Augmin-dependent microtubule self-organization drives kinetochore fiber maturation in mammals.

Chromosome segregation in mammals relies on the maturation of a thick bundle of kinetochore-attached microtubules known as k-fiber. How k-fibers mature from initial kinetochore microtubule attachments remains a fundamental question. By combining molecular perturbations and phenotypic analyses in Indian muntjac fibroblasts containing the lowest known diploid chromosome number in mammals (2N = 6) and distinctively large kinetochores, with fixed/live-cell super-resolution coherent-hybrid stimulated emission depletion (CH-STED) nanoscopy and laser microsurgery, we demonstrate a key role for augmin in kinetochore microtubule self-organization and maturation, regardless of pioneer centrosomal microtubules. In doing so, augmin promotes kinetochore and interpolar microtubule turnover and poleward flux. Tracking of microtubule growth events within individual k-fibers reveals a wide angular dispersion, consistent with augmin-mediated branched microtubule nucleation. Augmin depletion reduces the frequency of kinetochore microtubule growth events and hampers efficient repair after acute k-fiber injury by laser microsurgery. Together, these findings underscore the contribution of augmin-mediated microtubule amplification for k-fiber self-organization and maturation in mammals.

Acute BAF perturbation causes immediate changes in chromatin accessibility.

Cancer-associated, loss-of-function mutations in genes encoding subunits of the BRG1/BRM-associated factor (BAF) chromatin-remodeling complexes1-8 often cause drastic chromatin accessibility changes, especially in important regulatory regions9-19. However, it remains unknown how these changes are established over time (for example, immediate consequences or long-term adaptations), and whether they are causative for intracomplex synthetic lethalities, abrogating the formation or activity of BAF complexes9,20-24. In the present study, we use the dTAG system to induce acute degradation of BAF subunits and show that chromatin alterations are established faster than the duration of one cell cycle. Using a pharmacological inhibitor and a chemical degrader of the BAF complex ATPase subunits25,26, we show that maintaining genome accessibility requires constant ATP-dependent remodeling. Completely abolishing BAF complex function by acute degradation of a synthetic lethal subunit in a paralog-deficient background results in an almost complete loss of chromatin accessibility at BAF-controlled sites, especially also at superenhancers, providing a mechanism for intracomplex synthetic lethalities.

Functional Dissection of Mitosis Using Immortalized Fibroblasts from the Indian Muntjac, a Placental Mammal with Only Three Chromosomes.

During cell division in eukaryotes a microtubule-based network undergoes drastic changes and remodeling to assemble a mitotic spindle competent to segregate chromosomes. Several model systems have been widely used to dissect the molecular and structural mechanisms behind mitotic spindle assembly and function. These include budding and fission yeasts, which are ideal for genetic and molecular approaches, but show limitations in high-resolution live-cell imaging, while being evolutionarily distant from humans. On the other hand, systems that were historically used for their exceptional properties for live-cell imaging of mitosis (e.g., newt lung cells and Haemanthus endosperm cells) lack the necessary genomic tools for molecular studies. In a CRISPR-Cas9 era, human cultured cells have conquered the privilege to be positioned among the most powerful genetically manipulatable systems, but their high chromosome number remains a significant bottleneck for the molecular dissection of mitosis in mammals. We believe that we can significantly broaden this scenario by establishing a unique placental mammal model system that combines the powerful genetic tools and low chromosome number of fission yeast and Drosophila melanogaster, with the exceptional cytological features of a rat kangaroo cell. This system is based on hTERT-immortalized fibroblasts from a female Indian muntjac, a placental mammal with the lowest known chromosome number (n = 3). Here we describe a series of methodologies established in our laboratory for the study of mitosis in Indian muntjac. These include standard techniques such as immunofluorescence, western blotting, and FISH, but also several state-of-the-art methodologies, including live-cell imaging, cell confinement, RNAi, super-resolution STED microscopy, and laser microsurgery.

Chromosome Segregation Is Biased by Kinetochore Size.

Chromosome missegregation during mitosis or meiosis is a hallmark of cancer and the main cause of prenatal death in humans. The gain or loss of specific chromosomes is thought to be random, with cell viability being essentially determined by selection. Several established pathways including centrosome amplification, sister-chromatid cohesion defects, or a compromised spindle assembly checkpoint can lead to chromosome missegregation. However, how specific intrinsic features of the kinetochore-the critical chromosomal interface with spindle microtubules-impact chromosome segregation remains poorly understood. Here we used the unique cytological attributes of female Indian muntjac, the mammal with the lowest known chromosome number (2n = 6), to characterize and track individual chromosomes with distinct kinetochore size throughout mitosis. We show that centromere and kinetochore functional layers scale proportionally with centromere size. Measurement of intra-kinetochore distances, serial-section electron microscopy, and RNAi against key kinetochore proteins confirmed a standard structural and functional organization of the Indian muntjac kinetochores and revealed that microtubule binding capacity scales with kinetochore size. Surprisingly, we found that chromosome segregation in this species is not random. Chromosomes with larger kinetochores bi-oriented more efficiently and showed a 2-fold bias to congress to the equator in a motor-independent manner. Despite robust correction mechanisms during unperturbed mitosis, chromosomes with larger kinetochores were also strongly biased to establish erroneous merotelic attachments and missegregate during anaphase. This bias was impervious to the experimental attenuation of polar ejection forces on chromosome arms by RNAi against the chromokinesin Kif4a. Thus, kinetochore size is an important determinant of chromosome segregation fidelity.

