Loss of Drosophila melanogaster p21-activated kinase 3 suppresses defects in synapse structure and function caused by spastin mutations

Microtubules are dynamic structures that must elongate, disassemble, and be cleaved into smaller pieces for proper neuronal development and function. The AAA ATPase Spastin severs microtubules along their lengths and is thought to regulate the balance between long, stable filaments and shorter fragments that seed extension or are transported. In both Drosophila and humans, loss of Spastin function results in reduction of synaptic connections and disabling motor defects. To gain insight into how spastin is regulated, we screened the Drosophila melanogaster genome for deletions that modify a spastin overexpression phenotype, eye size reduction. One suppressor region deleted p21-activated kinase 3 (pak3), which encodes a member of the Pak family of actin-regulatory enzymes, but whose in vivo function is unknown. We show that pak3 mutants have only mild synaptic defects at the larval neuromuscular junction, but exhibit a potent genetic interaction with spastin mutations. Aberrant bouton morphology, microtubule distribution, and synaptic transmission caused by spastin loss of function are all restored to wild type when pak3 is simultaneously reduced. Neuronal overexpression of pak3 induces actin-rich thin projections, suggesting that it functions in vivo to promote filopodia during presynaptic terminal arborization. pak3 therefore regulates synapse development in vivo, and when mutated, suppresses the synaptic defects that result from spastin loss.     

Brain patterning defects caused by mutations of the twin of eyeless gene in Drosophila melanogaster

The Drosophila Pax6 genes, eyeless (ey) and twin of eyeless (toy), are expressed in both eyes and the brain. Previous studies have demonstrated that ey plays important roles in axonal outgrowth and differentiation of mushroom bodies (MBs), which are centers for associative learning and memory in flies. However, the functional significance of toy in brain development is poorly understood. Here, we describe the expression patterns of TOY, and show that TOY expression partially overlaps with EY expression in the embryonic, larval and adult brains. Mutations of toy perturb brain neuromere formation in the embryonic stages, and result in severe deformation of the MB lobes in pharate adult brains. Moreover, we also analyzed toy functions by gain-of-function experiments, and show that overexpression of toy results in degeneration of MB lobes. Thus, our results demonstrate the importance of toy in embryonic brain patterning as well as in post-embryonic development of the major brain structures such as MBs.     

The contribution of DNA and chromosome repair deficiencies to the radiosensitivity of ataxia-telangiectasia.

Cells derived from individuals with ataxia-telangiectasia (AT) are more sensitive to ionizing radiation and radiomimetic drugs, as evidenced by decreased survival and increased chromosome aberrations at mitosis when compared with normal cell lines. Our previous studies showed that, despite similar initial levels of DNA double-strand breaks (DSBs), AT cells express higher initial chromosome damage than do normal cells as demonstrated by the technique of premature chromosome condensation. However, this finding accounted for only a portion of the increased sensitivity (T. K. Pandita and W. N. Hittelman, Radiat. Res. 130, 94-103, 1992). The purpose of the study reported here was to examine the contribution of DNA and chromosome repair to the radiosensitivity of AT cells. Exponentially growing AT and normal lymphoblastoid cells were fractionated into cell cycle phase-enriched populations by centrifugal elutriation, and their DNA and chromosome repair characteristics were evaluated by DNA neutral filter elution (for DNA DSBs) and by premature chromosome condensation, respectively. AT cells exhibited a reduced fast-repair component in both G1- and G2-phase cells, as observed at the level of both DNA DSBs and the chromosome; however, S-phase cells showed nearly normal DNA DSB repair. The findings that AT cells exhibit an increased level of chromosome damage and a deficiency in the fast component (but not the slow component) of repair suggest that chromatin organization might play a major role in the observed sensitivity of AT cells. When survival was plotted as a function of the residual amount of chromosome damage in G1- and G2- phase cells after 90 min of repair, the curves for normal and AT cells approached each other but did not overlap. These results suggest that, although higher initial levels of chromosome damage and reduced chromosome repair capability can explain much of the radiosensitivity of AT cells, other differences in AT cells must also contribute to their sensitivity phenotype.     

Progressive loss of DNA sequences from terminal chromosome deficiencies in Drosophila melanogaster.

Terminal deficiencies at the tip of the X chromosome can be induced at a high frequency (0.2-0.3%) by irradiating Drosophila females carrying a homozygous mutator (mu-2) with low doses of X-rays. These terminal deficiencies are unstable, since over a period of 3 1/2 years DNA sequences were lost from their distal ends at a rate of 75 bp per generation, presumably due to the absence of a complete wild-type telomeric structure. Breakpoints of these deletions in the 5' upstream regulatory region of the yellow gene, giving rise to a mosaic cuticle pigmentation pattern typical of the y2 type, were used to define the location of tissue-specific cis-acting regulatory elements that are required for body, wing or bristle pigmentation.     

