Spatial and temporal resolution of mORC4 fluorescent variants reveals structural requirements for achieving higher order self-association and pronuclei entry

Abstract

The Origin Replication Complex (ORC), which is a multi-subunit protein complex composed of six proteins ORC1-6, is essential for initiating licensing at DNA replication origins. We have previously reported that ORC4 has an alternative function wherein it forms a cage surrounding the extruded chromatin in female meiosis and is required for polar body extrusion (PBE). As this is a highly unexpected finding for protein that normally binds DNA, we tested whether ORC4 can actually form larger, higher order structures, which would be necessary to form a cage-like structure. We generated two fluorescent constructs of mouse ORC4, mORC4-EGFP and mORC4-FlAsH, to examine its spatial dynamics during oocyte activation in live cells. We show that both constructs were primarily monomeric throughout the embryo but self-association into larger units was detected with both probes. However, mORC4-FlAsH clearly showed higher order selfassociation and unique spatial distribution while mORC4-EGFP failed to form large structures during Anaphase II. Interestingly, both variants were found in the pronuclei suggesting that its role in DNA licensing is still functional. Our results with both constructs support the prediction that ORC4 can form higher order structures in the cytoplasm, suggesting that it is possible to form a cage-like structure. The finding that FlAsH labeled ORC4 formed demonstrably larger higher order structures than ORC4-GFP suggests that ORC4 oligomerization is sensitive to the bulky addition of GFP at its carboxy terminus.

ArfGAP1 inhibits mTORC1 lysosomal localization and activation

“…Fluorescence lifetime imaging microscopy (FLIM) and raster imaging correlation spectroscopy (RICS) data were collected using an Alba fluorescence correlation spectrometer (ISS, Champaign, IL), connected to a Nikon TE2000-U inverted microscope (Nikon, Melville, NY) with x-y scanning mirror and a PlanApo VC 60 × 1.2 NA water objective as previously described (Goo et al, 2018;Nguyen et al, 2019). Two-photon excitation was provided by a Chameleon Ultra (Coherent, Santa Clara, CA) tuned to 920 nm for FLIM data collection, while 1,000 nm was used for RICS experiments to obtain optimal excitation of both fluorophores (laser power > 1 mM at plane of excitation).…”

AKAP13 couples GPCR signaling to mTORC1 inhibition

“…Fluorescence Lifetime Imaging Microscopy (FLIM) data were collected using an Alba fluorescence correlation spectrometer (ISS, Champaign, IL), connected to a Nikon TE2000-U inverted microscope (Nikon, Melville, NY) with x-y scanning mirror and a PlanApo VC 60 x 1.2 NA water objective as previously described [49,50]. Two-photon excitation was provided by a Chameleon Ultra (Coherent, Santa Clara, CA) tuned to 900 nm for FLIM data collection to minimize the amount of direct YFP excitation.…”

Fluctuation Imaging of LRRK2 Reveals that the G2019S Mutation Alters Spatial and Membrane Dynamics

“…Fluorescence lifetime imaging microscopy (FLIM) measurements were obtained with an ISS A320 FastFLIM box with photomultiplier detector joined to the Ti:Sapphire laser that created 80 fs pulses at a repetition rate of 80 MHz (H7422P-40, Hamamatsu, Hamamatsu City, Japan) as previously described [63]. The fluorescence signal was filtered from the excitation light using a 520 nm bandpass filter (FF01-520/35; Semrock Rochester, NY, USA) that was secured in front of the detector.…”