Sethi, S. | bioRxiv Science

bioRxiv2017

Postmitotic Nuclear Pore Assembly Proceeds By Radial Dilation Of Small ER Membrane Openings

The nuclear envelope has to be reformed after mitosis to create viable daughter cells with closed nuclei. How membrane sealing of DNA and assembly of nuclear pore complexes (NPCs) are achieved and coordinated is poorly understood. Here, we reconstructed nuclear membrane topology and structure of assembling NPCs in a correlative three dimensional electron microscopy time-course of dividing human cells. Our quantitative ultrastructural analysis shows that nuclear membranes form from highly fenestrated ER sheets, whose shrinking holes are stabilized and then dilated into NPCs during inner ring complex assembly, forming thousands of transport channels within minutes. This mechanism is fundamentally different from interphase NPC assembly and explains how mitotic cells can rapidly establish a closed nuclear compartment while making it transport-competent at the same time.

cell biology↗

bioRxiv2017

A Versatile Genetic Tool For Post-Translational Control Of Gene Expression With A Small Molecule In Drosophila melanogaster

Several techniques have been developed in Drosophila to control gene expression temporally. While some of these techniques are incompatible with existing GAL4 lines, others suffer from side effects on physiology or behavior. Here, we describe a method of post-translational temporal control of gene expression which is compatible with the current library of transgenic reagents. We adopted a strategy to regulate protein degradation by fusing a protein of interest to a destabilizing domain (DD) derived from the Escherichia coli dihydrofolate reductase (ecDHFR). Trimethoprim (TMP), a stabilizing small molecule, binds to DD and blocks degradation of the chimeric protein. With a GFP-DD reporter, we show that this system is effective across different tissues and developmental stages in the fly. Notably, feeding flies with TMP can increase the expression level of GFP-DD up to 34 times in a dosage-dependent and reversible manner without altering the lifespan or behavior of the animal. To broaden the utility of our method, we engineered GAL80-DD flies that can be crossed to the available GAL4 lines to control the temporal pattern of gene expression with TMP. We also developed an inducible recombinase, FLP-DD, for high-efficiency sparse labeling and intersectional lineage analysis. Finally, we demonstrated the utility of the DD system in manipulating neuronal activity of sensory neurons. In summary, we have developed a system to control in vivo gene expression levels with negligible background, large dynamic range, and in a reversible manner, all by feeding a small molecule to Drosophila melanogaster.

genetics↗