Intraflagellar transport is required in Drosophila to differentiate sensory cilia but not sperm

BACKGROUND: Intraflagellar transport (IFT) uses kinesin II to carry a multiprotein particle to the tips of eukaryotic cilia and flagella and a nonaxonemal dynein to return it to the cell body. IFT particle proteins and motors are conserved in ciliated eukaryotes, and IFT-deficient mutants in algae, nematodes, and mammals fail to extend or maintain cilia and flagella, including sensory cilia. In Drosophila, the only ciliated cells are sensory neurons and sperm. no mechanoreceptor potential (nomp) mutations have been isolated that affect the differentiation and function of ciliated sense organs. The nompB gene is here shown to encode an IFT protein. Its mutant phenotypes reveal the consequences of an IFT defect in an insect. RESULTS: Mechanosensory and olfactory neurons in nompB mutants have missing or defective cilia. nompB encodes the Drosophila homolog of the IFT complex B protein IFT88/Polaris/OSM-5. nompB is expressed in the ciliated sensory neurons, and a functional, tagged NOMPB protein is located in sensory cilia and around basal bodies. Surprisingly, nompB mutant males produce normally elongated, motile sperm. Neuronally restricted expression and male germline mosaic experiments show that nompB-deficient sperm are fully functional in transfer, competition, and fertilization. CONCLUSIONS: NOMPB, the Drosophila homolog of IFT88, is required for the assembly of sensory cilia but not for the extension or function of the sperm flagellum. Assembly of this extremely long axoneme is therefore independent of IFT.     

The actin-binding protein capulet genetically interacts with the microtubule motor kinesin to maintain neuronal dendrite homeostasis

Background

Neurons require precise cytoskeletal regulation within neurites, containing microtubule tracks for cargo transport in axons and dendrites or within synapses containing organized actin. Due to the unique architecture and specialized function of neurons, neurons are particularly susceptible to perturbation of the cytoskeleton. Numerous actin-binding proteins help maintain proper cytoskeletal regulation.

Methodology/Principal Findings

From a Drosophila forward genetic screen, we identified a mutation in capulet-encoding a conserved actin-binding protein-that causes abnormal aggregates of actin within dendrites. Through interaction studies, we demonstrate that simultaneous genetic inactivation of capulet and kinesin heavy chain, a microtubule motor protein, produces elongate cofilin-actin rods within dendrites but not axons. These rods resemble actin-rich structures induced in both mammalian neurodegenerative and Drosophila Alzheimer''s models, but have not previously been identified by loss of function mutations in vivo. We further demonstrate that mitochondria, which are transported by Kinesin, have impaired distribution along dendrites in a capulet mutant. While Capulet and Cofilin may biochemically cooperate in certain circumstances, in neuronal dendrites they genetically antagonize each other.

Conclusions/Significance

The present study is the first molecularly defined loss of function demonstration of actin-cofilin rods in vivo. This study suggests that simultaneous, seemingly minor perturbations in neuronal dendrites can synergize producing severe abnormalities affecting actin, microtubules and mitochondria/energy availability in dendrites. Additionally, as >90% of Alzheimer''s and Parkinson''s cases are sporadic this study suggests mechanisms by which multiple mutations together may contribute to neurodegeneration instead of reliance on single mutations to produce disease.     

The Drosophila homologue of the 64 kDa subunit of cleavage stimulation factor interacts with the 77 kDa subunit encoded by the suppressor of forked gene

During mRNA 3′ end formation, cleavage stimulation factor (CstF) binds to a GU-rich sequence downstream from the polyadenylation site and helps to stabilise the binding of cleavage-polyadenylation specificity factor (CPSF) to the upstream polyadenylation sequence (AAUAAA). The 64 kDa subunit of CstF (CstF-64) contains an RNA binding domain and is responsible for the RNA binding activity of CstF. It interacts with CstF-77, which in turn interacts with CPSF. The Drosophila suppressor of forked gene encodes a homologue of CstF-77, and mutations in it affect mRNA 3′ end formation in vivo. A Drosophila homologue for CstF-64 has now been isolated, both through homology with the human protein and through protein–protein interaction in yeast with the suppressor of forked gene product. Alignment of CstF-64 homologues shows that the proteins have a conserved N-terminal 200 amino acids, the first half of which is the RNA binding domain with the second half likely to contain the CstF-77 interaction domain; a central region variable in length and rich in glycine, proline and glutamine residues and containing an unusual degenerate repeat motif; and then a conserved C-terminal 50 amino acids. In Drosophila, the CstF-64 gene has a single 63 bp intron, is transcribed throughout development and probably corresponds to l(3)91Cd.     

