Eisosomes promote the ability of Sur7 to regulate plasma membrane organization in Candida albicans.

The plasma membrane of the fungal pathogen Candida albicans forms a protective barrier that also mediates many processes needed for virulence, including cell wall synthesis, invasive hyphal morphogenesis, and nutrient uptake. Because compartmentalization of the plasma membrane is believed to coordinate these diverse activities, we examined plasma membrane microdomains termed eisosomes or membrane compartment of Can1 (MCC), which correspond to ∼200-nm-long furrows in the plasma membrane. A pil1∆ lsp1∆ mutant failed to form eisosomes and displayed strong defects in plasma membrane organization and morphogenesis, including extensive cell wall invaginations. Mutation of eisosome proteins Slm2, Pkh2, and Pkh3 did not cause similar cell wall defects, although pkh2∆ cells formed chains of furrows and pkh3∆ cells formed wider furrows, identifying novel roles for the Pkh protein kinases in regulating furrows. In contrast, the sur7∆ mutant formed cell wall invaginations similar to those for the pil1∆ lsp1∆ mutant even though it could form eisosomes and furrows. A PH-domain probe revealed that the regulatory lipid phosphatidylinositol 4,5-bisphosphate was enriched at sites of cell wall invaginations in both the sur7∆ and pil1∆ lsp1∆ cells, indicating that this contributes to the defects. The sur7∆ and pil1∆ lsp1∆ mutants displayed differential susceptibility to various types of stress, indicating that they affect overlapping but distinct functions. In support of this, many mutant phenotypes of the pil1∆ lsp1∆ cells were rescued by overexpressing SUR7 These results demonstrate that C. albicans eisosomes promote the ability of Sur7 to regulate plasma membrane organization.

Nitroxide-loaded hexosomes provide MRI contrast in vivo.

The purpose of this work was to synthesize and screen, for their effectiveness to act as T1-enhancing magnetic resonance imaging (MRI) contrast agents, a small library of nitroxide lipids incorporated into cubic-phase lipid nanoparticles (cubosomes). The most effective nitroxide lipid was then formulated into lower-toxicity lipid nanoparticles (hexosomes), and effective MR contrast was observed in the aorta and spleen of live rats in vivo. This new class of lower-toxicity lipid nanoparticles allowed for higher relaxivities on the order of those of clinically used gadolinium complexes. The new hexosome formulation presented herein was significantly lower in toxicity and higher in relaxivity than cubosome formulations previously reported by us.

The MARVEL domain protein Nce102 regulates actin organization and invasive growth of Candida albicans.

UNLABELLED: Invasive growth of the fungal pathogen Candida albicans into tissues promotes disseminated infections in humans. The plasma membrane is essential for pathogenesis because this important barrier mediates morphogenesis and invasive growth, as well as secretion of virulence factors, cell wall synthesis, nutrient import, and other processes. Previous studies showed that the Sur7 tetraspan protein that localizes to MCC (membrane compartment occupied by Can1)/eisosome subdomains of the plasma membrane regulates a broad range of key functions, including cell wall synthesis, morphogenesis, and resistance to copper. Therefore, a distinct tetraspan protein found in MCC/eisosomes, Nce102, was investigated. Nce102 belongs to the MARVEL domain protein family, which is implicated in regulating membrane structure and function. Deletion of NCE102 did not cause the broad defects seen in sur7Δ cells. Instead, the nce102Δ mutant displayed a unique phenotype in that it was defective in forming hyphae and invading low concentrations of agar but could invade well in higher agar concentrations. This phenotype was likely due to a defect in actin organization that was observed by phalloidin staining. In support of this, the invasive growth defect of a bni1Δ mutant that mislocalizes actin due to lack of the Bni1 formin was also reversed at high agar concentrations. This suggests that a denser matrix provides a signal that compensates for the actin defects. The nce102Δ mutant displayed decreased virulence and formed abnormal hyphae in mice. These studies identify novel ways that Nce102 and the physical environment surrounding C. albicans regulate morphogenesis and pathogenesis. IMPORTANCE: The plasma membrane promotes virulence of the human fungal pathogen Candida albicans by acting as a protective barrier around the cell and mediating dynamic activities, such as morphogenesis, cell wall synthesis, secretion of virulence factors, and nutrient uptake. To better understand how the plasma membrane contributes to virulence, we analyzed a set of eight genes encoding MARVEL family proteins that are predicted to function in membrane organization. Interestingly, deletion of one gene, NCE102, caused a strong defect in formation of invasive hyphal growth in vitro and decreased virulence in mice. The nce102Δ mutant cells showed defects in actin organization that underlie the morphogenesis defect, since mutation of a known regulator of actin organization caused a similar defect. These studies identify a novel way in which the plasma membrane regulates the actin cytoskeleton and contributes to pathogenesis.

