Chromosome segregation requires sister chromatid resolution. Condensins are essential for this process since they organize an axial structure where topoisomerase II can work. How sister chromatid separation is coordinated with chromosome condensation and decatenation activity remains unknown. We combined four-dimensional (4D) microscopy, RNA interference (RNAi), and biochemical analyses to show that topoisomerase II plays an essential role in this process. Either depletion of topoisomerase II or exposure to specific anti-topoisomerase II inhibitors causes centromere nondisjunction, associated with syntelic chromosome attachments. However, cells degrade cohesins and timely exit mitosis after satisfying the spindle assembly checkpoint. Moreover, in topoisomerase II–depleted cells, Aurora B and INCENP fail to transfer to the central spindle in late mitosis and remain tightly associated with centromeres of nondisjoined sister chromatids. Also, in topoisomerase II–depleted cells, Aurora B shows significantly reduced kinase activity both in S2 and HeLa cells. Codepletion of BubR1 in S2 cells restores Aurora B kinase activity, and consequently, most syntelic attachments are released. Taken together, our results support that topoisomerase II ensures proper sister chromatid separation through a direct role in centromere resolution and prevents incorrect microtubule–kinetochore attachments by allowing proper activation of Aurora B kinase.
Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40-50 nm thick. On average, each S2 kinetochore binds 11±2 MTs, in contrast to the 4-6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly,
Significance The mitotic spindle, a structure composed primarily of microtubules, guides the segregation of DNA during cell division. In somatic animal cells, centrosomes (microtubule nucleating structures) reside near the mitotic spindle poles. However, germ cells lack centrosomes, and even somatic cells can execute cell division if centrosome function is compromised. This study on Drosophila cells reports a whole-genome RNAi screen for genes involved in spindle assembly in the absence of functional centrosomes. The results show that spindle assembly pathways with and without centrosomes involve an essentially identical set of genes, demonstrating the constitutive nature of centrosome-independent spindle assembly. However, certain gene knockdowns show distinct phenotypes when centrosomes are absent, thus revealing how spindles adapt to the presence or absence of centrosomes.
Cytoskeleton microtubules undergo a reversible metamorphosis as cells enter and exit mitosis to build a transient mitotic spindle required for chromosome segregation. Centrosomes play a dominant but dispensable role in microtubule (MT) organization throughout the animal cell cycle, supporting the existence of concurrent mechanisms that remain unclear. Here we investigated MT organization at the entry and exit from mitosis, after perturbation of centriole function in Drosophila S2 cells. We found that several MTs originate from acentriolar microtubule-organizing centers (aMTOCs) that contain gamma-tubulin and require Centrosomin (Cnn) for normal architecture and function. During spindle assembly, aMTOCs associated with peripheral MTs are recruited to acentriolar spindle poles by an Ncd/dynein-dependent clustering mechanism to form rudimentary aster-like structures. At anaphase onset, down-regulation of CDK1 triggers massive formation of cytoplasmic MTs de novo, many of which nucleated directly from aMTOCs. CDK1 down-regulation at anaphase coordinates the activity of Msps/XMAP215 and the kinesin-13 KLP10A to favor net MT growth and stability from aMTOCs. Finally, we show that microtubule nucleation from aMTOCs also occurs in cells containing centrosomes. Our data reveal a new form of cell cycle-regulated MTOCs that contribute for MT cytoskeleton remodeling during mitotic spindle assembly/disassembly in animal somatic cells, independently of centrioles.