Drosophila Cdk8, a kinase partner of cyclin C that interacts with the large subunit of RNA polymerase II.

A number of cyclins have been described, most of which act together with their catalytic partners, the cyclin-dependent kinases (Cdks), to regulate events in the eukaryotic cell cycle. Cyclin C was originally identified by a genetic screen for human and Drosophila cDNAs that complement a triple knock-out of the CLN genes in Saccharomyces cerevisiae. Unlike other cyclins identified in this complementation screen, there has been no evidence that cyclin C has a cell-cycle role in the cognate organism. Here we report that cyclin C is a nuclear protein present in a multiprotein complex. It interacts both in vitro and in vivo with Cdk8, a novel protein-kinase of the Cdk family, structurally related to the yeast Srb10 kinase. We also show that Cdk8 can interact in vivo with the large subunit of RNA polymerase II and that a kinase activity that phosphorylates the RNA polymerase II large subunit is present in Cdk8 immunoprecipitates. Based on these observations and sequence similarity to the kinase/cyclin pair Srb10/Srb11 in S. cerevisiae, we suggest that cyclin C and Cdk8 control RNA polymerase II function.     

Neuropathology in sensory,but not motor,brainstem nuclei of the rat whisker circuit after diffuse brain injury

Neurological dysfunction after traumatic brain injury (TBI) is associated with pathology in cortical, subcortical, and brainstem nuclei. Our laboratory has reported neuropathology and microglial activation in the somatosensory barrel cortex (S1BF) and ventral posterior medial thalamus (VPM) after diffuse TBI in the rat, which correlated with post-injury whisker sensory sensitivity. The present study extends our previous work by evaluating pathology in whisking-associated sensory and motor brainstem nuclei. Brains from adult, male rats were recovered over 1 month after midline fluid percussion or sham injury. The principal trigeminal nucleus (PrV, sensory nucleus) and facial nucleus (VIIN, motor nucleus) were examined for neuropathology (silver histochemistry) and microglial activation (Iba1). Significant neuropathology in PrV was evident at 2 and 7 days post-injury compared to sham. Iba1-labeled microglia showed swollen somata and thickened processes over 1 month post-injury. In contrast, the VIIN showed non-significant neuropathology and reduced labeling of activated Iba1 microglia over 1 month post-injury. Together with our previous data, neuropathology and neuroinflammation in the whisker somatosensory pathway may contribute to post-injury sensory sensitivity more than the motor pathway. Whether these findings are direct results of the mechanical injury or consequences of progressive degeneration remains to be determined.     

Caveolin interacts with the angiotensin II type 1 receptor during exocytic transport but not at the plasma membrane

The mechanisms involved in angiotensin II type 1 receptor (AT1-R) trafficking and membrane localization are largely unknown. In this study, we examined the role of caveolin in these processes. Electron microscopy of plasma membrane sheets shows that the AT1-R is not concentrated in caveolae but is clustered in cholesterol-independent microdomains; upon activation, it partially redistributes to lipid rafts. Despite the lack of AT1-R in caveolae, AT1-R.caveolin complexes are readily detectable in cells co-expressing both proteins. This interaction requires an intact caveolin scaffolding domain because mutant caveolins that lack a functional caveolin scaffolding domain do not interact with AT1-R. Expression of an N-terminally truncated caveolin-3, CavDGV, that localizes to lipid bodies, or a point mutant, Cav3-P104L, that accumulates in the Golgi mislocalizes AT1-R to lipid bodies and Golgi, respectively. Mislocalization results in aberrant maturation and surface expression of AT1-R, effects that are not reversed by supplementing cells with cholesterol. Similarly mutation of aromatic residues in the caveolin-binding site abrogates AT1-R cell surface expression. In cells lacking caveolin-1 or caveolin-3, AT1-R does not traffic to the cell surface unless caveolin is ectopically expressed. This observation is recapitulated in caveolin-1 null mice that have a 55% reduction in renal AT1-R levels compared with controls. Taken together our results indicate that a direct interaction with caveolin is required to traffic the AT1-R through the exocytic pathway, but this does not result in AT1-R sequestration in caveolae. Caveolin therefore acts as a molecular chaperone rather than a plasma membrane scaffold for AT1-R.     

TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking

The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c'. Subunit c' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.     

Drosophila Spd-2 recruits PCM to the sperm centriole, but is dispensable for centriole duplication

In C. elegans, genome-wide screens have identified just five essential centriole-duplication factors: SPD-2, ZYG-1, SAS-5, SAS-6, and SAS-4 [1-8]. These proteins are widely believed to comprise a conserved core duplication module [3, 9-14]. In worm embryos, SPD-2 is the most upstream component of this module, and it is also essential for pericentriolar material (PCM) recruitment to the centrioles [1, 4, 15, 16]. Here, we show that Drosophila Spd-2 (DSpd-2) is a component of both the centrioles and the PCM and has a role in recruiting PCM to the centrioles. DSpd-2 appears not, however, to be essential for centriole duplication in somatic cells. Moreover, PCM recruitment in DSpd-2 mutant somatic cells is only partially compromised, and mitosis appears unperturbed. In contrast, DSpd-2 is essential for proper PCM recruitment to the fertilizing sperm centriole, and hence for microtubule nucleation and pronuclear fusion. DSpd-2 therefore appears to have a particularly important role in recruiting PCM to the sperm centriole. We speculate that the SPD-2 family of proteins might only be absolutely essential for the recruitment of centriole duplication factors and PCM to the centriole(s) that enter the egg with the fertilizing sperm.     

The core planar cell polarity gene prickle interacts with flamingo to promote sensory axon advance in the Drosophila embryo

The atypical cadherin Drosophila protein Flamingo and its vertebrate homologues play widespread roles in the regulation of both dendrite and axon growth. However, little is understood about the molecular mechanisms that underpin these functions. Whereas flamingo interacts with a well-defined group of genes in regulating planar cell polarity, previous studies have uncovered little evidence that the other core planar cell polarity genes are involved in regulation of neurite growth. We present data in this study showing that the planar cell polarity gene prickle interacts with flamingo in regulating sensory axon advance at a key choice point — the transition between the peripheral nervous system and the central nervous system. The cytoplasmic tail of the Flamingo protein is not required for this interaction. Overexpression of another core planar cell polarity gene dishevelled produces a similar phenotype to prickle mutants, suggesting that this gene may also play a role in regulation of sensory axon advance.     

BAALC 1-6-8 protein is targeted to postsynaptic lipid rafts by its N-terminal myristoylation and palmitoylation, and interacts with alpha, but not beta, subunit of Ca/calmodulin-dependent protein kinase II

We cloned a rat BAALC 1-6-8 isoform cDNA (GenBank Accession No. AB073318) that encoded a 22-kDa protein, and identified endogenous BAALC 1-6-8 protein in the brain. The gene was expressed widely in the frontal part of the brain, and the protein was localized to the synaptic sites and was increased in parallel with synaptogenesis. The protein interacted with the alpha, but not beta, subunit of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIalpha). The interaction occurred between the N-terminal 35-amino-acid region of BAALC 1-6-8 protein and the C-terminal end of the regulatory domain of CaMKIIalpha, which contains alpha isoform-specific sequence. Thus, the interaction may be CaMKIIalpha-specific. We also found that BAALC 1-6-8 protein, as well as CaMKIIalpha, was localized to lipid rafts and that both myristoylation and palmitoylation of BAALC 1-6-8 N-terminal portion were required for targeting of the protein into lipid rafts. These findings suggest that BAALC 1-6-8 protein play a synaptic role at the postsynaptic lipid raft possibly through interaction with CaMKIIalpha.     

L63, the Drosophila PFTAIRE,interacts with two novel proteins unrelated to cyclins

L63 encodes a CDK-like protein homologous to the mammalian PFTAIRE. We showed previously that L63 provides a CDK-related function critical to development (Dev. Biol. 221 (2000) 23). We present here the first biochemical characterization of L63 kinase. In addition, we describe two novel Drosophila proteins, PIF-1 and PIF-2 (for PFTAIRE Interacting Factor-1 and -2), identified in a two-hybrid screen for their ability to interact with the amino-terminal region of L63. The full-length PIF-1 cDNA shows an unusual dicistronic organization. PIF-1A and PIF-1B (the L63 interactor) predicted proteins are expressed in vivo, and show a distinct expression profile during development. Interaction between L63 and PIF-1B was confirmed in vitro and in vivo. The role of this interaction remains to be demonstrated, but our data suggest that PIF-1B might serve as a regulator of L63.     