Metal-free and MRI visible theranostic lyotropic liquid crystal nitroxide-based nanoparticles.

The development of improved, low toxicity, clinically viable nanomaterials that provide MRI contrast have tremendous potential to form the basis of translatable theranostic agents. Herein we describe a class of MRI visible materials based on lyotropic liquid crystal nanoparticles loaded with a paramagnetic nitroxide lipid. These readily synthesized nanoparticles achieved enhanced proton-relaxivities on the order of clinically used gadolinium complexes such as Omniscan™ without the use of heavy metal coordination complexes. Their low toxicity, high water solubility and colloidal stability in buffer resulted in them being well tolerated in vitro and in vivo. The nanoparticles were initially screened in vitro for cytotoxicity and subsequently a defined concentration range was tested in rats to determine the maximum tolerated dose. Pharmacokinetic profiles of the candidate nanoparticles were established in vivo on IV administration to rats. The lyotropic liquid crystal nanoparticles were proven to be effective liver MRI contrast agents. We have demonstrated the effective in vivo performance of a T1 enhancing, biocompatible, colloidally stable, amphiphilic MRI contrast agent that does not contain a metal.

Sur7 promotes plasma membrane organization and is needed for resistance to stressful conditions and to the invasive growth and virulence of Candida albicans.

The human fungal pathogen Candida albicans causes lethal systemic infections because of its ability to grow and disseminate in a host. The C. albicans plasma membrane is essential for virulence by acting as a protective barrier and through its key roles in interfacing with the environment, secretion of virulence factors, morphogenesis, and cell wall synthesis. Difficulties in studying hydrophobic membranes have limited the understanding of how plasma membrane organization contributes to its function and to the actions of antifungal drugs. Therefore, the role of the recently discovered plasma membrane subdomains termed the membrane compartment containing Can1 (MCC) was analyzed by assessing the virulence of a sur7Δ mutant. Sur7 is an integral membrane protein component of the MCC that is needed for proper localization of actin, morphogenesis, cell wall synthesis, and responding to cell wall stress. MCC domains are stable 300-nm-sized punctate patches that associate with a complex of cytoplasmic proteins known as an eisosome. Analysis of virulence-related properties of a sur7Δ mutant revealed defects in intraphagosomal growth in macrophages that correlate with increased sensitivity to oxidation and copper. The sur7Δ mutant was also strongly defective in pathogenesis in a mouse model of systemic candidiasis. The mutant cells showed a decreased ability to initiate an infection and greatly diminished invasive growth into kidney tissues. These studies on Sur7 demonstrate that the plasma membrane MCC domains are critical for virulence and represent an important new target for the development of novel therapeutic strategies. IMPORTANCECandida albicans, the most common human fungal pathogen, causes lethal systemic infections by growing and disseminating in a host. The plasma membrane plays key roles in enabling C. albicans to grow in vivo, and it is also the target of the most commonly used antifungal drugs. However, plasma membrane organization is poorly understood because of the experimental difficulties in studying hydrophobic components. Interestingly, recent studies have identified a novel type of plasma membrane subdomain in fungi known as the membrane compartment containing Can1 (MCC). Cells lacking the MCC-localized protein Sur7 display broad defects in cellular organization and response to stress in vitro. Consistent with this, C. albicans cells lacking the SUR7 gene were more susceptible to attack by macrophages than cells with the gene and showed greatly reduced virulence in a mouse model of systemic infection. Thus, Sur7 and other MCC components represent novel targets for antifungal therapy.

The Candida albicans Sur7 protein is needed for proper synthesis of the fibrillar component of the cell wall that confers strength.

