latheo

Protein Interactions

In Drosophila, four unknown proteins from embryonic extracts copurify (and cosediment) with DmORC2 and DmORC5, suggesting that these form DmORC (Gossen, 1995). One 79 kDa protein component is similar to the molecular weight of Lat. Immunoprecipitation of DmORC2 from Schneider cells results in specific coimmunoprecipitation of Lat. Immunoprecipitation of Lat with anti-Lat antibodies also coimmunoprecipitates DmORC2. The sequence similarity with ScORC3, the association of Lat with DmORC2, and the cell proliferation defects of lat null mutants strongly argue that Lat functions as a subunit of Drosophila ORC (Pinto, 1999).

The origin recognition complex (ORC) is the DNA replication initiator protein in eukaryotes. A functional recombinant Drosophila ORC has been reconstituted and activities of the wild-type and several mutant ORC variants have been compared. Drosophila ORC is an ATPase, and the ORC1 subunit is essential for ATP hydrolysis and for ATP-dependent DNA binding. Moreover, DNA binding by ORC reduces its ATP hydrolysis activity. In vitro, ORC binds to chromatin in an ATP-dependent manner, and this process depends on the functional AAA+ nucleotide-binding domain of ORC1. Mutations in the ATP-binding domain of ORC1 are unable to support cell-free DNA replication. However, mutations in the putative ATP-binding domain of either the ORC4 or ORC5 subunits do not affect either of these functions. Evidence is provided that the Drosophila ORC6 subunit is directly required for all of these activities and that a large pool of ORC6 is present in the cytoplasm, cytologically proximal to the cell membrane. Studies reported here provide the first functional dissection of a metazoan initiator and highlight the basic conserved and divergent features between Drosophila and budding yeast ORC complexes (Chesnokov, 2001).

Six different mutant complexes and wild-type recombinant ORC were prepared. For each case, simultaneous expression of the wild-type or mutant genes in a baculovirus expression system resulted in complexes that could be purified to homogeneity through four chromatographic steps, and the mutant complexes assembled and exhibited no chromatographic differences during the purification. In a final step, the pooled peak fractions were subjected to glycerol-gradient sedimentation (Chesnokov, 2001).

The best understood functions of the yeast ORC are its DNA-binding and ATP hydrolysis functions. The bulk of recombinant (or purified embryonic) Drosophila ORC DNA binding activity is nonspecific and ATP-independent. However, this ATP-independent DNA binding activity can be titrated away with sufficient amount of carrier DNA when the carrier DNA is in a range 50-100 molar excess to the probe DNA. At physiologically relevant ATP concentrations (10 microM to 1 mM), the wild-type ORC binds to DNA 10-50-fold better than either the ORC1A or ORC1B mutant complex. Mutations in either the Walker A or B motif of ORC4 or the Walker A motif of ORC5 have no effect on the formation of ATP-dependent DNA-protein complex. These experiments supports the idea that the recombinant Drosophila ORC, like the recombinant S. cerevisiae homolog, requires only the ORC1 component of the complex to bind ATP for tight DNA interactions. However, the complex missing the ORC6 subunit does not form an ATP-dependent DNA-protein complex (Chesnokov, 2001).

Kinetic analysis of ATP hydrolysis with multiple independent wild-type (wt) ORC preparations shows a K_m of 1.92 µM and a V_max of 0.4 mol ATP hydrolyzed per min per mol of complex. Binding to DNA has a small (2-fold) but measurable effect on slowing the rate of ATP hydrolysis by ORC. In these experiments, ATP was not limiting, the mutant ORI complexed to DNA was titrated to its maximal effect. In the absence of any carrier DNA, the saturation is reached at an approximate 2.5-fold molar excess of DNA to ORC. Complexes harboring similar mutations in either ORC4 or ORC5 hydrolyzes ATP with equivalent kinetics to wild type, all displaying K_m values and V_max within the experimental error range of wild type. Consistent with the DNA-binding experiments, the ATP-hydrolysis rate for these mutant complexes is slowed by DNA similar to the effect observed for the wild-type ORC. In contrast, ORC1A or ORC1B mutants have severely crippled enzymatic activity, too close to background to measure any kinetic parameters. The ORC-6 complex is able to hydrolyze ATP at reduced levels, but this activity is unaffected by DNA, consistent with the finding that ORC6 is critical for formation of an ATP-dependent ternary complex (Chesnokov, 2001).