labial acts to initiate neuronal fate specification, but not axon pathfinding, in the embryonic brain of Drosophila

Drosophila embryos lacking the homeotic gene labial (lab) show two types of defects in brain development: (1) cells in the brain lab domain do not express neuronal markers or extend axons, and (2) axons originating from outside the lab domain stop at this region or project ectopically. A severe disruption of neuronal patterning and axon scaffolding is the net result. It is not clear how the absence of Lab can result in both neuronal fate defects and axon pathfinding defects. I have expressed Lab in short pulses in lab loss-of- function embryos, and this gave almost complete rescue; for example, the tritocerebral commissure was restored. Rescue only occurred when Lab was provided at the time when cells in the brain are adopting a neuronal fate. Lab expression later, when the first axons are seen in the lab domain, did not give rescue. I conclude that Lab expression helps to establish neuronal identity in the lab domain, and these neurons act as a permissive substrate for axon extension. However, Lab itself is not required at the time of axon pathfinding through this region. Received: 31 May 2000 / Accepted: 5 July 2000     

Courtship song recognition in the Drosophila melanogaster complex: heterospecific songs make females receptive in D. melanogaster, but not in D. sechellia

Abstract. The courtship song emitted by male wing vibration has been regarded as one of the most important signals in sexual isolation in the species of the Drosophila melanogaster complex. Inter- and intraspecific crosses were observed using males whose wings were removed (mute) or females whose aristae were removed (deaf). Females of D. melanogaster, D. simulans , and D. mauritiana mated with heterospecific males in the song-present condition (cross between normal females and winged males) more often than in the no-song condition (cross between normal females and wingless males or between aristaless females and winged males) or they showed no preference between the two conditions. It is possible that in these females heterospecific courtship songs play a role as if they were conspecific. In contrast, the females of D. sechellia mated with D. melanogaster or D. simulans males in the no-song condition more often than in the song-present condition, suggesting that they reject males with heterospecific song. Female mate recognition depending on the courtship song in D. melanogaster, D. simulans , and D. mauritiana is considered to be relatively broader and that in D. sechellia narrower.     

Aspirin, but not NO-releasing aspirin (NCX-4016), interacts with selective COX-2 inhibitors to aggravate gastric damage and inflammation

Aceylation of cyclooxygenase (COX)-2 by aspirin can trigger the formation of 15(R)-epilipoxin A4, or aspirin-triggered lipoxin (ATL). ATL exerts protective effects in the stomach. Selective COX-2 inhibitors block ATL synthesis and exacerbate aspirin-induced gastric damage. Nitric oxide-releasing aspirins, including NCX-4016, have antiplatelet effects similar to aspirin but do not cause gastric damage. In the present study, we examined whether or not NCX-4016 triggers ATL synthesis and/or upregulates gastric COX-2 expression and the effects of coadministration of NCX-4016 with a selective COX-2 inhibitor on gastric mucosal injury and inflammation. Rats were given aspirin or NCX-4016 orally and either vehicle or a selective COX-2 inhibitor (celecoxib) intraperitoneally. Gastric damage was blindly scored, and granulocyte infiltration into gastric tissue was monitored through measurement of myeloperoxidase activity. Gastric PG and ATL synthesis was measured as was COX-2 expression. Whereas celecoxib inhibited gastric ATL synthesis and increased the severity of aspirin-induced gastric damage and inflammation, coadministration of celecoxib and NCX-4016 did not result in damage or inflammation. NCX-4016 did not upregulate gastric COX-2 expression nor did it trigger ATL synthesis (in contrast to aspirin). Daily administration of aspirin for 5 days resulted in significantly less gastric damage than that seen with a single dose, as well as augmented ATL synthesis. Celecoxib reversed this effect. In contrast, repeated administration of NCX-4016 failed to cause gastric damage, whether given alone or with celecoxib. These studies support the notion that NCX-4016 may be an attractive alternative to aspirin for indications such as cardioprotection, including in individuals also taking selective COX-2 inhibitors.     

