The Kinesin-3, Unc-104 Regulates Dendrite Morphogenesis and Synaptic Development in Drosophila

Kinesin-based transport is important for synaptogenesis, neuroplasticity, and maintaining synaptic function. In an anatomical screen of neurodevelopmental mutants, we identified the exchange of a conserved residue (R561H) in the forkhead-associated domain of the kinesin-3 family member Unc-104/KIF1A as the genetic cause for defects in synaptic terminal- and dendrite morphogenesis. Previous structure-based analysis suggested that the corresponding residue in KIF1A might be involved in stabilizing the activated state of kinesin-3 dimers. Herein we provide the first in vivo evidence for the functional importance of R561. The R561H allele (unc-104^bris) is not embryonic lethal, which allowed us to investigate consequences of disturbed Unc-104 function on postembryonic synapse development and larval behavior. We demonstrate that Unc-104 regulates the reliable apposition of active zones and postsynaptic densities, possibly by controlling site-specific delivery of its cargo. Next, we identified a role for Unc-104 in restraining neuromuscular junction growth and coordinating dendrite branch morphogenesis, suggesting that Unc-104 is also involved in dendritic transport. Mutations in KIF1A/unc-104 have been associated with hereditary spastic paraplegia and hereditary sensory and autonomic neuropathy type 2. However, we did not observe synapse retraction or dystonic posterior paralysis. Overall, our study demonstrates the specificity of defects caused by selective impairments of distinct molecular motors and highlights the critical importance of Unc-104 for the maturation of neuronal structures during embryonic development, larval synaptic terminal outgrowth, and dendrite morphogenesis.     

ER remodeling by the large GTPase atlastin promotes vacuolar growth of Legionella pneumophila

The pathogenic bacterium Legionella pneumophila replicates in host cells within a distinct ER‐associated compartment termed the Legionella‐containing vacuole (LCV). How the dynamic ER network contributes to pathogen proliferation within the nascent LCV remains elusive. A proteomic analysis of purified LCVs identified the ER tubule‐resident large GTPase atlastin3 (Atl3, yeast Sey1p) and the reticulon protein Rtn4 as conserved LCV host components. Here, we report that Sey1/Atl3 and Rtn4 localize to early LCVs and are critical for pathogen vacuole formation. Sey1 overproduction promotes intracellular growth of L. pneumophila, whereas a catalytically inactive, dominant‐negative GTPase mutant protein, or Atl3 depletion, restricts pathogen replication and impairs LCV maturation. Sey1 is not required for initial recruitment of ER to PtdIns(4)P‐positive LCVs but for subsequent pathogen vacuole expansion. GTP (but not GDP) catalyzes the Sey1‐dependent aggregation of purified, ER‐positive LCVs in vitro. Thus, Sey1/Atl3‐dependent ER remodeling contributes to LCV maturation and intracellular replication of L. pneumophila.     

Mutations in neuromusculin,a gene encoding a cell adhesion molecule,cause nervous system defects

The Drosophila neuromusculin (nrm) gene encodes an immunoglobulin-like (Ig-like) cell adhesion molecule expressed in the precursors of the embryonic peripheral nervous system (PNS), in the midline precursors of the central nervous system (CNS), and in muscles. During the initial phases of CNS axonogenesis, nrm is expressed in cells involved in the development of commissures and longitudinal tracts. Mutations which alter expression of nrm mRNAs cause aberrant development of commissures and longitudinal axon pathways. Defects in the PNS and muscles of nrm mutants are also observed. In most nrm embryos, abnormal development can be detected in a subset of abdominal segments; however, in approximately 1 of 10 nrm embryos, the defects extend to all segments. Herein, we present evidence that nrm plays an important role in early morphogenesis, possibly by mediating or facilitating inductive cell contacts and movements.     

