Highly potent intracellular membrane-associated Aβ seeds

An early event in Alzheimer's disease (AD) pathogenesis is the formation of extracellular aggregates of amyloid-β peptide (Aβ), thought to be initiated by a prion-like seeding mechanism. However, the molecular nature and location of the Aβ seeds remain rather elusive. Active Aβ seeds are found in crude homogenates of amyloid-laden brains and in the soluble fraction thereof. To analyze the seeding activity of the pellet fraction, we have either separated or directly immunoisolated membranes from such homogenates. Here, we found considerable Aβ seeding activity associated with membranes in the absence of detectable amyloid fibrils. We also found that Aβ seeds on mitochondrial or associated membranes efficiently induced Aβ aggregation in vitro and seed β-amyloidosis in vivo. Aβ seeds at intracellular membranes may contribute to the spreading of Aβ aggregation along neuronal pathways and to the induction of intracellular pathologies downstream of Aβ

Autophagic tubes: vacuolar invaginations involved in lateral membrane sorting and inverse vesicle budding

Many intracellular compartments of eukaryotic cells do not adopt a spherical shape, which would be expected in the absence of mechanisms organizing their structure. However, little is known about the principles determining the shape of organelles. We have observed very defined structural changes of vacuoles, the lysosome equivalents of yeast. The vacuolar membrane can form a large tubular invagination from which vesicles bud off into the lumen of the organelle. Formation of the tube is regulated via the Apg/Aut pathway. Its lumen is continuous with the cytosol, making this inverse budding reaction equivalent to microautophagocytosis. The tube is highly dynamic, often branched, and defined by a sharp kink of the vacuolar membrane at the site of invagination. The tube is formed by vacuoles in an autonomous fashion. It persists after vacuole isolation and, therefore, is independent of surrounding cytoskeleton. There is a striking lateral heterogeneity along the tube, with a high density of transmembrane particles at the base and a smooth zone devoid of transmembrane particles at the tip where budding occurs. We postulate a lateral sorting mechanism along the tube that mediates a depletion of large transmembrane proteins at the tip and results in the inverse budding of lipid-rich vesicles into the lumen of the organelle

The bet Gene of Feline Foamy Virus Is Required for Virus Replication

AbstractFoamy viruses (FV) are complex retroviruses with additional bel genes located between env and the 3′ long-terminal repeat. The functions of the bel 2 and bet genes are unknown and both are dispensable for replication of the prototypic human foamy virus in cell cultures. We examined the function(s) of bel 2 and bet of the distantly related feline foamy virus (FFV) in the proviral context. Mutagenesis was used to alter the Bel 2 and Bet or to abrogate their expression. The Bel 2/Bet mutants showed a 1000-fold reduced viral titer in feline kidney cells; in human 293T cells, viral titer was only about 10-fold reduced compared to wild-type FFV. In both cell types, the Bel 2/Bet mutations resulted in a reduced release of FFV particles. The results indicate that FFV Bet is required for efficient virus replication. The functions of the Bel 2 and Bet proteins are discussed

Drosophila Kette coordinates myoblast junction dissolution and the ratio of Scar-to-WASp during myoblast fusion

The fusion of founder cells and fusion-competent myoblasts (FCMs) is crucial for muscle formation in Drosophila Characteristic events of myoblast fusion include the recognition and adhesion of myoblasts, and the formation of branched F-actin by the Arp2/3 complex at the site of cell-cell contact. At the ultrastructural level, these events are reflected by the appearance of finger-like protrusions and electron-dense plaques that appear prior to fusion. Severe defects in myoblast fusion are caused by the loss of Kette (a homolog of Nap1 and Hem-2, also known as NCKAP1 and NCKAP1L, respectively), a member of the regulatory complex formed by Scar or WAVE proteins (represented by the single protein, Scar, in flies). kette mutants form finger-like protrusions, but the electron-dense plaques are extended. Here, we show that the electron-dense plaques in wild-type and kette mutant myoblasts resemble other electron-dense structures that are known to function as cellular junctions. Furthermore, analysis of double mutants and attempts to rescue the kette mutant phenotype with N-cadherin, wasp and genes of members of the regulatory Scar complex revealed that Kette has two functions during myoblast fusion. First, Kette controls the dissolution of electron-dense plaques. Second, Kette controls the ratio of the Arp2/3 activators Scar and WASp in FCMs

Excretion of cytoplasmic proteins (ECP) in Staphylococcus aureus

Excretion of cytoplasmic proteins (ECP) is a common physiological feature in bacteria and eukaryotes. However, how these proteins without a typical signal peptide are excreted in bacteria is poorly understood. We studied the excretion pattern of cytoplasmic proteins using two glycolytic model enzymes, aldolase and enolase, and show that their excretion takes place mainly during the exponential growth phase in Staphylococcus aureus very similar to that of Sbi, an IgG-binding protein, which is secreted via the Sec-pathway. The amount of excreted enolase is substantial and is comparable with that of Sbi. For localization of the exit site, we fused aldolase and enolase with the peptidoglycan-binding motif, LysM, to trap the enzymes at the cell wall. With both immune fluorescence labeling and immunogold localization on electron microscopic thin sections aldolase and enolase were found apart from the cytoplasmic area particularly in the cross wall and at the septal cleft of dividing cells, whereas the non-excreted Ndh2, a soluble NADH:quinone oxidoreductase, is only seen attached to the inner side of the cytoplasmic membrane. The selectivity, the timing and the localization suggest that ECP is not a result of unspecific cell lysis but is mediated by an as yet unknown mechanism

Adenylate cyclase 5 is required for melanophore and male pattern development in the guppy (Poecilia reticulata)

