TRIM37 employs peptide motif recognition and substrate-dependent oligomerization to prevent ectopic spindle pole assembly.

Tightly controlled duplication of centrosomes, the major microtubule-organizing centers of animal cells, ensures bipolarity of the mitotic spindle and accurate chromosome segregation. The RBCC (RING-B-box-coiled coil) ubiquitin ligase TRIM37, whose loss is associated with elevated chromosome missegregation and the tumor-prone developmental human disorder Mulibrey nanism, prevents the formation of ectopic spindle poles that assemble around structured condensates containing the centrosomal protein centrobin. Here, we show that TRIM37's TRAF domain, unique in the extended TRIM family, engages peptide motifs in centrobin to suppress condensate formation.

53BP1-mediated activation of the tumor suppressor p53.

In recent years, the role of 53BP1 as a cell cycle regulator has come into the spotlight. 53BP1 is best understood for its role in controlling DNA double-strand break repair. However, 53BP1 was initially discovered as an interaction partner of the tumor suppressor p53, which proved to be independent of DNA repair.

Control of cell proliferation by memories of mitosis.

Mitotic duration is tightly constrained, and extended mitosis is characteristic of problematic cells prone to chromosome missegregation and genomic instability. We show here that mitotic extension leads to the formation of p53-binding protein 1 (53BP1)-ubiquitin-specific protease 28 (USP28)-p53 protein complexes that are transmitted to, and stably retained by, daughter cells. Complexes assembled through a Polo-like kinase 1-dependent mechanism during extended mitosis and elicited a p53 response in G that prevented the proliferation of the progeny of cells that experienced an approximately threefold extended mitosis or successive less extended mitoses.

TRIM37 prevents formation of condensate-organized ectopic spindle poles to ensure mitotic fidelity.

Centrosomes are composed of a centriolar core surrounded by pericentriolar material that nucleates microtubules. The ubiquitin ligase TRIM37 localizes to centrosomes, but its centrosomal roles are not yet defined. We show that TRIM37 does not control centriole duplication, structure, or the ability of centrioles to form cilia but instead prevents assembly of an ectopic centrobin-scaffolded structured condensate that forms by budding off of centrosomes.

Centriole-independent mitotic spindle assembly relies on the PCNT-CDK5RAP2 pericentriolar matrix.

Centrosomes, composed of centrioles that recruit a pericentriolar material (PCM) matrix assembled from PCNT and CDK5RAP2, catalyze mitotic spindle assembly. Here, we inhibit centriole formation and/or remove PCNT-CDK5RAP2 in RPE1 cells to address their relative contributions to spindle formation. While CDK5RAP2 and PCNT are normally dispensable for spindle formation, they become essential when centrioles are absent.

TRIM37 controls cancer-specific vulnerability to PLK4 inhibition.

Centrosomes catalyse the formation of microtubules needed to assemble the mitotic spindle apparatus. Centrosomes themselves duplicate once per cell cycle, in a process that is controlled by the serine/threonine protein kinase PLK4 (refs. ).

Centromere Dysfunction Compromises Mitotic Spindle Pole Integrity.

Centromeres and centrosomes are crucial mitotic players. Centromeres are unique chromosomal sites characterized by the presence of the histone H3-variant centromere protein A (CENP-A) [1]. CENP-A recruits the majority of centromere components, collectively named the constitutive centromere associated network (CCAN) [2].

NDF, a nucleosome-destabilizing factor that facilitates transcription through nucleosomes.

Our understanding of transcription by RNA polymerase II (Pol II) is limited by our knowledge of the factors that mediate this critically important process. Here we describe the identification of NDF, a nucleosome-destabilizing factor that facilitates Pol II transcription in chromatin. NDF has a PWWP motif, interacts with nucleosomes near the dyad, destabilizes nucleosomes in an ATP-independent manner, and facilitates transcription by Pol II through nucleosomes in a purified and defined transcription system as well as in cell nuclei.

The septin-associated kinase Gin4 recruits Gps1 to the site of cell division.

Cell cycle-dependent morphogenesis of unicellular organisms depends on the spatiotemporal control of cell polarity. Rho GTPases are the major players that guide cells through structural reorganizations such as cytokinesis (Rho1 dependent) and polarity establishment (Cdc42 dependent). In budding yeast, the protein Gps1 plays a pivotal role in both processes.

53BP1 and USP28 mediate p53 activation and G1 arrest after centrosome loss or extended mitotic duration.

In normal human cells, centrosome loss induced by centrinone-a specific centrosome duplication inhibitor-leads to irreversible, p53-dependent G1 arrest by an unknown mechanism. A genome-wide CRISPR/Cas9 screen for centrinone resistance identified genes encoding the p53-binding protein 53BP1, the deubiquitinase USP28, and the ubiquitin ligase TRIM37. Deletion of TP53BP1, USP28, or TRIM37 prevented p53 elevation in response to centrosome loss but did not affect cytokinesis failure-induced arrest or p53 elevation after doxorubicin-induced DNA damage.

Actomyosin ring driven cytokinesis in budding yeast.

Cytokinesis is the final process in the cell cycle that physically divides one cell into two. In budding yeast, cytokinesis is driven by a contractile actomyosin ring (AMR) and the simultaneous formation of a primary septum, which serves as template for cell wall deposition. AMR assembly, constriction, primary septum formation and cell wall deposition are successive processes and tightly coupled to cell cycle progression to ensure the correct distribution of genetic material and cell organelles among the two rising cells prior to cell division.