Chromatin binding assays were performed by using both mutant and wt ORC in extracts depleted of membranes. For these experiments Drosophila preblastula embryo extracts were immunodepleted of ORC by using antibody raised against ORC2 and ORC6. The effectiveness of immunodepletion was verified by immunoblotting. Demembranated sperm chromatin was added to the depleted extracts, and the binding activities of mutant and wild-type recombinant DmORC were compared with the endogenous Drosophila ORC. Treatment of the extracts with Apyrase abolishes ORC-chromatin binding, thus it is inferred that the binding process requires ATP. Endogenous ATP levels (which are estimated to be at 30-50 µM) were relied upon to mediate tight chromatin binding. Proteins associated with the chromosomes are separated from the unbound proteins by sedimentation. The results obtained via this assay parallel those obtained by the gel-shift experiments. Recombinant wt ORC, ORC4A, ORC4B, and ORC5A complexes associate with the chromatin with apparently the same efficiency as does endogenous protein, whereas the ORC1A, ORC1B, and ORC 6 complexes are severely crippled (Chesnokov, 2001).

Two independent measures of DNA replication competence were used for accessing the abilities of the mutant complexes to restore activity to depleted extracts. In the first assay, labeled precursor incorporation into high molecular DNA was detected by autoradiography of gels after electrophoresis or in a second assay after CsCl density gradient separation of DNA that was replicated in extracts with the density label precursor BrdUrd. As anticipated from the DNA and chromatin binding results, the ORC1A, ORC1B, and ORCdelta6 complexes were essentially inactive by at least 10-20-fold below the activity of wt recombinant ORC in restoring replication to the extracts. The ORC4A, ORC4B, and ORC5A mutants were effective in reconstitution but were in multiple experiments between 50% and 90% of wild-type complex (Chesnokov, 2001).

It has been concluded that the bulk of the subunits of the Drosophila ORC biochemically behave as a complex. ORC2 antibodies were used to track ORC in fractions from 0-12-h embryo extracts after gel-filtration chromatography. Two broad zones containing ORC were found. The highest apparent molecular weight fractions containing all ORC subunits were pooled and purified. A smaller complex was also detected that was apparently without ORC-1. However, when following ORC6 using ORC6-specific antibodies, a pool of ORC6 devoid of other ORC subunits is detected. No other ORC subunits were found in a form unassociated with other ORC proteins. It is estimated that this free pool is at least one-half of the total ORC6 protein present in these extracts. Given the important role that Drosophila ORC6 plays in cell-free replication and the other activities of ORC, it was of interest to ask whether this separate pool of ORC6 is localized with the other ORC subunits in the cell (Chesnokov, 2001).

Transient ectopic expression of ORC1 or ORC2 GFP-fusion proteins in cultured cells shows a distinct nuclear localization; in unexpected contrast, the GFP-ORC6 fusion protein was found both in the nucleus and cytoplasm. The ORC6 cytoplasmic signal seems to be closely associated, in various focal planes, with the cytoplasmic membranes. These experiments rely on overexpression: this issue was probed further by direct immunofluorescence of endogenous levels of the ORC proteins in Drosophila embryos. Before the onset of cellularization, ORC6 protein localizes only with ORC2 in the nuclear space of both interphase and mitotic cells. However, after cellularization, ORC6 seems to localize in the cytoplasm and nucleus. The signals for ORC6 can be blocked by preincubating the affinity-purified antibodies with recombinant ORC6 proteins and are clearly distinct from the ORC2 pattern. Further work will be required to judge whether the cytoplasmic pool of ORC6 is truly membrane associated, but it is worth noting that the carboxyl terminus of Drosophila ORC6 contains a predicted leucine-zipper region that could be involved in mediating multiple heterologous protein-protein interactions (Chesnokov, 2001).

