End Of Mitosis Research Articles

Cep57 and Cep57L1 maintain centriole engagement in interphase to ensure centriole duplication cycle.

Centrioles duplicate in interphase only once per cell cycle. Newly formed centrioles remain associated with their mother centrioles. The two centrioles disengage at the end of mitosis, which licenses centriole duplication in the next cell cycle. Therefore, timely centriole disengagement is critical for the proper centriole duplication cycle. However, the mechanisms underlying centriole engagement during interphase are poorly understood. Here, we show that Cep57 and Cep57L1 cooperatively maintain centriole engagement during interphase. Codepletion of Cep57 and Cep57L1 induces precocious centriole disengagement in interphase without compromising cell cycle progression. The disengaged daughter centrioles convert into centrosomes during interphase in a Plk1-dependent manner. Furthermore, the centrioles reduplicate and the centriole number increases, which results in chromosome segregation errors. Overall, these findings demonstrate that the maintenance of centriole engagement by Cep57 and Cep57L1 during interphase is crucial for the tight control of centriole copy number and thus for proper chromosome segregation.

The primordial germ line is refractory to perturbations of actomyosin regulator function in C. elegans L1 larvae.

Cytokinesis, the separation of daughter cells at the end of mitosis, relies on the coordinated activity of several regulators of actomyosin assembly and contractility (Green et al. 2012). These include the small GTPase RhoA (RHO-1) and its guanine-nucleotide exchange factor Ect2 (ECT-2), the scaffold protein Anillin (ANI-1), the non-muscle myosin II (NMY-2), the formin CYK-1 and the centralspindlin complex components ZEN-4 and CYK-4. These regulators were also shown to be required for maintenance of C. elegans germline syncytial organization by stabilizing intercellular bridges in embryos and adults (Amini et al. 2014; Goupil et al. 2017; Green et al. 2011; Priti et al. 2018; Zhou et al. 2013). We recently demonstrated that many of these regulators are enriched at intercellular bridges in the small rachis (proto-rachis) of L1-stage larvae (Bauer et al. 2021). We sought to assess whether these contractility regulators are functionally required for stability of intercellular bridges and maintenance of the primordial germ line syncytial architecture in L1-stage C. elegans animals. Here we report that temperature-sensitive alleles, RNAi-mediated depletion and latrunculin A treatment are largely ineffective to perturb actomyosin function in the L1-stage primordial germ line.

The Role of Nucleoporin Elys in Nuclear Pore Complex Assembly and Regulation of Genome Architecture

For a long time, the nuclear lamina was thought to be the sole scaffold for the attachment of chromosomes to the nuclear envelope (NE) in metazoans. However, accumulating evidence indicates that nuclear pore complexes (NPCs) comprised of nucleoporins (Nups) participate in this process as well. One of the Nups, Elys, initiates NPC reassembly at the end of mitosis. Elys directly binds the decondensing chromatin and interacts with the Nup107–160 subcomplex of NPCs, thus serving as a seeding point for the subsequent recruitment of other NPC subcomplexes and connecting chromatin with the re-forming NE. Recent studies also uncovered the important functions of Elys during interphase where it interacts with chromatin and affects its compactness. Therefore, Elys seems to be one of the key Nups regulating chromatin organization. This review summarizes the current state of our knowledge about the participation of Elys in the post-mitotic NPC reassembly as well as the role that Elys and other Nups play in the maintenance of genome architecture.

Rapid degradation of GRASP55 and GRASP65 reveals their immediate impact on the Golgi structure.

