ALFI: Cell cycle phenotype annotations of label-free time-lapse imaging data from cultured human cells.

Detecting and tracking multiple moving objects in a video is a challenging task. For living cells, the task becomes even more arduous as cells change their morphology over time, can partially overlap, and mitosis leads to new cells. Differently from fluorescence microscopy, label-free techniques can be easily applied to almost all cell lines, reducing sample preparation complexity and phototoxicity. In this study, we present ALFI, a dataset of images and annotations for label-free microscopy, made publicly available to the scientific community, that notably extends the current panorama of expertly labeled data for detection and tracking of cultured living nontransformed and cancer human cells. It consists of 29 time-lapse image sequences from HeLa, U2OS, and hTERT RPE-1 cells under different experimental conditions, acquired by differential interference contrast microscopy, for a total of 237.9 hours. It contains various annotations (pixel-wise segmentation masks, object-wise bounding boxes, tracking information). The dataset is useful for testing and comparing methods for identifying interphase and mitotic events and reconstructing their lineage, and for discriminating different cellular phenotypes.

The Aurora-A/TPX2 Axis Directs Spindle Orientation in Adherent Human Cells by Regulating NuMA and Microtubule Stability.

Mitotic spindle orientation is a crucial process that defines the axis of cell division, contributing to daughter cell positioning and fate, and hence to tissue morphogenesis and homeostasis.1,2 The trimeric NuMA/LGN/Gαi complex, the major determinant of spindle orientation, exerts pulling forces on the spindle poles by anchoring astral microtubules (MTs) and dynein motors to the cell cortex.3,4 Mitotic kinases contribute to correct spindle orientation by regulating nuclear mitotic apparatus protein (NuMA) localization,5-7 among which the Aurora-A centrosomal kinase regulates NuMA targeting to the cell cortex in metaphase.8,9 Aurora-A and its activator targeting protein for Xklp2 (TPX2) are frequently overexpressed in cancer,10-12 raising the question as to whether spindle orientation is among the processes downstream the Aurora-A/TPX2 signaling axis altered under pathological conditions. Here, we investigated the role of TPX2 in the Aurora-A- and NuMA-dependent spindle orientation. We show that, in cultured adherent human cells, the interaction with TPX2 is required for Aurora-A to exert this function. We also show that Aurora-A, TPX2, and NuMA are part of a complex at spindle MTs, where TPX2 acts as a platform for Aurora-A regulation of NuMA. Interestingly, excess TPX2 does not influence NuMA localization but induces a "super-alignment" of the spindle axis with respect to the substrate, although an excess of Aurora-A induces spindle misorientation. These opposite effects are both linked to altered MT stability. Overall, our results highlight the importance of TPX2 for spindle orientation and suggest that spindle orientation is differentially sensitive to unbalanced levels of Aurora-A, TPX2, or the Aurora-A/TPX2 complex.

Excess TPX2 Interferes with Microtubule Disassembly and Nuclei Reformation at Mitotic Exit.

The microtubule-associated protein TPX2 is a key mitotic regulator that contributes through distinct pathways to spindle assembly. A well-characterised function of TPX2 is the activation, stabilisation and spindle localisation of the Aurora-A kinase. High levels of TPX2 are reported in tumours and the effects of its overexpression have been investigated in cancer cell lines, while little is known in non-transformed cells. Here we studied TPX2 overexpression in hTERT RPE-1 cells, using either the full length TPX2 or a truncated form unable to bind Aurora-A, to identify effects that are dependent-or independent-on its interaction with the kinase. We observe significant defects in mitotic spindle assembly and progression through mitosis that are more severe when overexpressed TPX2 is able to interact with Aurora-A. Furthermore, we describe a peculiar, and Aurora-A-interaction-independent, phenotype in telophase cells, with aberrantly stable microtubules interfering with nuclear reconstitution and the assembly of a continuous lamin B1 network, resulting in daughter cells displaying doughnut-shaped nuclei. Our results using non-transformed cells thus reveal a previously uncharacterised consequence of abnormally high TPX2 levels on the correct microtubule cytoskeleton remodelling and G1 nuclei reformation, at the mitosis-to-interphase transition.

Aurora-A inactivation causes mitotic spindle pole fragmentation by unbalancing microtubule-generated forces.

BACKGROUND: Aurora-A is an oncogenic kinase playing well-documented roles in mitotic spindle organisation. We previously found that Aurora-A inactivation yields the formation of spindles with fragmented poles that can drive chromosome mis-segregation. Here we have addressed the mechanism through which Aurora-A activity regulates the structure and cohesion of spindle poles. RESULTS: We inactivated Aurora-A in human U2OS osteosarcoma cells either by RNA-interference-mediated silencing or treating cultures with the specific inhibitor MLN8237. We show that mitotic spindle pole fragmentation induced by Aurora-A inactivation is associated with microtubule hyperstabilisation. Silencing of the microtubule-stabilising factor ch-TOG prevents spindle pole fragmentation caused by inactivation of Aurora-A alone and concomitantly reduces the hyperstabilisation of microtubules. Furthermore, decreasing pole-directed spindle forces by inhibition of the Eg5 kinesin, or by destabilisation of microtubule-kinetochore attachments, also prevents pole fragmentation in Aurora-A-inactivated mitoses. CONCLUSIONS: Our findings indicate that microtubule-generated forces are imbalanced in Aurora-A-defective cells and exert abnormal pressure at the level of spindle poles, ultimately causing their fragmentation. This study therefore highlights a novel role of the Aurora-A kinase in regulating the balance between microtubule forces during bipolar spindle assembly.

Control of Aurora-A stability through interaction with TPX2.

The Aurora-A kinase has well-established roles in spindle assembly and function and is frequently overexpressed in tumours. Its abundance is cell cycle regulated, with a peak in G2 and M phases, followed by regulated proteolysis at the end of mitosis. The microtubule-binding protein TPX2 plays a major role in regulating the activity and localisation of Aurora-A in mitotic cells. Here, we report a novel regulatory role of TPX2 and show that it protects Aurora-A from degradation both in interphase and in mitosis in human cells. Specifically, Aurora-A levels decrease in G2 and prometaphase cells silenced for TPX2, whereas degradation of Aurora-A is impaired in telophase cells overexpressing the Aurora-A-binding region of TPX2. The decrease in Aurora-A in TPX2-silenced prometaphases requires proteasome activity and the Cdh1 activator of the APC/C ubiquitin ligase. Reintroducing either full-length TPX2, or the Aurora-A-binding region of TPX2, but not a truncated TPX2 mutant lacking the Aurora-A-interaction domain, restores Aurora-A levels in TPX2-silenced prometaphases. The control by TPX2 of Aurora-A stability is independent of its ability to activate Aurora-A and to localise it to the spindle. These results highlight a novel regulatory level impinging on Aurora-A and provide further evidence for the central role of TPX2 in regulation of Aurora-A.