An important finding of this study is that the Drosophila ORC complex likely uses mechanisms for binding DNA that are similar to those reported for the budding yeast homolog. Of the three potential ATP binding proteins in ORC, only ORC1 seems to be critical for establishing a tight ternary complex with DNA and for binding to chromatin. Similarly only mutations in the ATP binding domains of ORC1 critically affect a single round of DNA replication in cell-free extracts. Additional experimentation needs to be done to test the roles of the conserved domains in ORC4 and ORC5. Particularly intriguing is the wide conservation of the GKT (Walker A motif) and D (D/EE) (Walker B motif) in the ORC4 subunit. Such domains may be critical for recycling ORC for subsequent rounds of replication or for other activities of the complex in heterochromatin formation or putative check-point control. Drosophila ORC is an ATPase, and again ORC1 seems to play the critical role for ATP hydrolysis, since mutants in the putative ATPase domains of ORC4 and ORC5 do not affect the kinetic parameters of the mutant complex. Nevertheless, it is possible that in the presence of other bound factors, ATP binding or hydrolysis by the other subunits plays some critical role. ATP hydrolysis by any subunit does not seem important for DNA-binding activity. ADP could not mediate such a DNA-protein complex, and ATPgammaS is better at forming a ternary complex than ATP. X-ray crystallographic structure models for several AAA+ proteins have been solved, and a common fold has been observed. The crystal structure model of an archael Cdc6 ortholog was used as a guide for the ATP-binding structures of ORC1. In the nucleotide-binding domain of this protein family, both the GKT and the DE motifs contribute to nucleotide affinity. In fact, similar mutants in the amino-part of the Walker B motif of the S. cerevisiae ORC1 are defective for ATP binding, in contrast to mutations at the carboxyl end of the B motif that are competent for such activity. Moreover, the solvent-exposed surfaces present in these parts of the ORC1 protein may influence interactions with other partners, yielding a mutant complex with altered functions. These studies of the ATPase activity of DmORC indicate that turnover is slower when ORC is bound to DNA, but the effect is significantly less than that observed for the budding yeast complex. Divergence in the way in which these proteins interact with DNA is also highlighted by the critical role that the Drosophila ORC6 protein plays in ATP-dependent DNA binding. Perhaps, given the lack of amino acid homologies found between the ScORC6 and DmORC6 proteins, it is dangerous to consider each to be homologs (Chesnokov, 2001).

Overexpression of ORC1 trans-genes in Drosophila can alter DNA replication patterns. This overexpression leads to detectable levels of BrdUrd incorporation in normally quiescent cells or increased levels of replication in follicle cells normally amplifying the chorion genes. Similar ectopic expression of an ORC1A mutant (ORC1K604E) has no phenotype. The biochemical results with the ORC1A mutant K604A predict that their mutation might have a dominant negative effect on DNA replication in vivo. It is possible that the mutant gene would not be antimorphic by virtue of its not being able to compete with a wild-type ORC1 protein for assembly into complex. Leaving this point aside, one idea favored is that ORC1 is limiting for replication in some cellular environments and, for example, complexes containing solely ORC2-6 wait for ORC1 for activation. These pools may or may not be bound to chromosomal DNA. Recent work in mammalian systems indicates that ORC1 may be more loosely associated with chromatin than is ORC2. ORC2, presumably with some of the subunits, can be pelleted with the chromosomes. The results reveal that intact ORC needs ATP and functional ORC1 to bind tightly to chromatin. Are all of these data compatible, assuming a conservation in basic binding properties for ORC between mammals and Drosophila? Perhaps, in the absence of ORC1 other subunits mediate another sort of chromatin association. More complex notions are possible, including the interaction of unknown chromatin binding proteins that serve to tether a complex lacking ORC1 to the ori sites (Chesnokov, 2001).

It is suggested that ORC6 is another subunit that may play important and perhaps dynamic roles in regulating replication activity. The data show that ORC6 is an essential component of the complex per se and may be directly involved in DNA binding and other replication functions or needed for proper ORC assembly. In H. sapiens extracts, ORC6 is not found associated with other ORC subunits, but when expressed in the baculovirus system with the other ORC genes, the protein does join a six-subunit complex. The high levels of free ORC6 in embryonic and cultured cell extracts is intriguing. A considerable fraction of this pool as judged by cytological methods is cytoplasmic, and the protein is perhaps associated with or proximal to the cytoplasmic membranes. It is possible that this localization enables ORC6 to participate in functions unrelated to DNA replication per se, as has been suggested for the 'latheo' gene product, which is ORC3. Latheo seems to be involved in ion transport at neuromuscular junctions. Data now exist for both the budding yeast and for the Drosophila ORC, which directly indicate that all of the subunits are critical for DNA replication function, and complex models involving traffic of subsets of ORC subunits can be the subject of future work (Chesnokov, 2001).