Homozygosity for a Missense Mutation in SERPINH1, which Encodes the Collagen Chaperone Protein HSP47, Results in Severe Recessive Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is characterized by bone fragility and fractures that may be accompanied by bone deformity, dentinogenesis imperfecta, short stature, and shortened life span. About 90% of individuals with OI have dominant mutations in the type I collagen genes COL1A1 and COL1A2. Recessive forms of OI resulting from mutations in collagen-modifying enzymes and chaperones CRTAP, LEPRE1, PPIB, and FKBP10 have recently been identified. We have identified an autosomal-recessive missense mutation (c.233T>C, p.Leu78Pro) in SERPINH1, which encodes the collagen chaperone-like protein HSP47, that leads to a severe OI phenotype. The mutation results in degradation of the endoplasmic reticulum resident HSP47 via the proteasome. Type I procollagen accumulates in the Golgi of fibroblasts from the affected individual and a population of the secreted type I procollagen is protease sensitive. These findings suggest that HSP47 monitors the integrity of the triple helix of type I procollagen at the ER/cis-Golgi boundary and, when absent, the rate of transit from the ER to the Golgi is increased and helical structure is compromised. The normal 3-hydroxylation of the prolyl residue at position 986 of the triple helical domain of proα1(I) chains places the role of HSP47 downstream from the CRTAP/P3H1/CyPB complex that is involved in prolyl 3-hydroxylation. Identification of this mutation in SERPINH1 gives further insight into critical steps of the collagen biosynthetic pathway and the molecular pathogenesis of OI.     

COPI-independent Anterograde Transport: Cargo-selective ER to Golgi Protein Transport in Yeast COPI Mutants

The coatomer (COPI) complex mediates Golgi to ER recycling of membrane proteins containing a dilysine retrieval motif. However, COPI was initially characterized as an anterograde-acting coat complex. To investigate the direct and primary role(s) of COPI in ER/Golgi transport and in the secretory pathway in general, we used PCR-based mutagenesis to generate new temperature-conditional mutant alleles of one COPI gene in Saccharomyces cerevisiae, SEC21 (γ-COP). Unexpectedly, all of the new sec21 ts mutants exhibited striking, cargo-selective ER to Golgi transport defects. In these mutants, several proteins (i.e., CPY and α-factor) were completely blocked in the ER at nonpermissive temperature; however, other proteins (i.e., invertase and HSP150) in these and other COPI mutants were secreted normally. Nearly identical cargo-specific ER to Golgi transport defects were also induced by Brefeldin A. In contrast, all proteins tested required COPII (ER to Golgi coat complex), Sec18p (NSF), and Sec22p (v-SNARE) for ER to Golgi transport. Together, these data suggest that COPI plays a critical but indirect role in anterograde transport, perhaps by directing retrieval of transport factors required for packaging of certain cargo into ER to Golgi COPII vesicles. Interestingly, CPY–invertase hybrid proteins, like invertase but unlike CPY, escaped the sec21 ts mutant ER block, suggesting that packaging into COPII vesicles may be mediated by cis-acting sorting determinants in the cargo proteins themselves. These hybrid proteins were efficiently targeted to the vacuole, indicating that COPI is also not directly required for regulated Golgi to vacuole transport. Additionally, the sec21 mutants exhibited early Golgi-specific glycosylation defects and structural aberrations in early but not late Golgi compartments at nonpermissive temperature. Together, these studies demonstrate that although COPI plays an important and most likely direct role both in Golgi–ER retrieval and in maintenance/function of the cis-Golgi, COPI does not appear to be directly required for anterograde transport through the secretory pathway.     

