Mastering DNA Content Estimation by Flow Cytometry as an Efficient Tool for Plant Breeding and Biodiversity Research.

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.

Genome-wide CRISPR screen identifies noncanonical NF-κB signaling as a regulator of density-dependent proliferation.

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.

Cell Cycle-Dependent Dynamics of the Golgi-Centrosome Association in Motile Cells.

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.

Polarity proteins in oncogenesis.

Most human cancers arise from epithelial tissues, which are apical-basally polarized and possess intercellular adhesive junctions. Epithelial cells grow to characteristic densities, often from proliferative progenitors, which arrest as they mature. Homeostatic mechanisms can maintain this characteristic density if it is exceeded (crowding) or is too low (e.g. in response to wounding). During tumor initiation and progression this homeostatic mechanism is lost. Some aspects of cell polarity are also lost, although many carcinomas retain intercellular junctions and even apical domains. In other cases, and particularly in recurrent tumors, however, the cells become predominantly mesenchymal. A major question, still only incompletely answered, is whether the proteins that determine cell polarity function as tumor suppressors or tumor promoters. Here we discuss recent advances in understanding the role of polarity proteins and homeostasis in cancer.

Ice recovery assay for detection of Golgi-derived microtubules.

Proper organization of the microtubule cytoskeleton is essential for many cellular processes including maintenance of Golgi organization and cell polarity. Traditionally, the centrosome is considered to be the major microtubule organizing center (MTOC) of the cell; however, microtubule nucleation can also occur through centrosome-independent mechanisms. Recently, the Golgi has been described as an additional, centrosome-independent, MTOC with distinct cellular functions. Golgi-derived microtubules contribute to the formation of an asymmetric microtubule network, control Golgi organization, and support polarized trafficking and directed migration in motile cells. In this chapter, we present an assay using recovery from ice treatment to evaluate the potential of the Golgi, or other MTOCs, to nucleate microtubules. This technique allows for clear separation of distinct MTOCs and observation of newly nucleated microtubules at these locations, which are normally obscured by the dense microtubule network present at steady-state conditions. This type of analysis is important for discovery and characterization of noncentrosomal MTOCs and, ultimately, understanding of their unique cellular functions.