Traveling waves play an essential role in coordinating mitosis over large distances, but what determines the spatial origin of mitotic waves remains unclear. Here, we show that such waves initiate at pacemakers, regions which oscillate faster than their surroundings. In cell-free extracts of Xenopus laevis eggs, we find that nuclei define such pacemakers by concentrating cell cycle regulators. In computational models of diffusively coupled oscillators that account for nuclear import, nuclear positioning determines the pacemaker location. Furthermore, we find that the spatial dimensions of the oscillatory medium change the nuclear positioning and strongly influence whether a pacemaker is more likely to be at a boundary or an internal region. Finally, we confirm experimentally that increasing the system width increases the proportion of pacemakers at the boundary. Our work provides insight into how nuclei and spatial system dimensions can control local concentrations of regulators, influencing the emergent behavior of mitotic waves.
All the data generated during the study are summarized and provided in the manuscript and supporting files. Source files have been provided for Figure 1, Figure 1-Figure Supplement 3, Figure 2, Figure 5-Figure Supplement 1, Box 2, Video 1 and Video 2 in the format of microscopy videos. Additionally, representative microscopy videos of all different conditions are provided as a Zenodo dataset (http://doi.org/10.5281/zenodo.3736728). The numerical codes that were used, together with an overview table of the performed experiments, are available through GitHub (Nolet, 2020).
- Lendert Gelens
- Lendert Gelens
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Animal experimentation: This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the KU Leuven. All of the animals were handled according to approved institutional animal care and use committee (IACUC) protocols of the KU Leuven. The protocol was approved by the Committee on the Ethics of Animal Experiments of the KU Leuven (ECD permit Number: P165/2016 ).
- Stefano Di Talia, Duke University, United States
© 2020, Nolet et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Epifluorescence miniature microscopes ('miniscopes') are widely used for in vivo calcium imaging of neural population activity. Imaging data is typically collected during a behavioral task and stored for later offline analysis, but emerging techniques for online imaging can support novel closed-loop experiments in which neural population activity is decoded in real time to trigger neurostimulation or sensory feedback. To achieve short feedback latencies, online imaging systems must be optimally designed to maximize computational speed and efficiency while minimizing errors in population decoding. Here we introduce DeCalciOn, an open-source device for real-time imaging and population decoding of in vivo calcium signals that is hardware compatible with all miniscopes that use the UCLA Data Acquisition (DAQ) interface. DeCalciOn performs online motion stabilization, neural enhancement, calcium trace extraction, and decoding of up to 1024 traces per frame at latencies of <50 ms after fluorescence photons arrive at the miniscope image sensor. We show that DeCalciOn can accurately decode the position of rats (n=12) running on a linear track from calcium fluorescence in the hippocampal CA1 layer, and can categorically classify behaviors performed by rats (n=2) during an instrumental task from calcium fluorescence in orbitofrontal cortex (OFC). DeCalciOn achieves high decoding accuracy at short latencies using innovations such as field-programmable gate array (FPGA) hardware for real time image processing and contour-free methods to efficiently extract calcium traces from sensor images. In summary, our system offers an affordable plug-and-play solution for real-time calcium imaging experiments in behaving animals.
High-throughput sequencing of adaptive immune receptor repertoires is a valuable tool for receiving insights in adaptive immunity studies. Several powerful TCR/BCR repertoire reconstruction and analysis methods have been developed in the past decade. However, detecting and correcting the discrepancy between real and experimentally observed lymphocyte clone frequencies is still challenging. Here we discovered a hallmark anomaly in the ratio between read count and clone count-based frequencies of non-functional clonotypes in multiplex PCR-based immune repertoires. Calculating this anomaly, we formulated a quantitative measure of V- and J-genes frequency bias driven by multiplex PCR during library preparation called Over Amplification Rate (OAR). Based on the OAR concept, we developed an original software for multiplex PCR-specific bias evaluation and correction named iROAR: Immune Repertoire Over Amplification Removal (https://github.com/smiranast/iROAR). The iROAR algorithm was successfully tested on previously published TCR repertoires obtained using both 5' RACE (Rapid Amplification of cDNA Ends)-based and multiplex PCR-based approaches and compared with a biological spike-in-based method for PCR bias evaluation. The developed approach can increase the accuracy and consistency of repertoires reconstructed by different methods making them more applicable for comparative analysis.