GRASP55 and GRASP65 have been implicated in stacking of Golgi cisternae and lateral linking of stacks within the Golgi ribbon. However, RNAi or gene knockout approaches to dissect their respective roles have often resulted in conflicting conclusions. Here, we gene-edited GRASP55 and/or GRASP65 with a degron tag in human fibroblasts, allowing for induced rapid degradation by the proteasome. We show that acute depletion of either GRASP55 or GRASP65 does not affect the Golgi ribbon, while chronic degradation of GRASP55 disrupts lateral connectivity of the ribbon. Acute double depletion of both GRASPs coincides with the loss of the vesicle tethering proteins GM130, p115, and Golgin-45 from the Golgi and compromises ribbon linking. Furthermore, GRASP55 and/or GRASP65 is not required for maintaining stacks or de novo assembly of stacked cisternae at the end of mitosis. These results demonstrate that both GRASPs are dispensable for Golgi stacking but are involved in maintaining the integrity of the Golgi ribbon together with GM130 and Golgin-45.

Closed mitosis requires local disassembly of the nuclear envelope.

At the end of mitosis, eukaryotic cells must segregate the two copies of their replicated genome into two new nuclear compartments1. They do this either by first dismantling and later reassembling the nuclear envelope in an 'open mitosis' or by reshaping an intact nucleus and then dividing it into two in a 'closed mitosis'2,3. Mitosis has been studied in a wide variety of eukaryotes for more than a century4, but how the double membrane of the nuclear envelope is split into two at the end of a closed mitosis without compromising the impermeability of the nuclear compartment remains unknown5. Here, using the fission yeast Schizosaccharomyces pombe (a classical model for closed mitosis5), genetics, live-cell imaging and electron tomography, we show that nuclear fission is achieved via local disassembly of nuclear pores within the narrow bridge that links segregating daughter nuclei. In doing so, we identify the protein Les1, which is localized to the inner nuclear envelope and restricts the process of local nuclear envelope breakdown to the bridge midzone to prevent the leakage of material from daughter nuclei. The mechanism of local nuclear envelope breakdown in a closed mitosis therefore closely mirrors nuclear envelope breakdown in open mitosis3, revealing an unexpectedly high conservation of nuclear remodelling mechanisms across diverse eukaryotes.

Cell Biology: An Open Solution for Closed Mitosis

At the end of mitosis, cells must remodel their nuclear envelope to produce two identical daughter nuclei. Two new studies using Schizosaccharomyces pombe provide insight into how compartmentalized nuclear pore complex disassembly allows cells that undergo closed mitosis to achieve nuclear division.

Importin binding mediates the intramolecular regulation of anillin during cytokinesis.

Cytokinesis occurs by the ingression of an actomyosin ring that cleaves a cell into two daughters. This process is tightly controlled to avoid aneuploidy, and we previously showed that active Ran coordinates ring positioning with chromatin. Active Ran is high around chromatin, and forms an inverse gradient to cargo-bound importins. We found that the ring component anillin contains a nuclear localization signal (NLS) that binds to importin and is required for its function during cytokinesis. Here we reveal the mechanism whereby importin binding favors a conformation required for anillin’s recruitment to the equatorial cortex. Active RhoA binds to the RhoA-binding domain causing an increase in accessibility of the nearby C2 domain containing the NLS. Importin binding subsequently stabilizes a conformation that favors interactions for cortical recruitment. In addition to revealing a novel mechanism for the importin-mediated regulation of a cortical protein, we also show how importin binding positively regulates protein function.

Cytokinesis: An Anillin-RhoGEF Module Sets the Stage for Septin Double Ring Assembly.

In many eukaryotes septin filaments form an hourglass-like structure at the division site that rearranges into a double ring at cytokinesis. A new study elucidates how an anillin-RhoGEF complex guides the assembly of the septin double ring in budding yeast.

A kinesin Klp10A mediates cell cycle-dependent shuttling of Piwi between nucleus and nuage.

