this regulation are specific phosphoinositide 4-kinases and the phosphoinositide phosphatase Sac1. Growth-dependent translocation of Sac1 between the ER and Golgi modulates the levels of PtdIns4P and anterograde traffic at the Golgi. In yeast, this mechanism is largely dependent on the availability of glucose, but our recent results in mammalian cells suggest that Sac1 phosphatases play evolutionarily conserved roles in the growth control of secretion. Sac1 lipid phosphatase plays also an essential role in the spatial control of PtdIns4P at the Golgi complex. A restricted pool of PtdIns4P at the TGN (trans-Golgi network) is required for Golgi integrity and for proper lipid and protein sorting. In mammalian cells, the stress-activated MAPK (mitogen-activated protein kinase) p38 appears to play a critical role in transmitting nutrient signals to the phosphoinositide signalling machinery at the ER and Golgi. These results suggest that temporal and spatial integration of metabolic and lipid signalling networks at the Golgi is required for controlling the secretory pathway.

Characterization of Household Waste in Islamabad and Exploring Possible Reuse and Recycling Options

Increased solid waste generation creates additional environmental problems as many cities are unable to manage it. Unfortunately, none of the cities of Pakistan has a proper solid waste management system right from collection of waste up to its proper disposal. Islamabad, being a capital city of Pakistan with 90% literacy rate faces the similar solid waste management problems. In Islamabad around 91% of total municipal waste is composed of green and household waste which may support composting. Currently, there is no proper sorting system of municipal solid waste which indicates insufficient and inefficient recycling of organic/inorganic waste materials in Islamabad. The major portion of the recyclable household waste collected is gone wasted and buried at dumping sites in sectors H-12 and H-10. The paper is an initiative to determine reusable components of household solid waste in Islamabad and explore possible recycling/reuse options. By adopting stratified random sampling design, the primary research is carried out in sector G-7, Islamabad. Districts with in the sector have been made on the basis of income groups. 2% sample size is taken as waste generated within the income groups has high degree of similarity. The study concluded the possible reuse and recycling options for the major household waste in Islamabad.

Exome-sequencing confirms DNAJC5 mutations as cause of adult neuronal ceroid-lipofuscinosis.

We performed whole-exome sequencing in two autopsy-confirmed cases and an elderly unaffected control from a multigenerational family with autosomal dominant neuronal ceroid lipofuscinosis (ANCL). A novel single-nucleotide variation (c.344T>G) in the DNAJC5 gene was identified. Mutational screening in an independent family with autosomal dominant ANCL found an in-frame single codon deletion (c.346_348 delCTC) resulting in a deletion of p.Leu116del. These variants fulfill all genetic criteria for disease-causing mutations: they are found in unrelated families with the same disease, exhibit complete segregation between the mutation and the disease, and are absent in healthy controls. In addition, the associated amino acid substitutions are located in evolutionarily highly conserved residues and are predicted to functionally affect the encoded protein (CSPα). The mutations are located in a cysteine-string domain, which is required for membrane targeting/binding, palmitoylation, and oligomerization of CSPα. We performed a comprehensive in silico analysis of the functional and structural impact of both mutations on CSPα. We found that these mutations dramatically decrease the affinity of CSPα for the membrane. We did not identify any significant effect on palmitoylation status of CSPα. However, a reduction of CSPα membrane affinity may change its palmitoylation and affect proper intracellular sorting. We confirm that CSPα has a strong intrinsic aggregation propensity; however, it is not modified by the mutations. A complementary disease-network analysis suggests a potential interaction with other NCLs genes/pathways. This is the first replication study of the identification of DNAJC5 as the disease-causing gene for autosomal dominant ANCL. The identification of the novel gene in ANCL will allow us to gain a better understanding of the pathological mechanism of ANCLs and constitutes a great advance toward the development of new molecular diagnostic tests and may lead to the development of potential therapies.