Antibodies against the COOH-terminal undecapeptide of subunit II, but not those against the NH2-terminal decapeptide, immunoprecipitate the whole human cytochrome c oxidase complex

Antibodies against synthetic peptides derived from the DNA sequence of human cytochrome c oxidase subunit II (COII) have been tested for their capacity to immunoprecipitate the whole enzyme complex. Antibodies against the COOH-terminal undecapeptide of COII (anti-COII-C), when incubated with a Triton X-100 mitochondrial lysate from HeLa cells pulse-labeled with [35S]methionine under conditions selective for mitochondrial protein synthesis and chased for 18 h in unlabeled medium, precipitated the pulse-labeled three largest subunits (mitochondrially synthesized) of cytochrome c oxidase in proportions close to equimolarity. Antibodies against the NH2-terminal decapeptide of COII (anti-COII-N), although equally reactive as the anti-COII-C antibodies with the sodium dodecyl sulfate-solubilized COII, did not precipitate any of the three labeled subunits from the Triton X-100 mitochondrial lysate. In other experiments, all the 13 subunits which have been identified in the mammalian cytochrome c oxidase were immunoprecipitated from a Triton X-100 mitochondrial lysate of cells long-term labeled with [35S]methionine by anti-COII-C antibodies, but not by anti-COII-N antibodies. By contrast, in immunoblots of total mitochondrial proteins dissociated with sodium dodecyl sulfate, the anti-COII-C antibodies reacted specifically only with COII. These results strongly suggest that, in the native cytochrome c oxidase complex, the epitope recognized by the anti-COII-C antibodies is in the COII subunit and that, therefore, in such complex, the COOH-terminal peptide of COII is exposed to antibodies, whereas the NH2-terminal peptide is not accessible.     

Molecular analysis of the Chlamydomonas nuclear gene encoding PsbW and demonstration that PsbW is a subunit of photosystem II,but not photosystem I

PsbW is a nuclear-encoded protein located in the thylakoid membrane of the chloroplast. Studies in higher plants have provided substantial evidence that PsbW is a core component of photosystem II. However, recent data have been presented to suggest that PsbW is also a subunit of photosystem I. Such a sharing of subunits between the two photosystems would represent a novel phenomenon. To investigate this, we have cloned and characterized the psbW gene from the green alga Chlamydomonas reinhardtii. The gene is split by five introns and encodes a polypeptide of 115 residues comprising the 6.1 kDa mature PsbW protein preceded by a 59 amino acid bipartite transit sequence. Using antibodies raised to PsbW we have examined: (1) C. reinhardtii mutants lacking either photosystem and (2) purified photosystem preparations. We find that PsbW is a subunit of photosystem II, but not photosystem I.     

Aspartate 75 mutation in sensory rhodopsin II from Natronobacterium pharaonis does not influence the production of the K-like intermediate, but strongly affects its relaxation pathway

The early steps in the photocycle of the aspartate 75-mutated sensory rhodopsin II from Natrobacterium pharaonis (pSRII-D75N) were studied by time-resolved laser-induced optoacoustic spectroscopy combined with quantum yield determinations by flash photolysis with optical detection. Similar to the case of pSRII-WT, excitation of pSRII-D75N produces in subnanosecond time a K-like intermediate. Different to the case of K in pSRII-WT, in pSRII-D75N there are two K states. K(E) decays into K(L) with a lifetime of 400 ns (independent of temperature in the range 6.5-52 degrees C) which is optically silent under the experimental conditions of our transient absorption experiments. This decay is concomitant with an expansion of 6.5 ml/mol of produced intermediate. This indicates a protein relaxation not affecting the chromophore absorption. For pSRII-D75N reconstituted into polar lipids from purple membrane, the mutation of Asp-75 by the neutral residue Asn affects neither the K(E) production yield (PhiK(e) 0.51 +/- 0.05) nor the energy stored by this intermediate (E(E)K(E) = 91 +/- 11 kJ/mol), nor the expansion upon its production (DeltaV(R,1) = 10 +/- 0.3 ml/mol). All these values are very similar to those previously determined for K with pSRII-WT in the same medium. The millisecond transient species is attributed to K(L) with a lifetime corresponding to that determined by electronic absorption spectroscopy for K(565). The determined energy content of the intermediates as well as the structural volume changes for the various steps afford the calculation of the free energy profile of the phototransformation during the pSRII-D75N photocycle. These data offer insights regarding the photocycle in pSRII-WT. Detergent solubilization of pSRII-D75N affects the sample properties to a larger extent than in the case of pSRII-WT.