Model-based trajectory classification of anchored molecular motor-biopolymer interactions.

During zygotic mitosis in many species, forces generated at the cell cortex are required for the separation and migration of paternally provided centrosomes, pronuclear migration, segregation of genetic material, and cell division. Furthermore, in some species, force-generating interactions between spindle microtubules and the cortex position the mitotic spindle asymmetrically within the zygote, an essential step in asymmetric cell division. Understanding the mechanical and molecular mechanisms of microtubule-dependent force generation and therefore asymmetric cell division requires identification of individual cortical force-generating units in vivo. There is no current method for identifying individual force-generating units with high spatiotemporal resolution. Here, we present a method to determine both the location and the relative number of microtubule-dependent cortical force-generating units using single-molecule imaging of fluorescently labeled dynein. Dynein behavior is modeled to classify trajectories of cortically bound dynein according to whether they are interacting with a microtubule. The categorization strategy recapitulates well-known force asymmetries in C. elegans zygote mitosis. To evaluate the robustness of categorization, we used RNAi to deplete the tubulin subunit TBA-2. As predicted, this treatment reduced the number of trajectories categorized as engaged with a microtubule. Our technique will be a valuable tool to define the molecular mechanisms of dynein cortical force generation and its regulation as well as other instances wherein anchored motors interact with biopolymers (e.g., actin, tubulin, DNA).

Why scientific societies should involve more early-career researchers.

Early-career researchers (ECRs) make up a large portion of the academic workforce. Yet, most leadership positions in scientific societies are held by senior scientists, and ECRs have little to no say over the decisions that will shape the future of research. This article looks at the level of influence ECRs have in 20 scientific societies based in the US and UK, and provides guidelines on how societies can successfully include ECRs in leadership roles.

Human chromosome-specific aneuploidy is influenced by DNA-dependent centromeric features.

Intrinsic genomic features of individual chromosomes can contribute to chromosome-specific aneuploidy. Centromeres are key elements for the maintenance of chromosome segregation fidelity via a specialized chromatin marked by CENP-A wrapped by repetitive DNA. These long stretches of repetitive DNA vary in length among human chromosomes. Using CENP-A genetic inactivation in human cells, we directly interrogate if differences in the centromere length reflect the heterogeneity of centromeric DNA-dependent features and whether this, in turn, affects the genesis of chromosome-specific aneuploidy. Using three distinct approaches, we show that mis-segregation rates vary among different chromosomes under conditions that compromise centromere function. Whole-genome sequencing and centromere mapping combined with cytogenetic analysis, small molecule inhibitors, and genetic manipulation revealed that inter-chromosomal heterogeneity of centromeric features, but not centromere length, influences chromosome segregation fidelity. We conclude that faithful chromosome segregation for most of human chromosomes is biased in favor of centromeres with high abundance of DNA-dependent centromeric components. These inter-chromosomal differences in centromere features can translate into non-random aneuploidy, a hallmark of cancer and genetic diseases.

Centromeric SMC1 promotes centromere clustering and stabilizes meiotic homolog pairing.

During meiosis, each chromosome must selectively pair and synapse with its own unique homolog to enable crossover formation and subsequent segregation. How homolog pairing is maintained in early meiosis to ensure synapsis occurs exclusively between homologs is unknown. We aimed to further understand this process by examining the meiotic defects of a unique Drosophila mutant, Mcm5A7. We found that Mcm5A7 mutants are proficient in homolog pairing at meiotic onset yet fail to maintain pairing as meiotic synapsis ensues, causing seemingly normal synapsis between non-homologous loci. This pairing defect corresponds with a reduction of SMC1-dependent centromere clustering at meiotic onset. Overexpressing SMC1 in this mutant significantly restores centromere clustering, homolog pairing, and crossover formation. These data indicate that the initial meiotic pairing of homologs is not sufficient to yield synapsis exclusively between homologs and provide a model in which meiotic homolog pairing must be stabilized by centromeric SMC1 to ensure proper synapsis.

PP2A-B55/SUR-6 collaborates with the nuclear lamina for centrosome separation during mitotic entry.

Across most sexually reproducing animals, centrosomes are provided to the oocyte through fertilization and must be positioned properly to establish the zygotic mitotic spindle. How centrosomes are positioned in space and time through the concerted action of key mitotic entry biochemical regulators, including protein phosphatase 2A (PP2A-B55/SUR-6), biophysical regulators, including dynein, and the nuclear lamina is unclear. Here, we uncover a role for PP2A-B55/SUR-6 in regulating centrosome separation. Mechanistically, PP2A-B55/SUR-6 regulates nuclear size before mitotic entry, in turn affecting nuclear envelope-based dynein density and motor capacity. Computational simulations predicted the requirement of PP2A-B55/SUR-6 regulation of nuclear size and nuclear-envelope dynein density for proper centrosome separation. Conversely, compromising nuclear lamina integrity led to centrosome detachment from the nuclear envelope and migration defects. Removal of PP2A-B55/SUR-6 and the nuclear lamina simultaneously further disrupted centrosome separation, leading to unseparated centrosome pairs dissociated from the nuclear envelope. Taking these combined results into consideration, we propose a model in which centrosomes migrate and are positioned through the concerted action of PP2A-B55/SUR-6-regulated nuclear envelope-based dynein pulling forces and centrosome-nuclear envelope tethering. Our results add critical precision to models of centrosome separation relative to the nucleus during spindle formation in cell division.

PP2A-B55 promotes nuclear envelope reformation after mitosis in Drosophila.

As a dividing cell exits mitosis and daughter cells enter interphase, many proteins must be dephosphorylated. The protein phosphatase 2A (PP2A) with its B55 regulatory subunit plays a crucial role in this transition, but the identity of its substrates and how their dephosphorylation promotes mitotic exit are largely unknown. We conducted a maternal-effect screen in Drosophila melanogaster to identify genes that function with PP2A-B55/Tws in the cell cycle. We found that eggs that receive reduced levels of Tws and of components of the nuclear envelope (NE) often fail development, concomitant with NE defects following meiosis and in syncytial mitoses. Our mechanistic studies using Drosophila cells indicate that PP2A-Tws promotes nuclear envelope reformation (NER) during mitotic exit by dephosphorylating BAF and suggests that PP2A-Tws targets additional NE components, including Lamin and Nup107. This work establishes Drosophila as a powerful model to further dissect the molecular mechanisms of NER and suggests additional roles of PP2A-Tws in the completion of meiosis and mitosis.

Introduction to Modern Methods in Light Microscopy.

For centuries, light microscopy has been a key method in biological research, from the early work of Robert Hooke describing biological organisms as cells, to the latest in live-cell and single-molecule systems. Here, we introduce some of the key concepts related to the development and implementation of modern microscopy techniques. We briefly discuss the basics of optics in the microscope, super-resolution imaging, quantitative image analysis, live-cell imaging, and provide an outlook on active research areas pertaining to light microscopy.