A mutation in SCARB2 is a modifier in gaucher disease

Lysosomal integral membrane protein type 2 (LIMP-2) is responsible for proper sorting and lysosomal targeting of glucocerebrosidase, the enzyme deficient in Gaucher disease (GD). Mutations in the gene for LIMP-2, SCARB2, are implicated in inherited forms of myoclonic epilepsy, and myoclonic epilepsy is part of the phenotypic spectrum associated with GD. We investigated whether SCARB2 mutations impact the Gaucher phenotype focusing on patients with myoclonic epilepsy, including a pair of siblings with GD who were discordant for myoclonic seizures. Sequencing of SCARB2 genomic and cDNA identified a heterozygous, maternally inherited novel mutation, c.1412A>G (p.Glu471Gly), in the brother with GD and myoclonic epilepsy, absent from his sibling and controls. Glucocerebrosidase activity, Western blots, real-time PCR, and immunofluorescence studies demonstrated markedly decreased LIMP-2 and glucocerebrosidase in cells from the sibling with (p.Glu471Gly) LIMP-2, and diminished glucocerebrosidase in lysosomes. The cells secreted highly glycosylated enzyme and showed mistrafficking of glucocerebrosidase. Sequencing of SCARB2 in 13 other subjects with GD and myoclonic epilepsy and 40 controls failed to identify additional mutations. The study provides further evidence for the association of LIMP-2 and myoclonic epilepsy, explains the drastically different phenotypes encountered in the siblings, and demonstrates that LIMP-2 can serve as a modifier in GD.

Transautocrine Signaling by Membrane Neuregulins Requires Cell Surface Targeting, Which Is Controlled by Multiple Domains

The neuregulins (NRGs) play important roles in animal development and homeostasis, and their deregulation has been linked to diseases such as cancer and schizophrenia. The NRGs belong to the epidermal growth factor (EGF) family of transmembrane growth factors. Although NRGs may be synthesized as transmembrane proteins (the pro-NRGs), some of them lack an N-terminal signal sequence, raising the question of how these pro-NRGs are directed to the plasma membrane. Here we have explored the domains of pro-NRGs that are required for their membrane anchoring, cell surface exposure, and biological activity. We show that an internal hydrophobic region acts as a membrane-anchoring domain, but other regions of pro-NRG are required for proper sorting to the plasma membrane. Using mutants that are located in different subcellular compartments, we show that only plasma membrane-exposed pro-NRG is biologically active. At this location, the pro-NRGs may act as transautocrine molecules (i.e. as membrane factors able to activate receptors present in cells that are in physical contact with the pro-NRG-producing cells (in trans) or capable of activating receptors present in the pro-NRG-producing cells (in cis)).

Orexin neurons in hypothalamic slice cultures are vulnerable to endoplasmic reticulum stress

Narcolepsy results from disruption of orexin neurons in the hypothalamus that play a key role in maintenance of the arousal state. Underlying mechanisms leading to selective loss of orexin neurons remain unknown. On the other hand, endoplasmic reticulum stress, namely, conditions associated with impairment of endoplasmic reticulum functions such as proper folding and sorting of newly synthesized proteins, is implicated in pathogenesis of several types of neurodegenerative disorders. Here we found that application of endoplasmic reticulum stress inducers such as tunicamycin (that prevents protein N-glycosylation) and thapsigargin (that inhibits Ca²⁺-ATPase) to organotypic slice cultures of the hypothalamus caused preferential loss of orexin-immunoreactive neurons, as compared to melanin-concentrating hormone- or calcitonin gene-related peptide-immunoreactive neurons. The decrease in orexin-immunoreactive neurons at early time points (6-24 h) was not accompanied by induction of cell death as indicated by the absence of caspase-3 activation and no significant change in the number of NeuN-positive cells, whereas sustained treatment with tunicamycin for 72 h induced cell death. At 24-h treatment, tunicamycin and thapsigargin did not decrease expression of prepro-orexin mRNA, suggesting that post-transcriptional mechanisms were responsible for depletion of orexin peptides. In addition, inhibition of axonal transport by colchicine and inhibition of proteasomal activity by MG132 significantly prevented the decrease in orexin immunoreactivity by tunicamycin. Comparative examinations of expression of unfolded protein response-related proteins revealed that C/EBP-homologous protein (a transcription factor that promotes induction of apoptosis) as well as phosphorylated form of RNA-dependent protein kinase-like endoplasmic reticulum kinase (a protein kinase that mediates inhibition of protein translation) was expressed more prominently in orexin neurons than in melanin-concentrating hormone neurons, in response to tunicamycin. These results indicate that orexin neurons are particularly sensitive to endoplasmic reticulum stress, which may be relevant to pathogenic events in narcolepsy.

