UBAP2L ensures homeostasis of nuclear pore complexes at the intact nuclear envelope.

Assembly of macromolecular complexes at correct cellular sites is crucial for cell function. Nuclear pore complexes (NPCs) are large cylindrical assemblies with eightfold rotational symmetry, built through hierarchical binding of nucleoporins (Nups) forming distinct subcomplexes. Here, we uncover a role of ubiquitin-associated protein 2-like (UBAP2L) in the assembly and stability of properly organized and functional NPCs at the intact nuclear envelope (NE) in human cells. UBAP2L localizes to the nuclear pores and facilitates the formation of the Y-complex, an essential scaffold component of the NPC, and its localization to the NE. UBAP2L promotes the interaction of the Y-complex with POM121 and Nup153, the critical upstream factors in a well-defined sequential order of Nups assembly onto NE during interphase. Timely localization of the cytoplasmic Nup transport factor fragile X-related protein 1 (FXR1) to the NE and its interaction with the Y-complex are likewise dependent on UBAP2L. Thus, this NPC biogenesis mechanism integrates the cytoplasmic and the nuclear NPC assembly signals and ensures efficient nuclear transport, adaptation to nutrient stress, and cellular proliferative capacity, highlighting the importance of NPC homeostasis at the intact NE.

Cell cycle-dependent binding between Cyclin B1 and Cdk1 revealed by time-resolved fluorescence correlation spectroscopy.

Measuring the dynamics with which the regulatory complexes assemble and disassemble is a crucial barrier to our understanding of how the cell cycle is controlled that until now has been difficult to address. This considerable gap in our understanding is due to the difficulty of reconciling biochemical assays with single cell-based techniques, but recent advances in microscopy and gene editing techniques now enable the measurement of the kinetics of protein-protein interaction in living cells. Here, we apply fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy to study the dynamics of the cell cycle machinery, beginning with Cyclin B1 and its binding to its partner kinase Cdk1 that together form the major mitotic kinase. Although Cyclin B1 and Cdk1 are known to bind with high affinity, our results reveal that in living cells there is a pool of Cyclin B1 that is not bound to Cdk1. Furthermore, we provide evidence that the affinity of Cyclin B1 for Cdk1 increases during the cell cycle, indicating that the assembly of the complex is a regulated step. Our work lays the groundwork for studying the kinetics of protein complex assembly and disassembly during the cell cycle in living cells.

SARS-CoV-2 infection and cancer: Evidence for and against a role of SARS-CoV-2 in cancer onset.

Despite huge efforts towards understanding the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis, little is known about the long-term consequences of the disease. Here, we critically review existing literature about oncogenesis as a potential long-term effect of SARS-CoV-2 infection. Like other viral infections, SARS-CoV-2 may promote cancer onset by inhibiting tumor suppressor genes. We conclude that, although unlikely, such hypothesis cannot be excluded a priori and we delineate an experimental approach to address it. Also see the video abstract here: https://youtu.be/TBUTDSLR7vY.

UBAP2L Forms Distinct Cores that Act in Nucleating Stress Granules Upstream of G3BP1.

Stress granules (SGs) are membraneless organelles that form in eukaryotic cells after stress exposure [1] (reviewed in [2-4]). Following translation inhibition, polysome disassembly releases 48S preinitiation complexes (PICs). mRNA, PICs, and other proteins coalesce in SG cores [1, 5-7]. SG cores recruit a dynamic shell, whose properties are dominated by weak interactions between proteins and RNAs [8-10]. The structure and assembly of SGs and how different components contribute to their formation are not fully understood. Using super-resolution and expansion microscopy, we find that the SG component UBAP2L [11, 12] and the core protein G3BP1 [5, 11-13] occupy different domains inside SGs. UBAP2L displays typical properties of a core protein, indicating that cores of different compositions coexist inside the same granule. Consistent with a role as a core protein, UBAP2L is required for SG assembly in several stress conditions. Our reverse genetic and cell biology experiments suggest that UBAP2L forms granules independent of G3BP1 and 2 but does not interfere with stress-induced translational inhibition. We propose a model in which UBAP2L is an essential SG nucleator that acts upstream of G3BP1 and 2 and facilitates G3BP1 core formation and SG assembly and growth.

Cell polarity-dependent centrosome separation in the C. elegans embryo.

In animal cells, faithful chromosome segregation depends on the assembly of a bipolar spindle driven by the timely separation of the two centrosomes. Here we took advantage of the highly stereotypical cell divisions in Caenorhabditis elegans embryos to identify new regulators of centrosome separation. We find that at the two-cell stage, the somatic AB cell initiates centrosome separation later than the germline P1 cell. This difference is strongly exacerbated by the depletion of the kinesin-13 KLP-7/MCAK, resulting in incomplete centrosome separation at NEBD in AB but not P1. Our genetic and cell biology data indicate that this phenotype depends on cell polarity via the enrichment in AB of the mitotic kinase PLK-1, which itself limits the cortical localization of the dynein-binding NuMA orthologue LIN-5. We postulate that the timely separation of centrosomes is regulated in a cell type-dependent manner.