The Candida albicans plasma membrane plays important roles in interfacing with the environment, morphogenesis, and cell wall synthesis. The role of the Sur7 protein in cell wall structure and function was analyzed, since previous studies showed that this plasma membrane protein is needed to prevent abnormal intracellular growth of the cell wall. Sur7 localizes to stable patches in the plasma membrane, known as MCC (membrane compartment occupied by Can1), that are associated with eisosome proteins. The sur7Δ mutant cells displayed increased sensitivity to factors that exacerbate cell wall defects, such as detergent (SDS) and the chitin-binding agents calcofluor white and Congo red. The sur7Δ cells were also slightly more sensitive to inhibitors that block the synthesis of cell wall chitin (nikkomycin Z) and ß-1,3-glucan (caspofungin). In contrast, Fmp45, a paralog of Sur7 that also localizes to punctate plasma membrane patches, did not have a detectable role in cell wall synthesis. Chemical analysis of cell wall composition demonstrated that sur7Δ cells contain decreased levels of ß-glucan, a glucose polymer that confers rigidity on the cell wall. Consistent with this, sur7Δ cells were more sensitive to lysis, which could be partially rescued by increasing the osmolarity of the medium. Interestingly, Sur7 is present in static patches, whereas ß-1,3-glucan synthase is mobile in the plasma membrane and is often associated with actin patches. Thus, Sur7 may influence ß-glucan synthesis indirectly, perhaps by altering the functions of the cell signaling components that localize to the MCC and eisosome domains.

Membrane Compartment Occupied by Can1 (MCC) and Eisosome Subdomains of the Fungal Plasma Membrane.

Studies on the budding yeast Saccharomyces cerevisiae have revealed that fungal plasma membranes are organized into different subdomains. One new domain termed MCC/eisosomes consists of stable punctate patches that are distinct from lipid rafts. The MCC/eisosome domains correspond to furrows in the plasma membrane that are about 300 nm long and 50 nm deep. The MCC portion includes integral membrane proteins, such as the tetraspanners Sur7 and Nce102. The adjacent eisosome includes proteins that are peripherally associated with the membrane, including the BAR domains proteins Pil1 and Lsp1 that are thought to promote membrane curvature. Genetic analysis of the MCC/eisosome components indicates these domains broadly affect overall plasma membrane organization. The mechanisms regulating the formation of MCC/eisosomes in model organisms will be reviewed as well as the role of these plasma membrane domains in fungal pathogenesis and response to antifungal drugs.

Identification of GIG1, a GlcNAc-induced gene in Candida albicans needed for normal sensitivity to the chitin synthase inhibitor nikkomycin Z.

The amino sugar N-acetylglucosamine (GlcNAc) is known to be an important structural component of cells from bacteria to humans, but its roles in cell signaling are less well understood. GlcNAc induces two pathways in the human fungal pathogen Candida albicans. One activates cyclic AMP (cAMP) signaling, which stimulates the formation of hyphal cells and the expression of virulence genes, and the other pathway induces genes needed to catabolize GlcNAc. Microarray analysis of gene expression was carried out under four different conditions in order to characterize the transcriptional changes induced by GlcNAc. The most highly induced genes include those that encode a GlcNAc transporter (NGT1) and the GlcNAc catabolic enzymes (HXK1, DAC1, and NAG1). GlcNAc also activated most of the genes whose expression is increased when cells are triggered with other stimuli to form hyphae. Surprisingly, GlcNAc also induced a subset of genes that are regulated by galactose (GAL1, GAL7, and GAL10), which may be due to cross talk between signaling pathways. A novel GlcNAc-induced gene, GIG1, which is not essential for GlcNAc catabolism or the induction of hyphae, was identified. However, a Gig1-green fluorescent protein (GFP) fusion protein was specifically induced by GlcNAc, and not by other sugars. Gig1-GFP localized to the cytoplasm, where GlcNAc metabolism occurs. Significantly, a gig1Δ mutant displayed increased resistance to nikkomycin Z, which inhibits chitin synthase from converting UDP-GlcNAc into cell wall chitin. Gig1 is highly conserved in fungi, especially those that contain GlcNAc catabolic genes. These results implicate Gig1 in GlcNAc metabolism.

Deletion of the Parkin co-regulated gene causes defects in ependymal ciliary motility and hydrocephalus in the quakingviable mutant mouse.