Guppies (Poecilia reticulata) are colorful fish that have attracted the attention of pigmentation researchers for almost a century. Here, we report that the blond phenotype of the guppy is caused by a spontaneous mutation in the guppy ortholog of adenylate cyclase 5 (adcy5). Using double digest restriction site-associated DNA sequencing (ddRADseq) and quantitative trait locus (QTL) mapping, we linked the blond phenotype to a candidate region of 118 kb, in which we subsequently identified a 2-bp deletion in adcy5 that alters splicing and leads to a premature stop codon. We show that adcy5, which affects lifespan and melanoma growth in mouse, is required for melanophore development and formation of male orange pigmentation traits in the guppy. We find that some components of the male orange pattern are particularly sensitive to loss of Adcy5 function. Our work thus reveals a function for Adcy5 in patterning of fish color ornaments

Nup153 Recruits the Nup107-160 Complex to the Inner Nuclear Membrane for Interphasic Nuclear Pore Complex Assembly

In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase

The LRR receptor Islr2 is required for retinal axon routing at the vertebrate optic chiasm

Background: In the visual system of most binocular vertebrates, the axons of retinal ganglion cells (RGCs) diverge at the diencephalic midline and extend to targets on both ipsi- and contralateral sides of the brain. While a molecular mechanism explaining ipsilateral guidance decisions has been characterized, less is known of how RGC axons cross the midline. Results: Here, we took advantage of the zebrafish, in which all RGC axons project contralaterally at the optic chiasm, to characterize Islr2 as an RGC receptor required for complete retinal axon midline crossing. We used a systematic extracellular protein-protein interaction screening assay to identify two Vasorin paralogs, Vasna and Vasnb, as specific Islr2 ligands. Antibodies against Vasna and Vasnb reveal cellular populations surrounding the retinal axon pathway, suggesting the involvement of these proteins in guidance decisions made by axons of the optic nerve. Specifically, Vasnb marks the membranes of a cellular barricade located anteriorly to the optic chiasm, a structure termed the "glial knot" in higher vertebrates. Loss of function mutations in either vasorin paralog, individually or combined, however, do not exhibit an overt retinal axon projection phenotype, suggesting that additional midline factors, acting either independently or redundantly, compensate for their loss. Analysis of Islr2 knockout mice supports a scenario in which Islr2 controls the coherence of RGC axons through the ventral midline and optic tract. Conclusions: Although stereotypic guidance of RGC axons at the vertebrate optic chiasm is controlled by multiple, redundant mechanisms, and despite the differences in ventral diencephalic tissue architecture, we identify a novel role for the LRR receptor Islr2 in ensuring proper axon navigation at the optic chiasm of both zebrafish and mouse

Reprogramming of myeloid angiogenic cells by Bartonella henselae leads to microenvironmental regulation of pathological angiogenesis

The contribution of myeloid cells to tumour microenvironments is a decisive factor in cancer progression. Tumour-associated macrophages (TAMs) mediate tumour invasion and angiogenesis through matrix remodelling, immune modulation and release of pro-angiogenic cytokines. Nothing is known about how pathogenic bacteria affect myeloid cells in these processes. Here we show that Bartonella henselae, a bacterial pathogen causing vasculoproliferative diseases (bacillary angiomatosis), reprogrammes human myeloid angiogenic cells (MACs), a pro-angiogenic subset of circulating progenitor cells, towards a TAM-like phenotype with increased pro-angiogenic capacity. B. henselae infection resulted in inhibition of cell death, activation of angiogenic cellular programmes and induction of M2 macrophage polarization. MACs infected with B. henselae incorporated into endothelial sprouts and increased angiogenic growth. Infected MACs developed a vascular mimicry phenotype in vitro, and expression of B. henselae adhesin A was essential in inducing these angiogenic effects. Secretome analysis revealed that increased pro-angiogenic activities were associated with the creation of a tumour-like microenvironment dominated by angiogenic inflammatory cytokines and matrix remodelling compounds. Our results demonstrate that manipulation of myeloid cells by pathogenic bacteria can contribute to microenvironmental regulation of pathological tissue growth and suggest parallels underlying both bacterial infections and cancer

Functional Conservation of the Pathological Effects of Human Mutations In. KIF5A in a Drosophila Model of Autosomal-Dominant

Impairments in intracellular transport are the hallmark of many neurological diseases including hereditary spastic paraplegia (HSP). HSP is a genetically heterogeneous neurodegenerative disorder causing spastic weakness of the lower extremities. On the cellular level the disease is characterized by distal axonopathy that affects the longest axons in the corticospinal tract. At present at least 45 HSP loci have been described. Mutations in atlastin and spastin (accounting for around 50% of all HSP cases) as well as mutations in 6 other identified SPG genes: (kif5a, nipa, spatacsin, spastizin, spartin and maspardin) have been implicated in disturbance of axonal transport and membrane trafficking. The fact that spinal neurons with the longest axons are selectively affected might be due to their morphology. They have substantial dependence on efficient axonal transport of organelles, molecules and signals to and from nerve terminals. Disturbances in antero- grade and retrograde transport might interfere with efficient synaptic function/maintenance and maintenance of the axon itself. Perturbations of axonal transport emerge as a common patho- logical mechanism in many HSP cases. To test this hypothesis and to further probe the pathological mechanisms we generated a Drosophila model for SPG10, an autosomal dominant form of HSP. SPG10 is caused by mutations in KIF5A gene, which codes for the heavy chain of the neuronal microtubule motor Kinesin-1. The Kinesin-1 family represents the major anterograde motor complex