Effects of brefeldin A on the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum in a green alga,Scenedesmus acutus

Summary The effects of brefeldin A (BFA) on the structure of the Golgi apparatus, the nuclear envelope, and the endoplasmic reticulum (ER), and on the thiamine pyrophosphatase (TPPase) activity in these organelles were examined in a green alga,Scenedesmus acutus, to obtain evidence for the existence of a retrograde transport from the Golgi apparatus to the ER via the nuclear envelope. InScenedesmus, Golgi bodies are situated close to the nuclear envelope throughout the cell cycle and receive the transition vesicles not directly from the ER, but from the nuclear envelope. BFA induced the disassembly of Golgi bodies and an increase in the ER cisternae at the trans-side of decomposed Golgi bodies in interphase cells and multinuclear cells before septum formation. The accumulated ER cisternae connected to the nuclear envelope at one part. TPPase activity was detected in all cisternae of Golgi bodies, but not in the nuclear envelope or the ER in nontreated cells. On the contrary, in BFA-treated cells, TPPase activity was detected in the nuclear envelope and the ER in addition to the decomposed Golgi bodies. When septum-forming cells were treated with BFA, the disassembly of Golgi bodies was less than that in interphase cells, and TPPase activity was detected in the Golgi cisternae but not in the nuclear envelope or the ER. These results suggest mat BFA blocks the anterograde transport from the nuclear envelope to the Golgi bodies but does not block the retrograde transport from the Golgi bodies to the nuclear envelope in interphase and multinuclear cells.Abbreviations BFA brefeldin A - ER endoplasmic reticulum - TPPase thiamine pyrophosphatase     

A novel staphylococcal internalization mechanism involves the major autolysin Atl and heat shock cognate protein Hsc70 as host cell receptor

Staphylococcus aureus and Staphylococcus epidermidis can cause serious chronic and recurrent infections that are difficult to eradicate. An important pathogenicity factor in these infections caused by S. aureus is its ability to be internalized by non‐professional phagocytes thereby evading the host immune system and antibiotic treatment. Here, we report a novel mechanism involved in staphylococcal internalization by host cells, which is mediated by the major autolysin/adhesins Atl and AtlE from S. aureus and S. epidermidis respectively. In a flow cytometric internalization assay, atl and atlE mutants are significantly reduced in their capacities to be internalized by endothelial cells. Moreover, pre‐incubation of endothelial cells with recombinant Atl dose‐dependently inhibited internalization. As putative Atl‐host cell receptor, the heat shock cognate protein Hsc70 was identified by mass spectrometry. The importance of Hsc70 in internalization was demonstrated by the inhibition of S. aureus internalization with anti‐Hsc70 antibodies. In conclusion, this novel Atl‐ or AtlE‐mediated internalization mechanism may represent a ‘back‐up’ mechanism in S. aureus internalization, while it may represent the major or even sole mechanism involved in the internalization of coagulase‐negative staphylococci and thus may play an important role in the pathogenesis of chronic and relapsing infections with these serious pathogens.     

Mutations in CAPN1 Cause Autosomal-Recessive Hereditary Spastic Paraplegia

Hereditary spastic paraplegia (HSP) is a genetically and clinically heterogeneous disease characterized by spasticity and weakness of the lower limbs with or without additional neurological symptoms. Although more than 70 genes and genetic loci have been implicated in HSP, many families remain genetically undiagnosed, suggesting that other genetic causes of HSP are still to be identified. HSP can be inherited in an autosomal-dominant, autosomal-recessive, or X-linked manner. In the current study, we performed whole-exome sequencing to analyze a total of nine affected individuals in three families with autosomal-recessive HSP. Rare homozygous and compound-heterozygous nonsense, missense, frameshift, and splice-site mutations in CAPN1 were identified in all affected individuals, and sequencing in additional family members confirmed the segregation of these mutations with the disease (spastic paraplegia 76 [SPG76]). CAPN1 encodes calpain 1, a protease that is widely present in the CNS. Calpain 1 is involved in synaptic plasticity, synaptic restructuring, and axon maturation and maintenance. Three models of calpain 1 deficiency were further studied. In Caenorhabditis elegans, loss of calpain 1 function resulted in neuronal and axonal dysfunction and degeneration. Similarly, loss-of-function of the Drosophila melanogaster ortholog calpain B caused locomotor defects and axonal anomalies. Knockdown of calpain 1a, a CAPN1 ortholog in Danio rerio, resulted in abnormal branchiomotor neuron migration and disorganized acetylated-tubulin axonal networks in the brain. The identification of mutations in CAPN1 in HSP expands our understanding of the disease causes and potential mechanisms.     