The quakingviable mouse (qkv) is a spontaneous recessive mouse mutant with a deletion of approximately 1.1 Mb in the proximal region of chromosome 17. The deletion affects the expression of three genes; quaking (Qk), Parkin-coregulated gene (Pacrg) and parkin (Park2). The resulting phenotype, which includes dysmyelination of the central nervous system and male sterility, is due to reduced expression of Qk and a complete lack of Pacrg expression, respectively. Pacrg is required for correct development of the spermatozoan flagella, a specialized type of motile cilia. In vertebrates, motile cilia are required for multiple functions related to cellular movement or movement of media over a stationary cell surface. To investigate the potential role of PACRG in motile cilia we analysed qkv mutant mice for evidence of cilial dysfunction. Histological and magnetic resonance imaging analyses demonstrated that qkv mutant mice were affected by acquired, communicating hydrocephalus (HC). Structural analysis of ependymal cilia demonstrated that the 9 + 2 arrangement of axonemal microtubules was intact and that both the density of ciliated cells and cilia length was similar to wild-type littermates. Cilia function studies showed a reduction in ependymal cilial beat frequency and cilial mediated flow in qkv mutant mice compared with wild-type littermate controls. Moreover, transgenic expression of Pacrg was necessary and sufficient to correct this deficit and rescue the HC phenotype in the qkv mutant. This study provides novel in vivo evidence that Pacrg is required for motile cilia function and may be involved in the pathogenesis of human ciliopathies, such as HC, asthenospermia and primary ciliary dyskinesia.

Retrograde axonal tracing using manganese enhanced magnetic resonance imaging.

Manganese-enhanced magnetic resonance imaging (MEMRI) was used to investigate retrograde axonal tracing in the rat sciatic nerve model to assess its potential to examine peripheral nerve injury. The right sciatic nerve was exposed and crushed. After each recovery period, the distal part of the right sciatic nerve was injected with manganese (400 mM, 15 microl). After allowing 3 days for manganese transport the animals were subsequently scanned to visualize the sciatic nerve and its corresponding spinal cord and dorsal root ganglia with T1-weighted MRI. Thirty-four animals were randomly divided into 4 experimental groups according to their recovery period post-crush injury: 3 days (n=6), 2 weeks (n=6), 4 weeks (n=6) and 12 weeks (n=6); and two control groups: a non-crushed group (n=6) and a nerve cut group (n=4). In the no-injury group, the right sciatic nerve tract including its corresponding spinal cord and dorsal root ganglia showed significant T1 signal enhancement. In the animals with crush injury, the MR signal intensity was significantly reduced proximal to the injured site but gradually reappeared with increasing recovery period. The signal intensity of the sciatic tract was compared to the results of behavioral functional testing, retrograde axonal tracing with neural tracer fluorogold and histomorphometric analysis of the distal nerve. Significant correlations were observed between the MR signal intensity and the behavioral functional test (r=0.50, p<0.05), and the retrograde axonal tracing (r=0.88; p<0.05). Retrograde neuronal tract tracing with MEMRI can be used for the assessment of peripheral nerve damage and regeneration.

Identification of amino acids at two dimer interface regions of the alpha-factor receptor (Ste2).

The yeast alpha-factor pheromone receptor (Ste2) belongs to the large superfamily of G protein-coupled receptors (GPCRs) that characteristically contain seven transmembrane domains (TMs). A wide range of GPCRs are thought to exist as dimers or oligomers. To identify the interface regions that mediate oligomerization of Ste2, a set of 73 different mutants with Cys residues substituted near the extracellular ends of the transmembrane domains were screened for the ability to form intermolecular disulfide bonds. Disulfide bonds formed between Cys residues at six positions in Ste2. Cys substituted for Val-45 formed disulfide bonds, indicating contact between residues at the extracellular end of TM1. Disulfide bonds also formed with Cys residues substituted for five different residues clustered near the extracellular end of TM4 (Val-183, Val-186, Lys-187, Met-189, and Ile-190). Binding of the alpha-factor ligand to Ste2 did not change the sites at which cross-linking occurred in these TMs, but it did increase the efficiency of dimer formation for the Ste2-V183C mutant. Interestingly, oligomers of the class A family of vertebrate GPCRs are also thought to form homomeric contacts at TM1 and TM4. These results support the conclusion that GPCRs form oligomers and not just dimers, since TM1 and TM4 are too far apart in the class A GPCRs to form contacts in the same dimer moiety. Similar dimer interface sites in Ste2 and class A receptors provide further evidence that many aspects of structure and function are highly conserved across the divergent GPCR superfamily.

