Publications

2023

1. Light-induced trapping of endogenous proteins reveals spatiotemporal roles of microtubule and kinesin-1 in dendrite patterning of Drosophila sensory neurons

   Yineng Xu, Bei Wang, Inle Bush, Harriet AJ Saunders, Jill Wildonger, and Chun Han

   in submission, 2023

   Abs PDF

   Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.Competing Interest StatementThe authors have declared no competing interest.

2. A Neural Algorithm for Computing Bipartite Matchings

   Sanjoy Dasgupta, Yaron Meirovitch, Xingyu Zheng, Inle Bush, Jeff Lichtman, and Saket Navlakha

   in submission, 2023
3. Visualization and Measurement of Dendrite Arborization in Neurons

   Yineng Xu, Inle Bush, and Chun Han

   Neuronal Morphogenesis - Methods and Protocols, 2023

   Abs

   Animal development involves numerous molecular events, whose spatiotemporal properties largely determine the biological outcomes. Conventional methods for studying gene function lack the necessary spatiotemporal resolution for precise dissection of developmental mechanisms. Optogenetic approaches are powerful alternatives, but most existing tools rely on exogenous designer proteins that produce narrow outputs and cannot be applied to diverse or endogenous proteins. To address this limitation, we developed OptoTrap, a light-inducible protein trapping system that allows manipulation of endogenous proteins tagged with GFP or split GFP. This system turns on fast and is reversible in minutes or hours. We generated OptoTrap variants optimized for neurons and epithelial cells and demonstrate effective trapping of endogenous proteins of diverse sizes, subcellular locations, and functions. Furthermore, OptoTrap allowed us to instantly disrupt microtubules and inhibit the kinesin-1 motor in specific dendritic branches of Drosophila sensory neurons. Using OptoTrap, we obtained direct evidence that microtubules support the growth of highly dynamic dendrites. Similarly, targeted manipulation of Kinesin heavy chain revealed differential spatiotemporal requirements of kinesin-1 in the patterning of low- and high-order dendritic branches, suggesting that different cargos are needed for the growth of these branches. OptoTrap allows for precise manipulation of endogenous proteins in a spatiotemporal manner and thus holds great promise for studying developmental mechanisms in a wide range of cell types and developmental stages.Competing Interest StatementThe authors have declared no competing interest.

2020

1. Cells with loss-of-heterozygosity after exposure to ionizing radiation in Drosophila are culled by p53-dependent and p53-independent mechanisms

   Jeremy Brown, Inle Bush, Justine Bozon, and Tin Tin Su

   PLOS Genetics, Oct 2020

   Abs Bib PDF

   Loss of Heterozygosity (LOH) typically refers to a phenomenon in which diploid cells that are heterozygous for a mutant allele lose their wild type allele through mutations. LOH is implicated in oncogenesis when it affects the remaining wild type copy of a tumor suppressor. Drosophila has been a useful model to identify genes that regulate the incidence of LOH, but most of these studies use adult phenotypic markers such as multiple wing hair (mwh). Here, we describe a cell-autonomous fluorescence-based system that relies on the QF/QS transcriptional module to detect LOH, which may be used in larval, pupal and adult stages and in conjunction with the GAL4/UAS system. Using the QF/QS system, we were able to detect the induction of cells with LOH by X-rays in a dose-dependent manner in the larval wing discs, and to monitor their fate through subsequent development in pupa and adult stages. We tested the genetic requirement for changes in LOH, using both classical mutants and GAL4/UAS-mediated RNAi. Our results identify two distinct culling phases that eliminate cells with LOH, one in late larval stages and another in the pupa. The two culling phases are genetically separable, showing differential requirement for pro-apoptotic genes of the H99 locus and transcription factor Srp. A direct comparison of mwh LOH and QF/QS LOH suggests that cells with different LOH events are distinguished from each other in a p53-dependent manner and are retained to different degrees in the final adult structure. These studies reveal previously unknown mechanisms for the elimination of cells with chromosome aberrations.

   @article{10.1371/journal.pgen.1009056,
     doi = {10.1371/journal.pgen.1009056},
     author = {Brown, Jeremy and Bush, Inle and Bozon, Justine and Su, Tin Tin},
     journal = {PLOS Genetics},
     publisher = {Public Library of Science},
     title = {Cells with loss-of-heterozygosity after exposure to ionizing radiation in Drosophila are culled by p53-dependent and p53-independent mechanisms},
     year = {2020},
     month = oct,
     volume = {16},
     url = {https://doi.org/10.1371/journal.pgen.1009056},
     pages = {1-23},
     number = {10},
   }