Ira E. Clark

Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin

Parkinsons disease is the second most common neurodegenerative disorder and is characterized by the degeneration of dopaminergic neurons in the substantia nigra. Mitochondrial dysfunction has been implicated as an important trigger for Parkinsons disease-like pathogenesis because exposure to environmental mitochondrial toxins leads to Parkinsons disease-like pathology. Recently, multiple genes mediating familial forms of Parkinsons disease have been identified, including PTEN-induced kinase 1 (PINK1 ; PARK6 ) and parkin (PARK2 ), which are also associated with sporadic forms of Parkinsons disease. PINK1 encodes a putative serine/threonine kinase with a mitochondrial targeting sequence. So far, no in vivo studies have been reported for pink1 in any model system. Here we show that removal of Drosophila PINK1 homologue (CG4523; hereafter called pink1) function results in male sterility, apoptotic muscle degeneration, defects in mitochondrial morphology and increased sensitivity to multiple stresses including oxidative stress. Pink1 localizes to mitochondria, and mitochondrial cristae are fragmented in pink1 mutants. Expression of human PINK1 in the Drosophila testes restores male fertility and normal mitochondrial morphology in a portion of pink1 mutants, demonstrating functional conservation between human and Drosophila Pink1. Loss of Drosophila parkin shows phenotypes similar to loss of pink1 function. Notably, overexpression of parkin rescues the male sterility and mitochondrial morphology defects of pink1 mutants, whereas double mutants removing both pink1 and parkin function show muscle phenotypes identical to those observed in either mutant alone. These observations suggest that pink1 and parkin function, at least in part, in the same pathway, with pink1 functioning upstream of parkin. The role of the pink1–parkin pathway in regulating mitochondrial function underscores the importance of mitochondrial dysfunction as a central mechanism of Parkinsons disease pathogenesis.

Activation of the Yeast HO Gene by Release from Multiple Negative Controls

Transcription of the yeast HO gene requires five genes, SWI 1, 2, 3, 4, 5. We present evidence that some SWI products activate HO by antagonizing negative regulatory activities encoded by the SIN genes. sin- mutants (defining six genes) were identified because they express HO in the absence of particular SWI products. We argue that SWI5 activates HO by antagonizing SIN3 and that SWI4 activates HO by antagonizing SIN6. HO is expressed in sin3- daughter cells, hence we infer that the SIN3 product represses HO in wild-type daughter cells and that SWI5 and SIN3 are responsible for the cell-lineage-dependent expression of HO. HO is transcribed only when all types of repression are absent: in mother cells, where SWI5 antagonizes SIN3; in late G1, when SWI4 antagonizes SIN6; and in a or alpha cells, where a1-alpha 2 repression is absent.

nanos and pumilio Are Essential for Dendrite Morphogenesis in Drosophila Peripheral Neurons

Much attention has focused on dendritic translational regulation of neuronal signaling and plasticity. For example, long-term memory in adult Drosophila requires Pumilio (Pum), an RNA binding protein that interacts with the RNA binding protein Nanos (Nos) to form a localized translation repression complex essential for anterior-posterior body patterning in early embryogenesis. Whether dendrite morphogenesis requires similar translational regulation is unknown. Here we report that nos and pum control the elaboration of high-order dendritic branches of class III and IV, but not class I and II, dendritic arborization (da) neurons. Analogous to their function in body patterning, nos and pum require each other to control dendrite morphogenesis, a process likely to involve translational regulation of nos itself. The control of dendrite morphogenesis by Nos/Pum, however, does not require hunchback, which is essential for body patterning. Interestingly, Nos protein is localized to RNA granules in the dendrites of da neurons, raising the possibility that the Nos/Pum translation repression complex operates in dendrites. This work serves as an entry point for future studies of dendritic translational control of dendrite morphogenesis.

The Saccharomyces cerevisiae SIN3 gene, a negative regulator of HO, contains four paired amphipathic helix motifs.