The piRNA pathway protects germline genomes from selfish genetic elements (e.g. transposons) through their transcript cleavage in the cytoplasm and/or their transcriptional silencing in the nucleus. Here, we describe a mechanism by which the nuclear and cytoplasmic arms of the piRNA pathway are linked. We find that during mitosis of Drosophila spermatogonia, nuclear Piwi interacts with nuage, the compartment that mediates the cytoplasmic arm of the piRNA pathway. At the end of mitosis, Piwi leaves nuage to return to the nucleus. Dissociation of Piwi from nuage occurs at the depolymerizing microtubules of the central spindle, mediated by a microtubule-depolymerizing kinesin, Klp10A. Depletion of klp10A delays the return of Piwi to the nucleus and affects piRNA production, suggesting the role of nuclear-cytoplasmic communication in piRNA biogenesis. We propose that cell cycle-dependent communication between the nuclear and cytoplasmic arms of the piRNA pathway may play a previously unappreciated role in piRNA regulation.

Drosophila ELYS regulates Dorsal dynamics during development

Embryonic large molecule derived from yolk sac (ELYS) is a constituent protein of nuclear pores. It initiates assembly of nuclear pore complexes into functional nuclear pores toward the end of mitosis. Using cellular, molecular, and genetic tools, including fluorescence and Electron microscopy, quantitative PCR, and RNAi-mediated depletion, we report here that the ELYS ortholog (dElys) plays critical roles during Drosophila development. dElys localized to the nuclear rim in interphase cells, but during mitosis it was absent from kinetochores and enveloped chromatin. We observed that RNAi-mediated dElys depletion leads to aberrant development and, at the cellular level, to defects in the nuclear pore and nuclear lamina assembly. Further genetic analyses indicated that dElys depletion re-activates the Dorsal (NF-κB) pathway during late larval stages. Re-activated Dorsal caused untimely expression of the Dorsal target genes in the post-embryonic stages. We also demonstrate that activated Dorsal triggers apoptosis during later developmental stages by up-regulating the pro-apoptotic genes reaper and hid The apoptosis induced by Reaper and Hid was probably the underlying cause for developmental abnormalities observed upon dElys depletion. Moreover, we noted that dElys has conserved structural features, but contains a noncanonical AT-hook-like motif through which it strongly binds to DNA. Together, our results uncover a novel epistatic interaction that regulates Dorsal dynamics by dElys during development.

Division (Architecture 5), and: “Adopt” as in “a Program” (Architecture 11), and: The Second Idea of Absence (Architecture 12), and: Nest (Architecture 17), and: Nest III (Architecture 102)

Division (Architecture 5), and: “Adopt” as in “a Program” (Architecture 11), and: The Second Idea of Absence (Architecture 12), and: Nest (Architecture 17), and: Nest III (Architecture 102) John Gallaher (bio) Division (Architecture 5) Let’s watch the process one more time. During the first stageof mitosis, prophase, we see the classic chromosome structure.Notice the DNA condensing. Outside, my neighboris watering the new tree they plantedto replace the one they had to remove, and in both her and the tree,microtubules are appearing and the nuclear membraneis breaking down. So many places to go wrong:metaphase, when the chromosomes are alignedat the center of the cell, or anaphase,as the chromosomes are moving apart.Telophase is then marked by the appearanceof new nuclear membranes. And this is the endof mitosis. About 80 minutes, and two new cellsare ready to grow and perform their specialized functions. I wasn’t there when my mother died. My brother Richard and Ihad been there, called by our dad, when her organsstarted shutting down. But when we got there, she rallied,her organs started functioning again, and we had work,so we left, and then she died. I can see her, though, as she was then,wax statue, Alzheimer’s to a stroke to a coma, in and out.My father is still fighting, demanding it was dementia instead.It’s become his meal of rocks. He took care of herthat last year, and several things I was sayingabout Alzheimer’s care, playing music, etc.,he didn’t like or want and turned back, folding into himself. We want it to be our show, how we die, but it isn’t.My mother, younger, told us that if she went senile,we were to take her out to the backyard and shoot her.She didn’t mean it, but what someone means at a moment like this [End Page 41] is complicated, as the slow milling of the oceansand land, geese rising from a lakeand back down, some clock beneath these hills. [End Page 42] “Adopt” as in “a Program” (Architecture 11) Something falls from the sky. And we all know anything that fallsfrom the sky is a gift from the gods. Grape seeds maybe. Bread.Lottery checks. It’s the premise of the Cargo cultsdotted across the 20th century, deriving from the beliefthat ritualistic acts such as the building of an airplane runwaywill result in the appearance of wealth. It’s logical conclusionsfrom faulty premises. As one might imagine a questionof chickens and eggs a real question, formed under conditionsof social stress, often linked to an ancestral efficacythought to be recoverable by a returnto traditional morality, characterizing the present stateas a dismantling of the social order, that social hierarchyand ego boundaries have broken down,and we need to make whatever place great again. You have to be careful not to fall in love with the spectacle. Spectaclesare easy. There’s also the rest of the day to consider, as the torsoof Apollo whispers: how old and broken things can create in onea fondness for memes and tote bags. “The themeof this year’s Fall Harvest Ball is ‘Fall Harvest,’” the flyer proclaimsthat fell from the bulletin board at the community centeras I passed. The theme is theme. We write it on our masksand go running. We’re exotic aliens to ourselves. I visited the Metropolitan Museum of Art in 2008,wanting to get a little something to take back to the Midwest,and their floor-wide installation turned out to be tractors. It’s allin how you define it. The theme unfolds, cult to denominationto nation, adopting a program, adopting a pet, a child,from the Latin, adoptio, from ad- ‘to’ + optio ‘choosing.’ To choose.I’m on a fact-finding mission to the center of my soul, working outmy family tree, birth and adoption, 2,000 people so farand still no mother. I wishI believed in ghosts. Maybe I halfway believe in ghosts. [End Page...