The Endoplasmic Reticulum-Resident Chaperone Heat Shock Protein 47 Protects the Golgi Apparatus from the Effects of O-Glycosylation Inhibition

The Golgi apparatus is important for the transport of secretory cargo. Glycosylation is a major post-translational event. Recognition of O-glycans on proteins is necessary for glycoprotein trafficking. In this study, specific inhibition of O-glycosylation (Golgi stress) induced the expression of endoplasmic reticulum (ER)-resident heat shock protein (HSP) 47 in NIH3T3 cells, although cell death was not induced by Golgi stress alone. When HSP47 expression was downregulated by siRNA, inhibition of O-glycosylation caused cell death. Three days after the induction of Golgi stress, the Golgi apparatus was disassembled, many vacuoles appeared near the Golgi apparatus and extended into the cytoplasm, the nuclei had split, and cell death assay-positive cells appeared. Six hours after the induction of Golgi stress, HSP47-knockdown cells exhibited increased cleavage of Golgi-resident caspase-2. Furthermore, activation of mitochondrial caspase-9 and ER-resident unfolded protein response (UPR)-related molecules and efflux of cytochrome c from the mitochondria to the cytoplasm was observed in HSP47-knockdown cells 24 h after the induction of Golgi stress. These findings indicate that (i) the ER-resident chaperon HSP47 protected cells from Golgi stress, and (ii) Golgi stress-induced cell death caused by the inhibition of HSP47 expression resulted from caspase-2 activation in the Golgi apparatus, extending to the ER and mitochondria.     

Temperature-sensitive mutants of hsp82 of the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has two HSP90-related genes per haploid genome, HSP82 and HSC82. Random mutations were induced in vitro in the HSP82 gene by treatment of the plasmid with hydroxylamine. Four temperature-sensitive (ts) mutants and one simultaneously is and cold-sensitivie (cs) mutant were then selected in a yeast strain in which HSC82 had previously been disrupted. The mutants were found to have single base changes in the coding region, which caused single amino acid substitutions in the HSP82 protein. All of these mutations occurred in amino acid residues that are well conserved among HSP90-related proteins of various species from Escherichia coli to human. Various properties including cell morphology, macromolecular syntheses and thermosensitivity were examined in each mutant at both the permissive and nonpermissive temperatures. The mutations in HSP82 caused pleiotropic effects on these properties although the phenotypes exhibited at the nonpermissive temperature varied among the mutants.     

ER network formation and membrane fusion by atlastin1/SPG3A disease variants

At least 38 distinct missense mutations in the neuronal atlastin1/SPG3A GTPase are implicated in an autosomal dominant form of hereditary spastic paraplegia (HSP), a motor-neurological disorder manifested by lower limb weakness and spasticity and length-dependent axonopathy of corticospinal motor neurons. Because the atlastin GTPase is sufficient to catalyze membrane fusion and required to form the ER network, at least in nonneuronal cells, it is logically assumed that defects in ER membrane morphogenesis due to impaired fusion activity are the primary drivers of SPG3A-associated HSP. Here we analyzed a subset of established atlastin1/SPG3A disease variants using cell-based assays for atlastin-mediated ER network formation and biochemical assays for atlastin-catalyzed GTP hydrolysis, dimer formation, and membrane fusion. As anticipated, some variants exhibited clear deficits. Surprisingly however, at least two disease variants, one of which represents that most frequently identified in SPG3A HSP patients, displayed wild-type levels of activity in all assays. The same variants were also capable of co-redistributing ER-localized REEP1, a recently identified function of atlastins that requires its catalytic activity. Taken together, these findings indicate that a deficit in the membrane fusion activity of atlastin1 may be a key contributor, but is not required, for HSP causation.     