The SIN3 gene (also known as SDI1) is a negative regulator of the yeast HO gene. Mutations in SIN3 suppress the requirement for the SWI5 activator for expression of the yeast HO gene and change the normal asymmetric pattern of HO expression in mother and daughter cells. Furthermore, the in vitro DNA-binding activity of several DNA-binding proteins is reduced in extracts prepared from sin3 mutants. We have cloned the SIN3 gene and determined that a haploid strain with a SIN3 gene disruption is viable. We determined the sequence of the SIN3 gene, which is predicted to encode a 175-kDa polypeptide with four paired amphipathic helix motifs. These motifs have been identified in the myc family of helix-loop-helix DNA-binding proteins and in the TPR family of regulatory proteins. The SIN3 transcript was mapped, and it was determined that the SIN3 transcript was absent in stationary-phase cells. Immunofluorescence microscopy with anti-SIN3 antibody demonstrated that SIN3 protein was present in nuclei. A comparison of restriction map and sequence data revealed that SIN3 is the same as regulatory genes UME4 and RPD1.

Loss-of-Function Analysis Suggests That Omi/HtrA2 Is Not an Essential Component of the pink1/parkin Pathway In Vivo

Recently, a mutation in the mitochondrial protease Omi/HtrA2, G399S, was found in sporadic Parkinsons disease (PD) patients, leading to the designation of Omi/HtrA2 as PD locus 13 (PARK13). G399S reportedly results in reduced Omi protease activity. In vitro studies have suggested that Omi/HtrA2 acts downstream of PINK1, mutations in which mediate recessive forms of PD. We, as well as other, have previously shown that the Drosophila homologs of the familial PD genes, PINK1 (PARK6) and PARKIN (PARK2), function in a common genetic pathway to regulate mitochondrial integrity and dynamics. Whether Omi/HtrA2 regulates mitochondrial integrity and whether it acts downstream of PINK1 in vivo remain to be explored. Here, we show that Omi/HtrA2 null mutants in Drosophila, in contrast to pink1 or parkin null mutants, do not show mitochondrial morphological defects. Extensive genetic interaction studies do not provide support for models in which Omi/HtrA2 functions in the same genetic pathway as pink1, or carries out partially redundant functions with pink1, at least with respect to regulation of mitochondrial integrity and dynamics. Furthermore, Omi/HtrA2 G399S retains significant, if not full, function of Omi/HtrA2, compared with expression of protease-compromised versions of the protein. In light of recent findings showing that G399S can be found at comparable frequencies in PD patients and healthy controls, we do not favor a hypothesis in which Omi/HtrA2 plays an essential role in PD pathogenesis, at least with respect to regulation of mitochondrial integrity in the pink1/parkin pathway.

Temporal complexity within a translational control element in the nanos mRNA

Translational control of gene expression plays a fundamental role in the early development of many organisms. In Drosophila, selective translation of nanos mRNA localized to the germ plasm at the posterior of the embryo, together with translational repression of nanos in the bulk cytoplasm, is essential for development of the anteroposterior body pattern. We show that both components to spatial control of nanos translation initiate during oogenesis and that translational repression is initially independent of Smaug, an embryonic repressor of nanos. Repression during oogenesis and embryogenesis are mediated by distinct stem loops within the nanos 3′ untranslated region; the Smaug-binding stem-loop acts strictly in the embryo, whereas a second stem-loop functions in the oocyte. Thus, independent regulatory modules with temporally distinct activities contribute to spatial regulation of nanos translation. We propose that nanos evolved to exploit two different stage-specific translational regulatory mechanisms.

“Deconstructing” Scientific Research: A Practical and Scalable Pedagogical Tool to Provide Evidence-Based Science Instruction

Focused analysis of current research projects provides an effective platform for teaching early-stage undergraduates the logic of scientific inquiry.