Imaging data for: Midbody formation triggers asymmetric fate specification in neural stem cells

LaFoya, Bryce 1 ; Penkert, Rhiannon2; Prehoda, Kenneth2

Published Aug 03, 2026 on Dryad. https://doi.org/10.5061/dryad.t4b8gtjhf

Abstract

Fate determinants are segregated during asymmetric cell division to generate distinct sibling cell fates, but determinants must not be deployed until fate can be specified asymmetrically. Determinants could be deployed after division, once sibling cell cytoplasms are separated, but potentially long after mitotic exit, when the cell state is uniquely plastic. Here we show that the midbody prevents fate determinant diffusion across the cytokinetic pore, allowing asymmetric fate specification to begin before cell division completes. Fate determinants sequestered at the membrane during mitosis are released immediately following nuclear division via the cell cycle phosphatase String. Our results identify the midbody as a key facilitator of asymmetric cell division that allows fate determinants be deployed before division, when cell state can be readily influenced. These data may be reused for image analysis, quantitative reanalysis, or methodological development. Users must cite both this dataset and the associated publication in any use, presentation, or derivative work. Users are encouraged to contact the dataset authors for clarification, guidance, or collaboration.

Dataset DOI: 10.5061/dryad.t4b8gtjhf

Description of the data and file structure

The deposited dataset consists of time-lapse microscopy image files in .tif format generated from live imaging experiments of Drosophila neural stem cells. These files can be opened and analyzed using standard bioimaging software such as Fiji (ImageJ) or Imaris.

File names correspond to specific figures or panels in the manuscript to facilitate direct mapping between the raw data and the published results. The temporal resolution of the imaging (time between frames) is included in the file name.

These data may be reused for image analysis, quantitative reanalysis, or methodological development. Users must cite both this dataset and the associated publication in any use, presentation, or derivative work. Users are encouraged to contact the dataset authors for clarification, guidance, or collaboration.

abbreviations: NSC = Neural Stem Cell

Files and variables

File: Fig1C_D_Prospero_and_Membrane.zip

Description: Prospero dynamics during the late stages of NSC asymmetric division. Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. N = 10 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig1E_Membrane_and_Microtubules.zip

Description: Membrane dynamics during the late stages of NSC asymmetric division. Super resolution videos of NSCs expressing microtubules marked with mCherry and the membrane marker UAS-PLCδ-PH-GFP “membrane”. N = 5 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig1F_G_Deadpan_and_Membrane.zip

Description: Deadpan dynamics during the late stages of NSC asymmetric division. Super resolution videos of NSCs expressing Deadpan-GFP “Deadpan” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. To improve image quality, Gaussian blur was applied to the Deadpan channel. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig2A_D_His2a_and_Membrane.zip

Description: Chromatin dynamics during late NSC division. Super resolution videos of NSCs expressing the chromosome marker His2a-RFP “His2a” and the membrane marker UAS-PLCδ-PH-GFP “membrane”. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig2B_D_Klaroid_and_Membrane.zip

Description: Nuclear dynamics during late NSC division. Super resolution videos of NSCs expressing the nuclear envelope marker Klaroid-GFP “Klaroid” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig2C_D_NLS-DsRed_and_Membrane.zip

Description: Nuclear import dynamics during late NSC division. Super resolution videos of NSCs expressing the nuclear marker UAS-NLS-DsRed “NLS-DsRed” and the membrane marker UAS-PLCδ-PH-GFP “membrane”. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig3A_B_Brat_and_Membrane.zip

Description: Brat dynamics during late NSC division. Super resolution videos of NSCs expressing Brain Tumor (Brat)-GFP “Brat” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. N = 3 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig3C_D_Prospero_and_Membrane_treated_with_Ivermectin.zip

Description: Prospero dynamics when nuclear import is delayed. Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” treated with the inhibitor of nuclear import, ivermectin. N = 5 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig3E_F_FRAP_experiment_GFP_and_Membrane.zip

Description: Fluorescence recovery after photobleaching (FRAP) analysis of cytoplasmic GFP in dividing NSCs. Super resolution videos of NSCs expressing cytosolic GFP “GFP” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. The FRAP laser was focused near the apical pole of the nascent NSC cytoplasm. N = 5 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig4A_Jupiter_and_Membrane.zip

Description: Microtubule dynamics during late NSC division. Super resolution videos of NSCs expressing the microtubule marker Jupiter-GFP “microtubules” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. N = 3 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig4B_Fascetto_and_Membrane.zip

Description: Fascetto dynamics during late NSC division. Super resolution videos of NSCs expressing Fascetto-GFP “Fascetto” and the membrane marker UAS-PLCδ-PH-mCherry “membrane”. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig4C_Actin_and_Membrane_and_treated_with_Latrunculin_A.zip

Description: Membrane and actin dynamics in NSC with the actin cytoskeleton depolymerized by LatA before midbody formation. Super resolution videos of NSCs expressing the F-actin marker UAS-GMA-GFP “F-actin” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the F-actin inhibitor, Latrunculin A (LatA). The filename indicates the frame in which LatA was added immediately prior. Time resolution is indicated in the filenames.