A missense mutation in the vacuolar protein GOLD36 causes organizational defects in the ER and aberrant protein trafficking in the plant secretory pathway

A central question in cell biology is how the identity of organelles is established and maintained. Here, we report on GOLD36, an EMS mutant identified through a screen for partial displacement of the Golgi marker, ST‐GFP, to other organelles. GOLD36 showed partial distribution of ST‐GFP into a modified endoplasmic reticulum (ER) network, which formed bulges and large skein‐like structures entangling Golgi stacks. GOLD36 showed defects in ER protein export as evidenced by our observations that, besides the partial retention of Golgi markers in the ER, the trafficking of a soluble bulk‐flow marker to the cell surface was also compromised. Using a combination of classical mapping and next‐generation DNA sequencing approaches, we linked the mutant phenotype to a missense mutation of a proline residue in position 80 to a leucine residue in a small endomembrane protein encoded by the gold36 locus ( At1g54030 ). Subcellular localization analyses indicated that GOLD36 is a vacuolar protein and that its mutated form is retained in the ER. Interestingly also, a gold36 knock‐out mutant mirrored the GOLD36 subcellular phenotype. These data indicate that GOLD36 is a protein destined to post‐ER compartments and suggest that its export from the ER is a requirement to ensure steady‐state maintenance of the organelle’s organization and functional activity in relation to other secretory compartments. We speculate that GOLD36 may be a factor that is necessary for ER integrity because of its ability to limit deleterious effects of other secretory proteins on the ER.     

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking β-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.     

Presynaptic proteins involved in exocytosis inDrosophila melanogaster: A genetic analysis

Neuronal communication involves the fusion of neurotransmitter filled synaptic vesicles with the presynaptic terminal. This exocytotic event depends upon proteins present in three separate compartments: the synaptic vesicle, the synaptic cytosol, and the presynaptic membrane. Recent data indicate that the basic components of exocytotic pathways, including those used for neurotransmitter release, are conserved from yeast to human. Genetic dissection of the secretory pathway in yeast, identification of the target proteins cleaved by the clostridial neurotoxins and biochemical characterization of the interactions of synaptic proteins from vertebrates have converged to provide the SNARE (soluble NSF attachment protein receptor) hypothesis for vesicle trafficking. This model proposes that proteins present in the vesicle (v-SNAREs) interact with membrane receptors (t-SNAREs) to provide a molecular scaffold for cytosolic proteins involved in fusion. The hypothesis that these mechanisms function at the synapse relies largely uponin vitro evidence. Recently, genetic approaches in mice, C.elegans and the fruitfly,Drosophila melanagaster, have been used to dissect thein vivo function of numerous proteins involved in synaptic transmission. This review covers recent progress and insights provided by a genetic dissection of neurotransmitter release inDrosophila. In addition, we will provide evidence that the mechanisms for synaptic communication are highly conserved from invertebrates to vertebrates, makingDrosophila an ideal model system to further unravel the intricacies of synaptic transmission.     

The Drosophila BMP type II receptor Wishful Thinking regulates neuromuscular synapse morphology and function

Proper synaptic development is critical for establishing all aspects of neural function including learning, memory, and locomotion. Here, we describe the phenotypic consequences of mutations in the wishful thinking (wit) gene, the Drosophila homolog of the vertebrate BMP type II receptor. Mutations in wit result in pharate lethality that can be rescued by expression of a wit transgene in motor neurons but not in muscles. Mutant larvae exhibit small synapses, severe defects in evoked junctional potentials, a lower frequency of spontaneous vesicle release, and an alteration in the ultrastructure of synaptic active zones. These results reveal a novel role for BMP signaling in regulating Drosophila neuromuscular junction synapse assembly and activity and may indicate that similar pathways could govern vertebrate synapse development.     