DEVELOPMENTAL BIOLOGY

Larval

A polyclonal antiserum was used to assay Lat protein expression in the larval peripheral nervous system and body muscle. The antiserum is specific for the LAT protein in Western blots of third instar larval CNS extracts, recognizing a single 79 kDa protein from wild-type larvae that is not detectable in lat null mutant larvae. In wild-type third instar larvae, the Lat protein is immunologically detected at synaptic boutons at most NMJs on a wide variety of muscle fibers in the ventral abdomen. LAT immunostaining is often only weakly detectable at NMJs containing predominantly large type I boutons, which utilize glutamate as the primary neurotransmitter. Staining is nevertheless clearly evident at type I boutons, including both subtypes Ib and Is at various NMJs, including muscles 4 and 6/7. At many NMJs, LAT immunostaining is most distinct at morphologically smaller boutons, including those resembling type II and type III boutons. These boutons are believed to contain amines and neuropeptides, including octopamine (Monastirioti, 1995) and neuropeptide proctolin (Arg-Tyr-Leu-Pro-Thr) found in type II synapses (Anderson, 1988) and insulin-like peptide, found in type III synapses (Gorczyca, 1993). The amines and neuropeptides may serve as modulatory transmitters. In particular, the NMJ at muscle 12, which receives types I, II, and III innervation, consistently exhibits positive staining, commonly at multiple bouton types at the same NMJ. LAT thus appears not to be segregated to particular body segments, subsets of muscle fibers, or particular bouton subtypes distinguishable by either morphological or physiological criteria. LAT immunoreactivity is also detectable at the NMJ in second instar larvae, indicating the protein is present throughout most of the period of dramatic morphological and functional synaptic maturation (Rohrbough, 1999).

To gain more specific information on LAT synaptic localization, double-staining experiments were carried out at the wild-type NMJ with antibodies against LAT and other known pre- or postsynaptic proteins. Confocal microscopy of immunofluorescence reveals colocalization of LAT with the presynaptic vesicle-associated cysteine string protein (CSP) (Zinsmaier, 1994) at multiple bouton types, including both types I and II, suggesting LAT is located predominantly presynaptically and colocalizes with synaptic vesicles. LAT and CSP immunostaining often show nearly complete overlap at smaller boutons resembling type II and type III boutons, while at type I boutons, LAT staining usually appears less distinct and contained within a subarea of CSP expression. LAT localization was also examined in preparations double stained for postsynaptic glutamate receptors (DGluR2a) and the postsynaptic Discs-large (Dlg) and position-specific ß (ßPS) integrin proteins, which are localized in the subsynaptic reticulum of type I boutons. LAT staining typically occupies smaller areas contained within the broader staining pattern of the respective postsynaptic proteins, including the central area of boutons. These results confirm that LAT is localized at type I boutons and strongly suggest that the protein is expressed predominantly, if not exclusively, in the presynaptic compartment (Rohrbough, 1999).

Effects of Mutation or Deletion

Genetic dissection of learning and memory in Drosophila has been limited by the existence of ethyl methanesulfonate (EMS)-induced mutations in only a small number of X-linked genes. To remedy this shortcoming, a P element mutagenesis has been carried out to screen for autosomal mutations that disrupt associative learning and/or memory. The generation of 'P-tagged' mutant alleles will expedite molecular cloning of these new genes. A behavior-genetic characterization of latheoP1, a recessive, hypomorphic mutation of an essential gene, has been carried out. latheoP1 flies perform poorly in olfactory avoidance conditioning experiments. This performance deficit could not be attributed to abnormal olfactory acuity or shock reactivity, two task-relevant 'peripheral' behaviors used during classical conditioning. Thus, the latheoP1 mutation appears to affect learning/memory specifically. Consistent with chromosomal in situ localization of the P element insertion, deficiencies of the 49F region of the second chromosome fail to complement the behavioral effect of the latheoP1 mutation. Further complementation analyses between latheoP1 and lethal alleles, produced by excision of the latheoP1 insert or by EMS or gamma-rays, in the 49F region maps the latheo mutation to one vital complementation group. Flies heterozygous for latheoP1 and one of two EMS lethal alleles or one lethal excision allele also show the behavioral deficits, thereby demonstrating that the behavioral and lethal phenotypes co-map to the same locus (Boynton, 1992).