Who Needs a Contractile Actomyosin Ring? The Plethora of Alternative Ways to Divide a Protozoan Parasite.

Cytokinesis, or the division of the cytoplasm, following the end of mitosis or meiosis, is accomplished in animal cells, fungi, and amoebae, by the constriction of an actomyosin contractile ring, comprising filamentous actin, myosin II, and associated proteins. However, despite this being the best-studied mode of cytokinesis, it is restricted to the Opisthokonta and Amoebozoa, since members of other evolutionary supergroups lack myosin II and must, therefore, employ different mechanisms. In particular, parasitic protozoa, many of which cause significant morbidity and mortality in humans and animals as well as considerable economic losses, employ a wide diversity of mechanisms to divide, few, if any, of which involve myosin II. In some cases, cell division is not only myosin II-independent, but actin-independent too. Mechanisms employed range from primitive mechanical cell rupture (cytofission), to motility- and/or microtubule remodeling-dependent mechanisms, to budding involving the constriction of divergent contractile rings, to hijacking host cell division machinery, with some species able to utilize multiple mechanisms. Here, I review current knowledge of cytokinesis mechanisms and their molecular control in mammalian-infective parasitic protozoa from the Excavata, Alveolata, and Amoebozoa supergroups, highlighting their often-underappreciated diversity and complexity. Billions of people and animals across the world are at risk from these pathogens, for which vaccines and/or optimal treatments are often not available. Exploiting the divergent cell division machinery in these parasites may provide new avenues for the treatment of protozoal disease.

The midbody interactome reveals unexpected roles for PP1 phosphatases in cytokinesis

The midbody is an organelle assembled at the intercellular bridge between the two daughter cells at the end of mitosis. It controls the final separation of the daughter cells and has been involved in cell fate, polarity, tissue organization, and cilium and lumen formation. Here, we report the characterization of the intricate midbody protein-protein interaction network (interactome), which identifies many previously unknown interactions and provides an extremely valuable resource for dissecting the multiple roles of the midbody. Initial analysis of this interactome revealed that PP1β-MYPT1 phosphatase regulates microtubule dynamics in late cytokinesis and de-phosphorylates the kinesin component MKLP1/KIF23 of the centralspindlin complex. This de-phosphorylation antagonizes Aurora B kinase to modify the functions and interactions of centralspindlin in late cytokinesis. Our findings expand the repertoire of PP1 functions during mitosis and indicate that spatiotemporal changes in the distribution of kinases and counteracting phosphatases finely tune the activity of cytokinesis proteins.