Sec22 Regulates Endoplasmic Reticulum Morphology but Not Autophagy and Is Required for Eye Development in Drosophila

The endoplasmic reticulum (ER) is a highly dynamic organelle that plays a critical role in many cellular processes. Abnormal ER morphology is associated with some human diseases, although little is known regarding how ER morphology is regulated. Using a forward genetic screen to identify genes that regulated ER morphology in Drosophila, we identified a mutant of Sec22, the orthologs of which in yeast, plants, and humans are required for ER to Golgi trafficking. However, the physiological function of Sec22 has not been previously investigated in animal development. A loss of Sec22 resulted in ER proliferation and expansion, enlargement of late endosomes, and abnormal Golgi morphology in mutant larvae fat body cells. However, starvation-induced autophagy was not affected by a loss of Sec22. Mosaic analysis of the eye revealed that Sec22 was required for photoreceptor morphogenesis. In Sec22 mutant photoreceptor cells, the ER was highly expanded and gradually lost normal morphology with aging. The rhabdomeres in mutants were small and sometimes fused with each other. The morphology of Sec22 mutant eyes resembled the eye morphology of flies with overexpressed eyc (eyes closed). eyc encodes for a Drosophila p47 protein that is required for membrane fusion. A loss of Syntaxin5 (Syx5), encoding for a t-SNARE on Golgi, also phenocopied the Sec22 mutant. Sec22 formed complexes with Syx5 and Eyc. Thus, we propose that appropriate trafficking between the ER and Golgi is required for maintaining ER morphology and for Drosophila eye morphogenesis.     

Disturbance of the secretory pathway inMicrasterias denticulata by tunicamycin and cyclopiazonic acid

Summary Both tunicamycin, an inhibitor of N-linked glycosylation of proteins, and cyclopiazonic acid, which inhibits the Ca²⁺-dependent ATPase in the ER, influence the secretory pathway at the ER level and lead to a cessation of cell growth inMicrasterias. Electron microscopical investigations reveal that the mode of action of the two inhibitors differs. While tunicamycin treatment results in a disintegration of the Golgi bodies into small vesicles, cyclopiazonic acid prevents products being supplied from the ER, resulting in the dilatation of ER cisternae and a reduction in the number of Golgi cisternae, combined with a loss of dictyosomal activity. The disturbed cell wall formation under tunicamycin indicates that N-linked glycosylation of proteins is required for normal cell growth inMicrasterias. Moreover, our studies reveal that changes in cytoplasmic free calcium concentration, as a consequence of ATPase inhibition in the ER by cyclopiazonic acid, may inhibit wall material secretion by interrupting the normal ER-dictyosome association.Abbreviations CPA cyclopiazonic acid - ER endoplasmic reticulum - TM tunicamycin     

Light and Electron Microscope Observations on Nosema nelsoni Sprague, 1950 (Microsporida,Nosematidae) with Particular Reference to its Golgi Complex*

SYNOPSIS. A species of Nosema in the muscles of the North American white shrimp, generally known as Penaeus setiferus but also known as P. fluviatilis, appears identical with type specimens of N. nelsoni Sprague, 1950, in P. aztecus. Its Golgi apparatus, as seen in the sporoblast, is a complex system of cisternae, small vesicles and expanded sacs which plays a major role in spore morphogenesis. It transforms directly into the polaroplast complex, certain membranous investments of the polar filament, the polar sac and perhaps part of the posterior vacuolar system. Probably the polar sac contains the polar cap. The PAS-positive material in both the cap and the filament may be a component of the Golgi complex. This new concept of the Golgi complex supplements our earlier view of spore morphogenesis according to which the polar filament is of nuclear origin. It also reconciles the idea with Vávra's identification of Golgi vesicles associated with the developing polar filament.