SummaryIn animal cells the centrosome is commonly viewed as the main cellular structure driving microtubule (MT) assembly into the mitotic spindle apparatus. However, additional pathways, such as those mediated by chromatin and augmin, are involved in the establishment of functional spindles. The molecular mechanisms involved in these pathways remain poorly understood, mostly due to limitations inherent to current experimental systems available. To overcome these limitations we have developed six new Drosophila cell lines derived from Drosophila homozygous mutants for DSas-4, a protein essential for centriole biogenesis. These cells lack detectable centrosomal structures, astral MT, with dispersed pericentriolar proteins D-PLP, Centrosomin and γ-tubulin. They show poorly focused spindle poles that reach the plasma membrane. Despite being compromised for functional centrosome, these cells could successfully undergo mitosis.Live-cell imaging analysis of acentriolar spindle assembly revealed that nascent MTs are nucleated from multiple points in the vicinity of chromosomes. These nascent MTs then grow away from kinetochores allowing the expansion of fibers that will be part of the future acentriolar spindle. MT repolymerization assays illustrate that acentriolar spindle assembly occurs “inside-out” from the chromosomes. Colchicine-mediated depolymerization of MTs further revealed the presence of a functional Spindle Assembly Checkpoint (SAC) in the acentriolar cells. Finally, pilot RNAi experiments open the potential use of these cell lines for the molecular dissection of anastral pathways in spindle and centrosome assembly.
In order to perpetuate their genetic content, eukaryotic cells have developed a microtubule-based machine known as the mitotic spindle. Independently of the system studied, mitotic spindles share at least one common characteristic -the dynamic nature of microtubules. This property allows the constant plasticity needed to assemble a bipolar structure, make proper kinetochoremicrotubule attachments, segregate chromosomes and finally disassemble the spindle and reform an interphase microtubule array. Here we describe a variety of experimental approaches currently used in our laboratory to study microtubule dynamics during mitosis using Drosophila melanogaster S2 cells as a model. By using quantitative live-cell imaging microscopy in combination with an advantageous labeling background, we illustrate how several cooperative pathways are used to build functional mitotic spindles. We illustrate different ways of perturbing spindle microtubule dynamics, including pharmacological inhibition and RNA interference of proteins that directly or indirectly impair microtubule dynamics. Additionally, we demonstrate the advantage of using fluorescent speckle microscopy (FSM) to investigate an intrinsic property of spindle microtubules known as poleward flux. Finally, we developed a set of laser microsurgery-based experiments that allow, with unique spatiotemporal resolution, the study of specific spindle-structures (e.g. centrosomes, microtubules and kinetochores) and their respective roles during mitosis.3
A 39-year-old woman with systemic lupus erythematosus treated with anti-CD20 monoclonal antibody rituximab was admitted to our hospital with COVID-19 pneumonia. Despite receiving dexamethasone, she developed hypoxaemia and persistent lung opacities. As bronchoalveolar lavage was suggestive of cryptogenic organising pneumonia, high-dose corticosteroid was administered, and she received antimicrobial therapy for opportunistic infections without improvement. Reverse transcription PCR was repeatedly positive for SARS-CoV-2, and virus replication was confirmed in cell cultures. As no anti-SARS-CoV-2 antibodies were detected more than 100 days after symptom onset, she was treated with convalescent plasma with fast clinical improvement, returning home days later. Our case shows that persistent SARS-CoV-2 infection in an immunocompromised patient may be overturned with the appropriate treatment.
Systemic sclerosis is an autoimmune disease that can result in lung fibrosis, and is strongly associated with the presence of serum anti-topoisomerase-I autoantibodies. A young man with genetic muscular dystrophy caused by titin-cap/telethonin (TCAP) gene mutation, developed a severe restrictive lung disease due to a fibrosing interstitial pneumonia secondary to systemic sclerosis with positive anti-topoisomerase-I antibodies. Using amino acid sequence alignment and protein structure modelling, we found that mutant telethonin exposes an amino acid sequence with significant homology to an immunodominant site of topoisomerase-I. Abnormal telethonin results in a loss of integrity of the sarcomere structure, which might result in rhabdomyolysis and abnormal protein exposure to the immune system. Our preliminary analysis suggests a possible role for mutant sarcomere protein telethonin as an immunogenic target recognised by anti-topoisomerase-I antibodies, which could explain the development of systemic sclerosis in this particular patient.
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