Loss-of-function mutations in the Arabidopsis heterotrimeric G-protein alpha subunit enhance the developmental defects of brassinosteroid signaling and biosynthesis mutants

Loss-of-function alleles of the sole heterotrimeric G-protein alpha subunit in Arabidopsis, GPA1, display defects in cell proliferation throughout plant development. Previous studies indicated that GPA1 is involved in brassinosteroid (BR) response. Here we provide genetic evidence that loss-of-function mutations in GPA1, gpa1-2 and gpa1-4, enhance the developmental defects of bri1-5, a weak allele of a BR receptor mutant, and det2-1, a BR-deficient mutant in Arabidopsis. gpa1-2 bri1-5 and gpa1-4 det2-1 double mutants had shorter hypocotyls, shorter roots and fewer lateral roots, and displayed more severe dwarfism than bri1-5 and det2-1 single mutants, respectively. By using the Arabidopsis hypocotyl as a model system where the parameters of cell division and cell elongation can be simultaneously measured, we found that gpa1 can specifically enhance the cell division defects of bri1-5 and det2-1 mutants. Similarly, gpa1 specifically enhances cell division defects in the primary roots of bri1-5 and det2-1 mutants. Furthermore, an additive effect on cell division between gpa1 and bri1-5 or det2-1 mutations was observed in the hypocotyls, whereas a synergistic effect was observed in the roots. Taken together, these results provided the first genetic evidence that G-protein- and BR-mediated pathways may be converged to modulate cell proliferation in a cell/tissue-specific manner.     

Tumor suppressor p53 dependent recruitment of nucleotide excision repair factors XPC and TFIIH to DNA damage

Functional tumor suppressor p53 is mainly required for efficient global genomic repair (GGR), a subpathway of nucleotide excisions repair (NER). In this study, the regulatory effect of p53, on the spaciotemporal recruitment of XPC and TFIIH to DNA damage sites, was investigated in repair-proficient and -deficient human cells in situ. Photoproducts were induced through micropore UV irradiation of discrete subnuclear areas of intact cells and the specific lesions, as well as recruited repair factors, were detected by immunofluorescent intensity and density of the damaged DNA subnuclear spots (SNS). Both cyclobutane pyrimidine dimers (CPD) and 6-4 photoproducts (6-4PP) were visualized in situ at SNS within irradiated nuclear foci. The in situ repair kinetics revealed that p53-WT normal fibroblasts are proficient for the repair of both CPD and 6-4PP, whereas, p53-Null Li-Fraumeni syndrome (LFS) fibroblasts fail to efficiently repair CPD but not 6-4PP. Colocalization experiments of the NER factors showed that in normal human cells, XPC and TFIIH are rapidly and efficiently recruited to DNA damage within SNS. By contrast, recruitment of both XPC and TFIIH to DNA damage in SNS occurred much less efficiently in p53-Null or p53-compromised cells. The total cellular levels of XPC and XPB were similar in both p53-WT and -Null cells and remained unchanged up to 24h following UV irradiation. The results also showed that dispersal of recruited XPC and TFIIH from DNA damage SNS occurs within a short period after DNA damage. Such dispersal requires functional XPA, XPF and XPG proteins. Taken together, the results demonstrated that p53 plays a pronounced role in the damage recognition and subsequent assembly of repair machinery during GGR and the recruitment of XPC and TFIIH to CPD is p53-dependent. Most likely mechanism of this p53 action is through its downstream effector protein, DDB2.     

DNA repair defects and other (mus)takes in Drosophila melanogaster.

Preservation of the structural integrity of DNA in any organism is crucial to its health and survival. Such preservation is achieved by an extraordinary cellular arsenal of damage surveillance and repair functions, many of which are now being defined at the gene and protein levels. Mutants hypersensitive to the killing effects of DNA-damaging agents have been instrumental in helping to identify DNA repair-related genes and to elucidate repair mechanisms. In Drosophila melanogaster, such strains are generally referred to as mutagen-sensitive (mus) mutants and currently define more than 30 genetic loci. Whereas most mus mutants have been recovered on the basis of hypersensitivity to the monofunctional alkylating agent methyl methanesulfonate, they nevertheless constitute a phenotypically diverse group, with many mutants having effects beyond mutagen sensitivity. These phenotypes include meiotic dysfunctions, somatic chromosome instabilities, chromatin abnormalities, and cell proliferation defects. Within the last few years numerous mus and other DNA repair-related genes of Drosophila have been molecularly cloned, providing new insights into the functions of these genes. This article outlines strategies for isolating mus mutations and reviews recent advances in the Drosophila DNA repair field, emphasizing mutant analysis and gene cloning.     

Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent. Received: 4 October 1999 / Accepted: 10 January 2000