An intimate link between centrosome function and neurogenesis is revealed by the identification of many genes with centrosome-associated functions mutated in microcephaly disorders. Consistent with the major role of the centrosome in mitosis, mutations in these centrosome-related microcephaly (CRM) genes are thought to affect neurogenesis by depleting the pool of neural progenitor cells, primarily through apoptosis as a consequence of mitotic failure, or premature differentiation as a consequence of cell cycle delay and randomization of spindle orientation. However, as suggested by the wide range of microcephaly phenotypes and the multifunctional nature of many CRM proteins, this picture of CRM gene function is incomplete. Our lab is exploring several CRM genes that appear to function through multiple pathways that contribute to microcephaly, including regulation of cell cycle signaling, actin cytoskeleton, and Hippo pathway proteins, as well as functions in post-mitotic neurons and glia. As these examples are likely just the tip of the iceberg, further exploration of the roles for microcephaly-related genes are certain to reveal additional unforeseen functions important for neurodevelopment. As part of a large screen for neurodevelopmental defects, we homed in on TRAIP, an MPD gene encoding an E3 ubiquitin ligase known to regulate DDR and apoptosis. Interestingly, the Drosophila melanogaster ortholog nopo (no poles) was named for its loss-of-function phenotype of acentrosomal spindles, suggesting that this DDR gene might function at centrosomes. We performed yeast two-hybrid analysis to reveal extensive interactions between Nopo and several core centrosome proteins, including Sas4, Ana2 and Plk4. Thus, we have established another potential link between a DDR gene and the centrosome. To explore the role of nopo in neurodevelopment, we analyzed both larval and adult brains from nopo mutant animals. We discovered that loss of nopo leads to defects in the mushroom body (MB), a brain region critical for memory formation. Nopo mutant and MB lobes are thin, fused, and are often missing. Axon guidance is also abnormal in nopo mutants as we find many misguided MB axons. Our studies of mutants for bendless, which encodes an E2 conjugating enzyme previously shown to interact with Nopo, reveal that 100% of MBs are fused but otherwise normal, suggesting that Nopo functions with Bendless and other E2 conjugating enzymes to ensure proper brain development and prevent MPD. We are currently focused on identifying the ubiquitination substrates of Nopo required for MB development; candidates include Nopo direct binding partners at the centrosome. Together, this work reveals an exciting new link between the DDR and the centrosome bridged by nopo. Furthermore, we have established D. melanogaster as a new model for understanding the role of TRAIP in neurodevelopment. Understanding how events at the molecular and cellular scales contribute to tissue form and function is key to uncovering mechanisms driving animal development, physiology and disease. Elucidating these mechanisms has been enhanced through the study of model organisms and the use of sophisticated genetic, biochemical and imaging tools. Here we highlight how non-destructive imaging of Drosophila melanogaster at high resolution using micro computed tomography (-CT) allows for visualization of development and tissue morphogenesis at an unprecedented level of detail. To demonstrate the power of -CT, we characterized the developing brain from larval to adult stages, including the stages of pupation that have been understudied by current light microscopy methods due to the delicate nature of metamorphosing tissue. We uncover a series of novel morphogenetic changes in brain volume as pupation proceeds during normal development. We then probed a series of microcephaly genes to determine when brain development might go awry. For example, we show that mutations inspc105r (the fly ortholog of the kinetochore protein KNL1) leads to massively reduced head structures due to complete failure in pupal brain development. To demonstrate how -CT can be incorporated into existing experimental workflows and combined with traditional light microscopy and molecular genetic approaches, we characterized two models of human microcephaly by evaluating mutations in the Abnormal Spindle (asp) and WD Repeat Domain 62 (wdr62). Loss of asp leads to significant reduction in brain size, coupled with severe morphology defects of the visual processing center of the fly (retina, lamina, and optic lobes), which can be enhanced through the loss of wdr62. Structure function analysis of Asp transgenes revealed that the N-terminal of Asp (AspMF) is sufficient to rescue both the volume and morphology defects of the asp mutant. Interestingly, live imaging of mitotic divisions within developing larval brains from asp mutant animals revealed that Asp is critical for maintaining centrosome-spindle pole cohesion and spindle pole focusing of microtubule minus ends in neuroblasts, whose loss severely disrupts the ability of these stem cells to divide correctly to generate neurons and glia. Surprisingly, identical cell division defects were observed in AspMF rescue brains, suggesting that Asp contributes to proper brain development through novel mechanisms independent of its role as a mitotic regulator. Our work demonstrates that -CT is a versatile and accessible tool that complements standard imaging techniques, capable of uncovering novel biological mechanisms that have remained undocumented due to limitations of current methods.
| O'Neill, Ryan S; Schoborg, Todd A; Rusan, Nasser M (2018) Same but different: pleiotropy in centrosome-related microcephaly. Mol Biol Cell 29:241-246 |
| Lerit, Dorothy A; Shebelut, Conrad W; Lawlor, Kristen J et al. (2017) Germ Cell-less Promotes Centrosome Segregation to Induce Germ Cell Formation. Cell Rep 18:831-839 |
| Varadarajan, Ramya; Hammer, John A; Rusan, Nasser M (2017) A centrosomal scaffold shows some self-control. J Biol Chem 292:20410-20411 |
| Galletta, Brian J; Jacobs, Katherine C; Fagerstrom, Carey J et al. (2016) Asterless is required for centriole length control and sperm development. J Cell Biol 213:435-50 |
| Schoborg, Todd A; Rusan, Nasser M (2016) Taking Centrioles to the Elimination Round. Dev Cell 38:10-2 |
| Lerit, Dorothy A; Jordan, Holly A; Poulton, John S et al. (2015) Interphase centrosome organization by the PLP-Cnn scaffold is required for centrosome function. J Cell Biol 210:79-97 |
| Schoborg, Todd; Zajac, Allison L; Fagerstrom, Carey J et al. (2015) An Asp-CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells. J Cell Biol 211:987-98 |
| Pronobis, Mira I; Rusan, Nasser M; Peifer, Mark (2015) A novel GSK3-regulated APC:Axin interaction regulates Wnt signaling by driving a catalytic cycle of efficient ?catenin destruction. Elife 4:e08022 |
| Smyth, Jeremy T; Schoborg, Todd A; Bergman, Zane J et al. (2015) Proper symmetric and asymmetric endoplasmic reticulum partitioning requires astral microtubules. Open Biol 5: |
| Lerit, Dorothy A; Plevock, Karen M; Rusan, Nasser M (2014) Live imaging of Drosophila larval neuroblasts. J Vis Exp : |
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