Histological analyses of late third instar larvae reveal the absence of all imaginal discs and an undersized CNS in lat mutants. In contrast, the gross morphology of imaginal discs and CNS appear normal in second instar mutants. During the pupal stage in Drosophila, larval structures outside of the CNS are histolyzed, and adult structures are generated from proliferating clusters of cells in imaginal discs. Hence, degeneration of imaginal discs in late third instar larvae likely produces the pupal lethality associated with these lat mutants. These morphological defects are accompanied by an apparent defect in DNA replication. Proliferating cells in normal and mutant lat larvae CNSs were examined by labeling their chromosomes with bromo deoxyuridine (BrdU) during DNA replication. In late third instar larvae (96 hr posthatching), cell proliferation in a normal CNS is maximal. Lateral portions of the brain hemispheres, where cells of the optic lobes are proliferating, show particularly high levels of BrdU incorporation. In contrast to this high level of cell proliferation occurring in normal larvae, virtually no BrdU incorporation can be seen in the CNS of homozygous lethal lat mutants. Consistent with this observation, the mutant CNS appears smaller at this late stage of larval development (Pinto, 1999).

Given the defect in neuroblast (Nb) proliferation in the CNS of pupal lethal lat larvae, the gross morphology of several anatomical regions of the adult brain in homozygous viable lat mutants was evaluated. Homozygous lat^P1 mutants (which display defective olfactory associative learning but normal sensorimotor responses) show a 20% reduction only in mushroom body neuropillar volume, while flies hemizygous for lat^P1 and a chromosomal deficiency (Df) (which display performance deficits in olfactory associative learning and in sensorimotor response) show an 80% reduction in mushroom bodies and a 20% reduction in central complex. These observations suggest a development etiology for the learning defect of adult viable lat^P1 mutants (aberrant mushroom bodies) and for the more severe performance deficits of lat^P1/Df hemizygotes (aberrant mushroom bodies and central complex (Pinto, 1999).

Morphological and functional examinations of three homozygous lethal lat genotypes were examined. Homozygous lat- animals of all three genotypes develop relatively normally through the first and second instars and exhibit coordinated locomotion similar to wild-type larvae. Thereafter, their movement and feeding behavior, though functional, becomes progressively less vigorous. Many mutant larvae grow to normal size but usually remain in the food past the normal wandering stage (5-6 days after egg laying [AEL]) and delay pupation for several days beyond the normal period. Lethality in lat- larvae and early pupae eventually results from a loss of postembryonic cell division, leading to a complete absence of CNS cell proliferation and imaginal development in late third instar (Boynton, 1993; Pinto, 1999). All three lethal lat- alleles appear to be strong protein hypomorphs or nulls, based on the near or complete absence of detectable LAT protein immunostaining at lat- NMJs in immunohistological assays (Rohrbough, 1999).

The neuromuscular morphology of lat- mutant larvae, including the stereotypic muscle and innervation patterns, appears largely normal. Mutant NMJs are present at the normal synaptic locations and exhibit morphological terminal elaborations, similar to wild-type NMJs. Presynaptic boutons at lat- NMJs are normal in size and at the light microscope level appear to possess a normal level of transmitter vesicle proteins, such as CSP. Alterations in synaptic terminal morphology have been described for other Drosophila learning and memory mutants. The number of terminal branches and synaptic boutons at lat- NMJs were examined, using anti-CSP to visualize presynaptic terminals. The terminal branching pattern at the muscle 12 NMJ, which receives multiple innervation and forms boutons of three to four subtypes, is similar to normal for mutant larvae. However, both lat- mutant strains have about 20% fewer terminal branches than do wild-type terminals, due to fewer higher order branch segments at mutant terminals. Consistent with reduced terminal branching, lat- NMJs also have about 20% fewer synaptic boutons than do wild-type NMJs at both muscle 12 and muscle 6/7. Similar but statistically insignificant decreases are observed at lat- muscle 12 and 6/7 NMJs, which show more variability in bouton number. However, parallel morphological changes are not observed at the muscle 4 NMJ, where lat- NMJs have a statistically insignificant increased bouton number compared to normal In summary, lat- mutant NMJs exhibit only mild alterations in synaptic morphology. At the more complex muscle 12 NMJ, mutant terminal complexity appears to be slightly reduced on the basis of terminal branching and bouton number. In contrast, at the simpler muscle 4 NMJ, mutant terminals have normal or slightly increased bouton numbers and terminal complexity. These morphological differences are completely insufficient to account for the observed functional alterations in transmission at mutant synapses (Rohrbough, 1999).

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date revised: 20 April 2002

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