File: Fig4D_Actin_and_Membrane_and_treated_with_Latrunculin_A.zip

File: Fig4E_G_Brat_and_Membrane_treated_with_Latrunculin_A.zip

Description: Brat dynamics in NSCs with the actin cytoskeleton depolymerized by LatA after midbody formation. Super resolution videos of NSCs expressing Brain Tumor (Brat)-GFP “Brat” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the F-actin inhibitor, Latrunculin A (LatA). The drug was added just after midbody formation. N = 3 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig4F_G_H_I_Brat_and_Membrane_treated_with_Latrunculin_A_and_Colcemid.zip

Description: Brat dynamics in NSC with midbody disrupted by LatA + Colcemid treatment after midbody formation. Super resolution videos of NSCs expressing Brain Tumor (Brat)-GFP “Brat” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the F-actin inhibitor, Latrunculin A (LatA), and the microtubule inhibitor, Colcemid. The drug cocktail was added just after midbody formation. N = 3 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5A_C_Fascetto_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Fascetto dynamics in NSCs with Aurora B inhibited after midbody formation. Super resolution videos of NSCs expressing Fascetto-GFP “Fascetto” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5B_C_Fascetto_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Fascetto dynamics in NSCs with Aurora B inhibited before midbody formation. Super resolution videos of NSCs expressing Fascetto-GFP “Fascetto” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5D_NLS-DsRed_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Nuclear import dynamics in NSCs with Aurora B inhibited before midbody formation. Super resolution videos of NSCs expressing the nuclear marker UAS-NLS-DsRed “NLS-DsRed” and the membrane marker UAS-PLCδ-PH-GFP “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 3 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5E_G_H_Prospero_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Prospero dynamics in NSCs with Aurora B inhibited after midbody formation. Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5F_G_H_Prospero_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Prospero dynamics in NSCs with Aurora B inhibited before midbody formation. Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5I_K_Brat_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Brat dynamics in NSCs with Aurora B inhibited after midbody formation. Super resolution videos of NSCs expressing Brain Tumor (Brat)-GFP “Brat” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig5J_K_Brat_and_Membrane_treated_with_AuroraB_inhibitor.zip

Description: Brat dynamics in NSCs with Aurora B inhibited before midbody formation. Super resolution videos of NSCs expressing Brain Tumor (Brat)-GFP “Brat” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the Aurora B inhibitor, Binucleine 2. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig6A_C_Prospero_and_Membrane_treated_with_Cdc25_(String)_inhibitor.zip

Description: Prospero dynamics with String inhibited. Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of the String (Cdc25 in mammals) inhibitor, NSC 663284. The drug was added just prior to midbody formation. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig6B_C_Prospero_and_Membrane_treated_with_DMSO.zip

Description: Prospero dynamics in NSCs treated with DMSO (vehicle control). Super resolution videos of NSCs expressing Prospero-GFP “Prospero” and the membrane marker UAS-PLCδ-PH-mCherry “membrane” in the presence of 2% DMSO. DMSO was added just prior to midbody formation. N = 6 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig6D_F_Prospero_and_String_RNAi.zip

Description: Prospero dynamics in NSCs expressing String RNAi. Videos of NSCs expressing Prospero-GFP “Prospero” and RNAi directed again String. N = 13 dividing NSCs are shown. Time resolution is indicated in the filenames.

File: Fig6E_F_Prospero_(wild-type_control).zip

Description: Prospero dynamics in NSCs (wild-type control). Videos of NSCs expressing Prospero-GFP “Prospero”. Time relative to midbody formation is indicated. N = 7 dividing NSCs are shown. Time resolution is indicated in the filenames.

If this ZIP file does not open correctly using the default program on your computer, please use 7-Zip or another ZIP extraction program to access the files. After extraction, the files can be accessed normally.

Live Imaging

To obtain brain explants, third instar Drosophila larvae were dissected in Schneider’s Insect Media (SIM) and the central nervous system was isolated. Next, larval brain explants were mounted on sterile poly-D-lysine coated 35mm glass bottom dish (ibidi Cat#81156) containing modified minimal hemolymph-like solution (HL3.1). Then, brain explants were imaged using a Nikon Eclipse Ti-2 Yokogawa CSU-W1 SoRa spinning disk microscope equipped dual Photometrics Prime BSI sCMOS cameras using a 60x H₂O objective. 488 nm light was used to illuminate GFP tagged proteins and 561 nm light was used to illuminate DsRed and mCherry tagged proteins. Super resolution imaging was achieved by using SoRa (super resolution through optical photon reassignment) optics (45). NSCs were identified by their large size, location in the central nervous system, and the use of NSC specific tissue driver lines. Time lapse imaging of midbody dynamics was achieved by refocusing the imaging plane on the medial plane of the cleavage furrow, and subsequently the midbody, along the apical-basal axis just before capturing each frame. Pharmacological inhibition of Aurora B was performed using 15 μM Binucleine 2 solubilized in DMSO. Pharmacological depolymerization of microtubules was performed using 1 mM Colcemid solubilized in DMSO. Pharmacological depolymerization of F-actin was performed using 50 μM Latrunculin A (LatA) solubilized in DMSO. Nuclear import was delayed using 57 μM Ivermectin solubilized in DMSO. Pharmacological inhibition of String was performed using 750 μM of the Cdc25 inhibitor NSC 663284 solubilized in DMSO.

FRAP (Fluorescence Recovery After Photobleaching)

Images were acquired every 500 milliseconds in n = 5 dividing NSCs expressing cytoplasmic GFP and UAS-PLCδ-PH-mCherry to label the plasma membrane. A brief 100-millisecond pulse of 405 nm light was used to photobleach a region of the nascent NSC cytoplasm near the apical pole, while the nascent sibling cells were connected by an intercellular bridge during late cytokinesis. GFP fluorescence was then recorded over time at multiple cytoplasmic locations to monitor its redistribution following photobleaching. Quantification details are provided in the corresponding section.

Image Processing and Analysis

Imaging data was processed using ImageJ (FIJI package). For some movies, the bleach correction tool was used to correct for photobleaching. To reduce noise in Deadpan images, Gaussian blur was applied. Image deconvolution was applied to both the String RNAi and the corresponding wild-type videos (Fig 6D,E in associated manuscript).