CD5 expression is regulated during human T-cell activation by alternative polyadenylation, PTBP1, and miR-204.

T lymphocytes stimulated through their antigen receptor (TCR) preferentially express mRNA isoforms with shorter 3´ untranslated regions (3´-UTRs) derived from alternative pre-mRNA cleavage and polyadenylation (APA). However, the physiological relevance of APA programs remains poorly understood. CD5 is a T-cell surface glycoprotein that negatively regulates TCR signaling from the onset of T-cell activation. CD5 plays a pivotal role in mediating outcomes of cell survival or apoptosis, and may prevent both autoimmunity and cancer. In human primary T lymphocytes and Jurkat cells we found three distinct mRNA isoforms encoding CD5, each derived from distinct poly(A) signals (PASs). Upon T-cell activation, there is an overall increase in CD5 mRNAs with a specific increase in the relative expression of the shorter isoforms. 3´-UTRs derived from these shorter isoforms confer higher reporter expression in activated T cells relative to the longer isoform. We further show that polypyrimidine tract binding protein (PTB/PTBP1) directly binds to the proximal PAS and PTB siRNA depletion causes a decrease in mRNA derived from this PAS, suggesting an effect on stability or poly(A) site selection to circumvent targeting of the longer CD5 mRNA isoform by miR-204. These mechanisms fine-tune CD5 expression levels and thus ultimately T-cell responses.

Polar Ejection Forces Promote the Conversion from Lateral to End-on Kinetochore-Microtubule Attachments on Mono-oriented Chromosomes.

Chromosome bi-orientation occurs after conversion of initial lateral attachments between kinetochores and spindle microtubules into stable end-on attachments near the cell equator. After bi-orientation, chromosomes experience tension from spindle forces, which plays a key role in the stabilization of correct kinetochore-microtubule attachments. However, how end-on kinetochore-microtubule attachments are first stabilized in the absence of tension remains a key unanswered question. To address this, we generated Drosophila S2 cells undergoing mitosis with unreplicated genomes (SMUGs). SMUGs retained single condensed chromatids that attached laterally to spindle microtubules. Over time, laterally attached kinetochores converted into end-on attachments and experienced intra-kinetochore stretch/structural deformation, and SMUGs eventually exited a delayed mitosis with mono-oriented chromosomes after satisfying the spindle-assembly checkpoint (SAC). Polar ejection forces (PEFs) generated by Chromokinesins promoted the conversion from lateral to end-on kinetochore-microtubule attachments that satisfied the SAC in SMUGs. Thus, PEFs convert lateral to stable end-on kinetochore-microtubule attachments, independently of chromosome bi-orientation.

Selective tracking of template DNA strands after induction of mitosis with unreplicated genomes (MUGs) in Drosophila S2 cells.

According to the "immortal" DNA strand hypothesis (Cairns Nature 255:197-200, 1975), stem cells would keep their template strands in order to prevent the accumulation of mutations, which could occur during DNA replication. Despite the growing number of studies that attempt to test this hypothesis, the conclusions remain highly controversial. In the base of this controversy lie the current limitations of available methodology to selectively and faithfully track the fate of template DNA strands throughout and upon cell division. Here, we developed a method that allows the unequivocal tracking of single chromatids containing template DNA strands in Drosophila S2 cells in culture. This method consists in the induction of mitosis with unreplicated genomes (MUGs) in which cells are allowed to enter mitosis without prior DNA replication. This is achieved by RNAi-mediated knockdown of Double parked, a conserved protein required for the initiation of DNA replication and post-replication checkpoint response. The advantages of this system when compared with MUGs generated in mammalian cells is the preservation of chromatid morphology, the ease of loss-of-function studies and the possibility of in vivo applications. Altogether, this approach allows for the readily visualization and tracking of template DNA strands by simply monitoring cells stably expressing GFP-fusions with either Histone H2B or the centromeric Histone variant CID/CENP-A by time-lapse fluorescence microscopy. This might be useful for the dissection of the molecular mechanism behind asymmetric DNA strand segregation.