LAP (LC3-associated phagocytosis): phagocytosis or autophagy?

Phagocytosis and macroautophagy, named here autophagy, are two essential mechanisms of lysosomal degradation of diverse cargos into membrane structures. Both mechanisms are involved in immune regulation and cell survival. However, phagocytosis triggers degradation of extracellular material whereas autophagy engulfs only cytoplasmic elements. Furthermore, activation and maturation of these two processes are different. LAP (LC3-associated phagocytosis) is a form of phagocytosis that uses components of the autophagy pathway. It can eliminate (i) pathogens, (ii) immune complexes, (iii) threatening neighbouring cells, dead or alive, and (iv) cell debris, such as POS (photoreceptor outer segment) and the midbody released at the end of mitosis. Cells have thus optimized their means of elimination of dangerous components by sharing some fundamental elements coming from the two main lysosomal degradation pathways.

A two-step mechanism for the inactivation of microtubule organizing center function at the centrosome.

The centrosome acts as a microtubule organizing center (MTOC), orchestrating microtubules into the mitotic spindle through its pericentriolar material (PCM). This activity is biphasic, cycling through assembly and disassembly during the cell cycle. Although hyperactive centrosomal MTOC activity is a hallmark of some cancers, little is known about how the centrosome is inactivated as an MTOC. Analysis of endogenous PCM proteins in C. elegans revealed that the PCM is composed of partially overlapping territories organized into an inner and outer sphere that are removed from the centrosome at different rates and using different behaviors. We found that phosphatases oppose the addition of PCM by mitotic kinases, ultimately catalyzing the dissolution of inner sphere PCM proteins at the end of mitosis. The nature of the PCM appears to change such that the remaining aging PCM outer sphere is mechanically ruptured by cortical pulling forces, ultimately inactivating MTOC function at the centrosome.

Nuclear formation induced by DNA-conjugated beads in living fertilised mouse egg

Reformation of a functional nucleus at the end of mitosis is crucial for normal cellular activity. Reconstitution approaches using artificial beads in frog egg extracts have clarified the molecules required for nuclear formation in vitro. However, the spatiotemporal regulation of these components, which is required for the formation of a functional nucleus in living embryos, remains unknown. Here we demonstrate that exogenous DNA introduced in the form of DNA-conjugated beads induces the assembly of an artificial nucleus in living mouse cleavage-stage embryos. Live-cell imaging and immunofluorescence studies revealed that core histones and regulator of chromosome condensation 1 (RCC1) assembled on the DNA, suggesting that nucleosomes were formed. Electron microscopy showed that double-membrane structures, partly extended from annulate lamellae, formed around the beads. Nuclear pore complex-like structures indistinguishable from those of native nuclei were also formed, suggesting that this membranous structure resembled the normal nuclear envelope (NE). However, the reconstituted NE had no nuclear import activity, probably because of the absence of Ras-related nuclear protein (Ran). Thus, DNA is necessary for NE reassembly in mouse embryos but is insufficient to form a functional nucleus. This approach provides a new tool to examine factors of interest and their spatiotemporal regulation in nuclear formation.

Roles of Nup133, Nup153 and membrane fenestrations in assembly of the nuclear pore complex at the end of mitosis.

