Here, we characterize spatial distribution of the Golgi complex in human cells. In contrast to the prevailing view that the Golgi compactly surrounds the centrosome throughout interphase, we observe characteristic differences in the morphology of Golgi ribbons and their association with the centrosome during various periods of the cell cycle. The compact Golgi complex is typical in G1; during S-phase, Golgi ribbons lose their association with the centrosome and extend along the nuclear envelope to largely encircle the nucleus in G2. Interestingly, pre-mitotic separation of duplicated centrosomes always occurs after dissociation from the Golgi. Shortly before the nuclear envelope breakdown, scattered Golgi ribbons reassociate with the separated centrosomes restoring two compact Golgi complexes. Transitions between the compact and distributed Golgi morphologies are microtubule-dependent. However, they occur even in the absence of centrosomes, which implies that Golgi reorganization is not driven by the centrosomal microtubule asters. Cells with different Golgi morphology exhibit distinct differences in the directional persistence and velocity of migration. These data suggest that changes in the radial distribution of the Golgi around the nucleus define the extent of cell polarization and regulate cell motility in a cell cycle-dependent manner.

Flow cytometry gives a unique opportunity to analyze thousands of individual cells for multiple parameters in a course of minutes. The most commonly used flow cytometry application in plant biology is estimation of nuclear DNA content. This becomes an indispensable tool in different areas of plant research, including breeding, taxonomy, plant development, evolutionary biology, populational studies and others. DNA content analysis can provide an insight into natural ploidy changes that reflect evolutionary processes, such as interspecific hybridization and polyploidization. It is also widely used for processing samples with biotechnologically induced ploidy changes, for instance, plants produced by doubled haploid technology. Absolute genome size data produced by cytometric analysis serve as useful taxon-specific markers since genome size vary between different taxa. It often allows the distinguishing of species within a genus or even different subspecies. Introducing flow cytometry method in the lab is extremely appealing, but new users face a significant challenge of learning instrument management, quality sample preparation and data processing. Not only is flow cytometry a complex method, but plant samples have unique features that make plants a demanding research subject. Without proper training, researchers risk damaging the expensive instrument or publishing poor quality data, artifacts or unreproducible results. We bring together information from our experience, key papers and online resources to provide step by step protocols and give a starting point for exploring the abundant cytometry literature.

Plant ploidy manipulation is often required for breeding purposes. However, there is no comprehensive review covering genome doubling in vegetable crops despite the abundance of data for a large number of vegetable species. Similar to other species, genome doubling is required in vegetable crops to obtain doubled haploids (DHs). It is also utilized for the production of polyploids to overcome interspecific hybrid sterility and improve agricultural traits. Spontaneous haploid genome duplication (SHGD) occurs in many Apiaceae, Brassicaceae, Cucurbitaceae, and Solanaceae crops, allowing for the laborious treatment with antimitotic agents to be bypassed. SHGD mechanisms are not fully understood, but existing data suggest that SHGD can occur via nuclear fusion, endoreduplication, or other mechanisms during microspore or ovule early embryogenic development. Other studies show that SHGD can occur at later developmental stages during extended plant growth in vitro or ex vitro, possibly due to the presence of phytohormones in the medium and/or diploid cell competitive advantage. For unresponsive accessions and species with rare SHGD, such as onion (Allium cepa L.) and beet cultivars (Beta vulgaris subsp. vulgaris L.), antimitotic agent treatment has to be applied. Antimitotic agent application efficiency depends on the treatment conditions, especially the agent concentration and exposure time. Also, plant developmental stage is critical for agent accessibility and plant survival. The existing methods can be used to further improve genome doubling methodology for major vegetable crops and other species.

Epithelial cells possess intrinsic mechanisms to maintain an appropriate cell density for normal tissue morphogenesis and homeostasis. Defects in such mechanisms likely contribute to hyperplasia and cancer initiation. To identify genes that regulate the density-dependent proliferation of murine mammary epithelial cells, we developed a fluorescence-activated cell sorting assay based on fluorescence ubiquitination cell cycle indicator, which marks different stages of the cell cycle with distinct fluorophores. Using this powerful assay, we performed a genome-wide CRISPR/Cas9 knockout screen, selecting for cells that proliferate normally at low density but continue to divide at high density. Unexpectedly, one top hit was Traf3, a negative regulator of NF-κB signaling that has never previously been linked to density-dependent proliferation. We demonstrate that loss of Traf3 specifically activates noncanonical NF-κB signaling. This in turn triggers an innate immune response and drives cell division independently of known density-dependent proliferation mechanisms, including YAP/TAZ signaling and cyclin-dependent kinase inhibitors, by blocking entry into quiescence.