Detection of Phosphorylation Status of Cytokinetic Components.

Yeast cells can be easily cultured, synchronized, and genetically modified making them a convenient model system to study molecular mechanisms underlying cytokinesis. Here, we describe simple methods that allow the analysis of the phosphorylation profile of cytokinetic proteins, both in vivo and in vitro, using standard laboratory equipment. In addition, we compare the ability of three different, standard, and optimized acrylamide gel conditions to separate phosphorylated forms, using the protein Inn1 as an example.

Visualization of Cytokinesis Events in Budding Yeast by Transmission Electron Microscopy.

In yeast cells, cytokinesis is accompanied by morphological changes due to cell wall growth during furrow ingression and abscission. The characteristics of the growing cell wall can be used as an indicator for the function of the contractile actomyosin ring, the Rho-GTPases Rho1 and Cdc42 and/or other factors that drive cytokinesis. The ultrastructural information of the cell wall can be easily acquired by transmission electron microscopy, which makes this technique an invaluable tool to analyze cell division in yeast cells.

A memory system of negative polarity cues prevents replicative aging.

Cdc42 is a highly conserved master regulator of cell polarity. Here, we investigated the mechanism by which yeast cells never re-establish polarity at cortical sites (cytokinesis remnants [CRMs]) that have previously supported Cdc42-mediated growth as a paradigm to mechanistically understand how Cdc42-inhibitory polarity cues are established. We revealed a two-step mechanism of loading the Cdc42 antagonist Nba1 into CRMs to mark these compartments as refractory for a second round of Cdc42 activation.

Lre1 directly inhibits the NDR/Lats kinase Cbk1 at the cell division site in a phosphorylation-dependent manner.

Background: The nuclear Dbf2 related (NDR) family of protein kinases play important roles in cell-cycle regulation, apoptosis, cell morphogenesis, and development in a variety of organisms. In budding yeast, the NDR kinase complex composed of Cbk1 and its regulatory subunit, Mob2, have an established role in the control of cell separation/abscission that follows cytokinesis. Whereas the activators of Cbk1-Mob2 have been more extensively described, the mechanisms that restrict or inhibit Cbk1-Mob2 catalytic activity remain largely unknown.

A safeguard mechanism regulates Rho GTPases to coordinate cytokinesis with the establishment of cell polarity.

The spatiotemporal control of cell polarity is crucial for the development of multicellular organisms and for reliable polarity switches during cell cycle progression in unicellular systems. A tight control of cell polarity is especially important in haploid budding yeast, where the new polarity site (bud site) is established next to the cell division site after cell separation. How cells coordinate the temporal establishment of two adjacent polarity sites remains elusive.

Dual function of the NDR-kinase Dbf2 in the regulation of the F-BAR protein Hof1 during cytokinesis.

The conserved NDR-kinase Dbf2 plays a critical role in cytokinesis in budding yeast. Among its cytokinesis-related substrates is the F-BAR protein Hof1. Hof1 colocalizes at the cell division site with the septin complex and, as mitotic exit progresses, moves to the actomyosin ring (AMR).

Cdc14-dependent dephosphorylation of Inn1 contributes to Inn1-Cyk3 complex formation.

In Saccharomyces cerevisiae the Cdc14 phosphatase plays a well-established role in reverting phosphorylation events on substrates of the mitotic cyclin-dependent kinase (M-Cdk1), thereby promoting mitotic exit and downregulation of M-Cdk1 activity. Cdc14 localizes at the site of cell cleavage after M-Cdk1 inactivation, suggesting that Cdc14 may perform a crucial, yet ill-defined, role during cytokinesis. Here, we identified Inn1, as a novel direct substrate of both M-Cdk1 and Cdc14.

The power of MEN in cytokinesis.

In budding yeast, the mitotic exit network (MEN) is a signaling pathway best known for its role in driving cells out of mitosis through activation of the conserved phosphatase Cdc14. However, work over the past few years show that MEN components and Cdc14 also have a direct role in promoting cytokinesis by acting upon components of the contractile actomyosin ring and cell separation machineries. In this review, we discuss the current view on the role of MEN kinases and Cdc14 in cytokinesis and comment on the cytokinesis-related function of MEN and Cdc14 orthologs in higher eukaryotes.

Phosphorylation-dependent regulation of the F-BAR protein Hof1 during cytokinesis.

Spatial and timely coordination of cytokinesis is crucial for the maintenance of organelle inheritance and genome integrity. The mitotic exit network (MEN) pathway controls both the timely initiation of mitotic exit and cytokinesis in budding yeast. Here we identified the conserved F-BAR protein Hof1 as a substrate of the MEN kinase complex Dbf2-Mob1 during cytokinesis.

Targeted localization of Inn1, Cyk3 and Chs2 by the mitotic-exit network regulates cytokinesis in budding yeast.

The mitotic-exit network (MEN) is a signaling pathway that is essential for the coordination of mitotic exit and cytokinesis. Whereas the role of the MEN in mitotic exit is well established, the molecular mechanisms by which MEN components regulate cytokinesis remain poorly understood. Here, we show that the MEN controls components involved in septum formation, including Inn1, Cyk3 and Chs2.