CIN85 Modulates the Down-regulation of FcγRIIa Expression and Function by c-Cbl in a PKC-dependent Manner in Human Neutrophils

We previously described a non-classical mechanism that arrests FcγRIIa signaling in human neutrophils once engaged by immune complexes or opsonized pathogens. The engagement of FcγRIIa leads to its ubiquitination by the ubiquitin ligase c-Cbl and degradation by the proteasome. Herein, we further examined some of the events regulating this novel pathway. The adaptor protein CIN85 was described in other systems to be involved in the regulation of the c-Cbl-dependent pathway. We found that CIN85 is expressed in human neutrophils and that it translocates like c-Cbl from the cytosol to the plasma membrane following receptor cross-linking. CIN85 was also recruited to the same subset of high density detergent-resistant membrane fractions in which stimulated FcγRIIa partitioned with c-Cbl. The integrity of these microdomains is essential to the FcγRIIa degradation process because the cholesterol-depleting agent methyl-β-cyclodextrin inhibits this event. Silencing the expression of CIN85 by siRNA in dibutyryl cyclic AMP-differentiated PLB 985 cells prevented FcγRIIa degradation and increased IgG-mediated phagocytosis. Confocal microscopy revealed that the presence of CIN85 is essential to the proper sorting of FcγRIIa during endocytosis. We also provide direct evidence that CIN85 is a substrate of serine/threonine kinase PKCs. Classical PKCs positively regulate FcγRIIa ubiquitination and degradation because these events were inhibited by Gö6976, a classical PKC inhibitor. We conclude that the ubiquitination and degradation of stimulated FcγRIIa mediated by c-Cbl are positively regulated by the adaptor protein CIN85 in a PKC-dependent manner and that these events contribute to the termination of FcγRIIa signaling.

Regulation of Trafficking of Activated TrkA Is Critical for NGF‐Mediated Functions

Upon activation by nerve growth factor (NGF), TrkA is internalized, trafficked and sorted through different endosomal compartments. Proper TrkA trafficking and sorting are crucial events as alteration of these processes hinders NGF-mediated functions. However, it is not fully known which proteins are involved in the trafficking and sorting of TrkA. Here we report that Nedd4-2 regulates the trafficking of TrkA and NGF functions in sensory neurons. Depletion of Nedd4-2 disrupts the correct sorting of activated TrkA at the early and late endosome stages, resulting in an accumulation of TrkA in these compartments and, as a result of the reduced trafficking to the degradative pathway, TrkA is either reverted to the cell surface through the recycling pathway or retrogradely transported to the cell body. In addition, Nedd4-2 depletion enhances TrkA signaling and the survival of NGF-dependent dorsal root ganglion neurons, but not those of brain-derived neurotrophic factor-dependent neurons. Furthermore, neurons from a knock-in mouse expressing a TrkA mutant that does not bind Nedd4-2 protein exhibit increased NGF-mediated signaling and cell survival. Our data indicate that TrkA trafficking and sorting are regulated by Nedd4-2 protein.