Reassembly of the nuclear pore complex (NPC) at the end of mitosis is an important event for eukaryotic nuclear function. In this study, we examined the dynamic behaviors of the endoplasmic reticulum (ER) by "Live CLEM" imaging. In metaphase, numerous fenestrations on the ER membrane were observed around chromosomes. In telophase, these fenestrations became filled at the region attached to chromosomes, whereas they remained open at the region unattached to chromosomes, suggesting that NPC assembly takes place at fenestrations on the membrane. To determine the roles of nucleoporins in postmitotic NPC formation, we used artificial beads conjugated with anti-GFP antibody, which captures GFP-fused proteins on the beads when incorporated into cells. Live CLEM imaging of telophase cells containing Nup133-coated beads or Nup153-coated beads showed that Nup133 and Nup153, as the sole effector molecules, assembled the NPC-like structure on the membrane fenestrations. Indirect immunofluorescence staining of the Nup133-coated beads showed that Nup133 effectively assembled Nup107 and ELYS, whereas minimal assembly of Nup98 and Nup62 was observed; the Nup153-coated bead effectively assembled Nup98, Nup62 and Pom121, but assembled neither Nup107 nor ELYS. Our results suggest that Nup133 and Nup153 play different roles in assembling the NPC on membrane fenestrations.

Centriole assembly at a glance.

The centriole organelle consists of microtubules (MTs) that exhibit a striking 9-fold radial symmetry. Centrioles play fundamental roles across eukaryotes, notably in cell signaling, motility and division. In this Cell Science at a Glance article and accompanying poster, we cover the cellular life cycle of this organelle - from assembly to disappearance - focusing on human centrioles. The journey begins at the end of mitosis when centriole pairs disengage and the newly formed centrioles mature to begin a new duplication cycle. Selection of a single site of procentriole emergence through focusing of polo-like kinase 4 (PLK4) and the resulting assembly of spindle assembly abnormal protein 6 (SAS-6) into a cartwheel element are evoked next. Subsequently, we cover the recruitment of peripheral components that include the pinhead structure, MTs and the MT-connecting A-C linker. The function of centrioles in recruiting pericentriolar material (PCM) and in forming the template of the axoneme are then introduced, followed by a mention of circumstances in which centrioles form de novo or are eliminated.

Cell cycle regulation of condensin Smc4.

The condensin complex is a conserved ATPase which promotes the compaction of chromatin during mitosis in eukaryotic cells. Condensin complexes have in addition been reported to contribute to interphase processes including sister chromatid cohesion. It is not understood how condensins specifically become competent to facilitate chromosome condensation in preparation for chromosome segregation in anaphase. Here we describe evidence that core condensin subunits are regulated at the level of protein stability in budding yeast. In particular, Smc2 and Smc4 abundance is cell cycle regulated, peaking at mitosis and falling to low levels in interphase. Smc4 degradation at the end of mitosis is dependent on the Anaphase Promoting Complex/Cyclosome and is mediated by the proteasome. Overproduction of Smc4 results in delayed decondensation, but has a limited ability to promote premature condensation in interphase. Unexpectedly, the Mad2 spindle checkpoint protein is required for mitotic Smc4 degradation. These studies have revealed the novel finding that condensin protein levels are cell cycle regulated and have identified the factors necessary for Smc4 proteolysis.

SIRT2 deacetylates GRASP55 to facilitate post-mitotic Golgi assembly.

Sirtuin 2 (SIRT2) is an NAD-dependent sirtuin deacetylase that regulates microtubule and chromatin dynamics, gene expression and cell cycle progression, as well as nuclear envelope reassembly. Recent proteomic analyses have identified Golgi proteins as SIRT2 interactors, indicating that SIRT2 may also play a role in Golgi structure formation. Here, we show that SIRT2 depletion causes Golgi fragmentation and impairs Golgi reassembly at the end of mitosis. SIRT2 interacts with the Golgi reassembly stacking protein GRASP55 (also known as GORASP2) in mitosis when GRASP55 is highly acetylated on K50. Expression of wild-type and the K50R acetylation-deficient mutant of GRASP55, but not the K50Q acetylation-mimetic mutant, in GRASP55 and GRASP65 (also known as GORASP1) double-knockout cells, rescued the Golgi structure and post-mitotic Golgi reassembly. Acetylation-deficient GRASP55 exhibited a higher self-interaction efficiency, a property required for Golgi structure formation. These results demonstrate that SIRT2 regulates Golgi structure by modulating GRASP55 acetylation levels.