Head growth in preterm infants: correlation with magnetic resonance imaging and neurodevelopmental outcome.

OBJECTIVE: Extremely preterm birth is associated with adverse neurodevelopmental sequelae. Head circumference has been used as a measure of brain growth. There are limited data relating head circumference to MRI. The purpose of this work was to establish the relationship between head circumference with brain MRI at term-equivalent age and to relate head circumference with neurodevelopmental outcome at 2 years. PATIENTS AND METHODS: Two hundred and twenty-seven preterm infants (birth weight of <1250 g or <30 weeks' gestation) were recruited. Head circumference was measured at birth, term, and 2 years' corrected age, and z scores were computed. Microcephaly was defined as a head circumference z score of less than -2 SDs for age and gender. MRI scans at term (n = 214) were graded for white and gray matter abnormalities, and segmented volumes were calculated for different tissue types. Outcome at 2 years' corrected age (n = 202) included scores on the Bayley Scales of Infant Development II. RESULTS: Microcephaly increased from 7.5% at term to 29.7% at 2 years. There was no significant relationship between head circumference and white or gray matter abnormalities on MRI. There was a strong correlation between head circumference and brain volume at term. At term, microcephalic infants had significantly decreased volumes for total brain tissue and most segmented volumes compared with infants with normal head circumference, but only deep nuclear gray matter volume remained significantly lower when adjusted for total intracranial volume. At 2 years, microcephaly was associated with poorer cognitive and motor development and an increased rate of cerebral palsy. CONCLUSIONS: Brain volume is a determinant of head size at term. Microcephaly is associated with a reduction of brain tissue volumes, especially deep nuclear gray matter, which suggests a selective vulnerability. Poor postnatal head growth in preterm infants becomes more evident by 2 years and is strongly associated with poor neurodevelopmental outcome and cerebral palsy.

Perinatal risk factors altering regional brain structure in the preterm infant.

Neuroanatomical structure appears to be altered in preterm infants, but there has been little insight into the major perinatal risk factors associated with regional cerebral structural alterations. MR images were taken to quantitatively compare regional brain tissue volumes between term and preterm infants and to investigate associations between perinatal risk factors and regional neuroanatomical alterations in a large cohort of preterm infants. In a large prospective longitudinal cohort study of 202 preterm and 36 term infants, MR scans at term equivalent were undertaken for volumetric estimates of cortical and deep nuclear grey matter, unmyelinated and myelinated white matter (WM) and CSF within 8 parcellated regions for each hemisphere of the brain. Perinatal correlates analysed in relation to regional brain structure included gender, gestational age, intrauterine growth restriction, bronchopulmonary dysplasia, white matter injury (WMI) and intraventricular haemorrhage. Results revealed region-specific reductions in brain volumes in preterm infants compared with term controls in the parieto-occipital (preterm mean difference: -8.1%; 95% CI = -13.8--2.3%), sensorimotor (-11.6%; -18.2--5.0%), orbitofrontal (-30.6%; -49.8--11.3%) and premotor (-7.6%; -14.2--0.9%) regions. Within the sensorimotor and orbitofrontal regions cortical grey matter and unmyelinated WM were most clearly reduced in preterm infants, whereas deep nuclear grey matter was reduced mainly within the parieto-occipital and subgenual regions. CSF (ventricular and extracerebral) was doubled in volume within the superior regions in preterm infants compared with term controls. Cerebral WMI and intrauterine growth restriction were both associated with a more posterior reduction in brain volumes, whereas bronchopulmonary dysplasia was associated with a more global reduction across all regions. In contrast degree of immaturity was not related to regional brain structure among preterm infants. In summary, preterm birth is associated with regional cerebral tissue reductions, with the adverse pattern varying between risk factors. These findings add to our understanding of the potential pathways leading to altered brain structure and outcome in the preterm infant.