In metazoa, nuclear pore complexes (NPCs) are assembled from constituent nucleoporins by two distinct mechanisms: in the re-forming nuclear envelope at the end of mitosis and into the intact nuclear envelope during interphase. Here, we show that the nucleoporin Nup153 is required for NPC assembly during interphase but not during mitotic exit. It functions in interphasic NPC formation by binding directly to the inner nuclear membrane via an N-terminal amphipathic helix. This binding facilitates the recruitment of the Nup107-160 complex, a crucial structural component of the NPC, to assembly sites. Our work further suggests that the nuclear transport receptor transportin and the small GTPase Ran regulate the interaction of Nup153 with the membrane and, in this way, direct pore complex assembly to the nuclear envelope during interphase.

Chromatin undergoes extensive structural changes during the cell cycle. Upon mitotic entry, metazoan chromatin undergoes tremendous condensation, creating mitotic chromosomes with 50-fold greater compaction relative to interphase chromosomes. At the end of mitosis, chromosomes reestablish functional interphase chromatin competent for replication and transcription through a decondensation process that is cytologically well described. However, the underlying molecular events and factors remain unidentified. We describe a cell-free system that recapitulates chromatin decondensation based on purified mitotic chromatin and Xenopus egg extracts. Using biochemical fractionation, we identify RuvB-like ATPases as chromatin decondensation factors and demonstrate that their ATPase activity is essential for decondensation. Our results show that decompaction of metaphase chromosomes is not merely an inactivation of known chromatin condensation factors but rather an active process requiring specific molecular machinery. Our cell-free system provides an important tool for further molecular characterization of chromatin decondensation and its coordination with concomitant processes.

Live-cell imaging has become state of the art to accurately identify the nature of mitotic and cell cycle defects. Low- and high-throughput microscopy setups have yield huge data amounts of cells recorded in different experimental and pathological conditions. Tailored semi-automated and automated image analysis approaches allow the analysis of high-content screening data sets, saving time and avoiding bias. However, they were mostly designed for very specific experimental setups, which restricts their flexibility and usability. The general need for dedicated experiment-specific user-annotated training sets and experiment-specific user-defined segmentation parameters remains a major bottleneck for fully automating the analysis process. In this work we present LiveCellMiner, a highly flexible open-source software tool to automatically extract, analyze and visualize both aggregated and time-resolved image features with potential biological relevance. The software tool allows analysis across high-content data sets obtained in different platforms, in a quantitative and unbiased manner. As proof of principle application, we analyze here the dynamic chromatin and tubulin cytoskeleton features in human cells passing through mitosis highlighting the versatile and flexible potential of this tool set.

The mitotic spindle, essential for segregating the sister chromatids into the two evolving daughter cells, is composed of highly dynamic cytoskeletal filaments, the microtubules. The dynamics of microtubules are regulated by numerous microtubule associated proteins. We identify here Developmentally regulated GTP binding protein 1 (DRG1) as a microtubule binding protein with diverse microtubule-associated functions. In vitro, DRG1 can diffuse on microtubules, promote their polymerization, drive microtubule formation into bundles, and stabilize microtubules. HeLa cells with reduced DRG1 levels show delayed progression from prophase to anaphase because spindle formation is slowed down. To perform its microtubule-associated functions, DRG1, although being a GTPase, does not require GTP hydrolysis. However, all domains are required as truncated versions show none of the mentioned activities besides microtubule binding.

RECQL4, which is mutated in the Rothmund–Thomson syndrome characterized by premature aging and cancer susceptibility, is a microtubule-associated protein required for mitotic chromosome alignment.

The metazoan nucleus breaks down and reassembles during each cell division. Upon mitotic exit, the successful reestablishment of an interphase nucleus requires the coordinated reorganization of chromatin and formation of a functional nuclear envelope. Here, we report that the histone demethylase LSD1 (also known as KDM1A) plays a crucial role in nuclear assembly at the end of mitosis. Downregulation of LSD1 in cells extends telophase and impairs nuclear pore complex assembly. In vitro, LSD1 demethylase activity is required for the recruitment of MEL28 (also known as ELYS and AHCTF1) and nuclear envelope precursor vesicles to chromatin, crucial steps in nuclear reassembly. Accordingly, the formation of a closed nuclear envelope and nuclear pore complex assembly are impaired upon depletion of LSD1 or inhibition of its activity. Our results identify histone demethylation by LSD1 as a new regulatory mechanism linking the chromatin state and nuclear envelope formation at the end of mitosis.

The eukaryotic nucleus remodels extensively during mitosis. Upon mitotic entry, the nuclear envelope breaks down and chromosomes condense into rod-shaped bodies, which are captured by the spindle apparatus and segregated during anaphase. Through telophase, chromosomes decondense and the nuclear envelope reassembles, leading to a functional interphase nucleus. While the molecular processes occurring in early mitosis are intensively investigated, our knowledge about molecular mechanisms of nuclear reassembly is rather limited. Using cell free and cellular assays, we identify the histone variant H2A.Z and its chaperone VPS72/YL1 as important factors for reassembly of a functional nucleus after mitosis. Live-cell imaging shows that siRNA-mediated downregulation of VPS72 extends the telophase in HeLa cells. In vitro, depletion of VPS72 or H2A.Z results in malformed and nonfunctional nuclei. VPS72 is part of two chromatin-remodeling complexes, SRCAP and EP400. Dissecting the mechanism of nuclear reformation using cell-free assays, we, however, show that VPS72 functions outside of the SRCAP and EP400 remodeling complexes to deposit H2A.Z, which in turn is crucial for formation of a functional nucleus.

Cell division, or mitosis, guarantees the accurate inheritance of the genomic information kept in the cell nucleus. Malfunctions in this process cause a threat to the health and life of the organism, including cancer and other manifold diseases. It is therefore crucial to study in detail the cell-cycle in general and mitosis in particular. Consequently, a large number of manual and semi-automated time-lapse microscopy image analyses of mitosis have been carried out in recent years. In this paper, we propose a method for automatic detection of cell-cycle stages using a recurrent neural network (RNN). An end-to-end model with center-cell focus tracker loss, and classification loss is trained. The evaluation was conducted on two time-series datasets, with 6-stages and 3-stages of cell splitting labeled. The frame-to-frame accuracy was calculated and precision, recall, and F1 Score were measured for each cell-cycle stage. We also visualized the learned feature space. Image reconstruction from the center-cell focus module was performed which shows that the network was able to focus on the center cell and classify it simultaneously. Our experiments validate the superior performance of the proposed network compared to a classifier baseline.