Selective Sorting of Cargo Proteins into Bacterial Membrane Vesicles

In contrast to the well established multiple cellular roles of membrane vesicles in eukaryotic cell biology, outer membrane vesicles (OMV) produced via blebbing of prokaryotic membranes have frequently been regarded as cell debris or microscopy artifacts. Increasingly, however, bacterial membrane vesicles are thought to play a role in microbial virulence, although it remains to be determined whether OMV result from a directed process or from passive disintegration of the outer membrane. Here we establish that the human oral pathogen Porphyromonas gingivalis has a mechanism to selectively sort proteins into OMV, resulting in the preferential packaging of virulence factors into OMV and the exclusion of abundant outer membrane proteins from the protein cargo. Furthermore, we show a critical role for lipopolysaccharide in directing this sorting mechanism. The existence of a process to package specific virulence factors into OMV may significantly alter our current understanding of host-pathogen interactions.

Identification and classification of defects on highly reflective textile machinery ring components

This paper deals with inspection and sorting of highly reflective chrome coated textile machinery rings using machine vision. To improve inspection speed and to ensure 100& quality inspection, it was absolutely essential to improve the complete inspection process and it was also required as well to classify defective and non-defective components by a proper sorting algorithm. In the present study, algorithm based on auto-median and single step thresholding approaches were used for defect detection and then their performances were compared with reference to efficiency of defect classification and speed. Bright field and dark field illumination have been used in the imaging setup. Once the defect detection and image segmentation is done the image details/data will be supplied to classification software. The description, analysis and the results of these techniques used in defect detection are reported and discussed in this paper.

Identification of defects on highly reflective coated ring components using dark field illumination and image segmentation using simple thresholding technique

This paper deals with inspection of highly reflective chrome coated rings used in textile machinery using machine vision. These rings are mass produced in very large numbers in a textile machinery manufacturing company and the inspection was carried out manually using optical methods. Introduction of automated vision inspection had not helped the industry fully to achieve 100% quality inspection. Moreover, the whole procedure had to be made quicker in view of the number of components manufactured which is of the order of hundreds of thousands and to be inspected 100% thereafter. Also in order to improve inspection speed and to ensure 100% quality inspection, it was absolutely essential to improve the complete inspection process. Also a proper sorting algorithm was to be considered to classify defective and non-defective components. The effect of curvature, reflective nature of material and the real-time inspection make the imaging and defect detection and classification difficult. The use of dark field illumination has been tried out in this work to quicken the process of defect detection and also to improve the accuracy of detection. The images of the defects captured using a CCD camera of the inner and outer surfaces of the coated ring components have been analysed. The images were subjected to a simple thresholding technique to segment out defects, thus avoiding the use of complex image processing algorithms. It was observed that the classification of defects using dark field illumination was very effective for this particular case and the results as well as analysis of the same are presented in this paper.

Ubiquitination of α5β1 Integrin Controls Fibroblast Migration through Lysosomal Degradation of Fibronectin-Integrin Complexes

Cell migration requires endocytosis and recycling of integrins, but it is not known whether degradation of these membrane proteins is involved. Here we demonstrate that in migrating cells, a fraction of the endocytosed fibronectin receptor, alpha 5 beta 1 integrin, is sorted into multivesicular endosomes together with fibronectin and degraded in lysosomes. This sorting requires fibronectin-induced ubiquitination of the alpha 5 subunit, and the activity of the endosomal sorting complex required for transport (ESCRT) machinery, which interacts with alpha 5 beta 1 integrin. Importantly, we demonstrate that both alpha 5 ubiquitination and ESCRT functions are required for proper migration of fibroblasts. We propose that ligand-mediated degradation of alpha 5 beta 1 integrin via the ESCRT pathway is required in order to prevent endosomal accumulation of ligand-bound integrins that might otherwise form nonproductive adhesion sites. Fibronectin and alpha 5 beta 1 integrin therefore are trafficked to lysosomes in a similar way to growth factors and their receptors.