The Elephant in the Room: The Role of Microtubules in Cancer.

Microtubules are the backbone of all eukaryotic cells cytoskeleton. Their dynamic behaviour constitutes the basis for many biological processes such as cellular motility, cytoplasmic transport and cell division. Some the most effective chemotherapeutics, such as the taxanes, are microtubule interfering drugs. Moreover, many studies suggest that microtubule dynamics are altered in cancer cell divisions and linked to chromosomal instability, aneuploidy and development of drug resistances. The elephant in the room, however, is that despite all these evidences, the exact role of microtubules in malignancies remains elusive, partially due to the lack of clear genetic alterations linking microtubules to cancer. This review will discuss the molecular mechanisms that might alter microtubule dynamics in cancer cells, the pro and cons of the different theories linking these alterations to cancer progression, and the possible directions to address future key questions.

Mitotic entry: The interplay between Cdk1, Plk1 and Bora.

Polo-like kinase 1 (Plk1) is an important mitotic kinase that is crucial for entry into mitosis after recovery from DNA damage-induced cell cycle arrest. Plk1 activation is promoted by the conserved protein Bora (SPAT-1 in C. elegans), which stimulates the phosphorylation of a conserved residue in the activation loop by the Aurora A kinase. In a recent article published in Cell Reports, we show that the master mitotic kinase Cdk1 contributes to Plk1 activation through SPAT-1/Bora phosphorylation. We identified 3 conserved Sp/Tp residues that are located in the N-terminal, most conserved part, of SPAT-1/Bora. Phosphorylation of these sites by Cdk1 is essential for Plk1 function in mitotic entry in C. elegans embryos and during DNA damage checkpoint recovery in mammalian cells. Here, using an untargeted Förster Resonance Energy Transfer (FRET) biosensor to monitor Plk1 activation, we provide additional experimental evidence supporting the importance of these phosphorylation sites for Plk1 activation and subsequent mitotic entry after DNA damage. We also briefly discuss the mechanism of Plk1 activation and the potential role of Bora phosphorylation by Cdk1 in this process. As Plk1 is overexpressed in cancer cells and this correlates with poor prognosis, understanding how Bora contributes to Plk1 activation is paramount for the development of innovative therapeutical approaches.

BORA-dependent PLK1 regulation: A new weapon for cancer therapy?

The mitotic kinase polo like kinase 1 (PLK1) is overexpressed in many cancers and its inhibition slows down proliferation and increases apoptosis in cancer cell lines. Understanding how PLK1 is activated is therefore crucial for the development of novel PLK1 inhibitors with anticancer properties. We recently identified a conserved regulatory loop leading to PLK1 activation that involves cyclin-dependent kinase 1 (CDK1).

Cdk1 Phosphorylates SPAT-1/Bora to Promote Plk1 Activation in C. elegans and Human Cells.

The conserved Bora protein is a Plk1 activator, essential for checkpoint recovery after DNA damage in human cells. Here, we show that Bora interacts with Cyclin B and is phosphorylated by Cyclin B/Cdk1 at several sites. The first 225 amino acids of Bora, which contain two Cyclin binding sites and three conserved phosphorylated residues, are sufficient to promote Plk1 phosphorylation by Aurora A in vitro. Mutating the Cyclin binding sites or the three conserved phosphorylation sites abrogates the ability of the N terminus of Bora to promote Plk1 activation. In human cells, Bora-carrying mutations of the three conserved phosphorylation sites cannot sustain mitotic entry after DNA damage. In C. elegans embryos, mutation of the three conserved phosphorylation sites in SPAT-1, the Bora ortholog, results in a severe mitotic entry delay. Our results reveal a crucial and conserved role of phosphorylation of the N terminus of Bora for Plk1 activation and mitotic entry.

PIM2 kinase is induced by cisplatin in ovarian cancer cells and limits drug efficacy.

Platinum-based chemotherapy is widely used to treat various cancers, but many patients ultimately relapse due to drug resistance. We employed phosphoproteomic analysis and functional assays of the response of SK-OV-3 ovarian cancer cells to cisplatin as a strategy to identify kinases as candidate druggable targets to sensitize cells to platinum. A SILAC-based approach combined with TiO2-based phosphopeptide enrichment allowed the direct identification of ERK1/2, p90RSK, and ERBB2 as kinases whose phosphorylation is regulated by cisplatin. Bioinformatic analysis revealed enrichment in linear phosphorylation motifs predicted to be targets of p38MAPK, CDK2, and PIM2. All three PIM kinases were found expressed in a panel of 10 ovarian cancer cell lines, with the oncogenic PIM2 being the most commonly induced by cisplatin. Targeting PIM2 kinase by either biochemical inhibitors or RNA interference impaired cell growth, decreased cisplatin-triggered BAD phosphorylation, and sensitized ovarian cancer cells to drug-induced apoptosis. Overexpression of PIM2 triggered anchorage-independent growth and resulted in increased BAD phosphorylation and cell resistance to DNA damaging agents. The data show that the PIM2 kinase plays a role in the response of ovarian cancer cells to platinum drugs and suggest that PIM inhibitors may find clinical application as an adjunct to platinum-based therapies.