Cell division is a tightly regulated mechanism, notably in tissues where malfunctions can lead to tumour formation or developmental defects. This is particularly true in land plants, where cells cannot relocate and therefore cytokinesis determines tissue topology. In plants, cell division is executed in radically different manners than in animals, with the appearance of new structures and the disappearance of ancestral mechanisms. Whilst F-actin and microtubules closely co-exist, recent studies mainly focused on the involvement of microtubules in this key process. Here, we used a root tracking system to image the spatio-temporal dynamics of both F-actin reporters and cell division markers in dividing cells embedded in their tissues. In addition to the F-actin accumulation at the phragmoplast, we observed and quantified a dynamic apico-basal enrichment of F-actin from the prophase/metaphase transition until the end of the cytokinesis.

Temperature is a key parameter in most biological and physical systems. It is, however, an inherent technological challenge to precisely track and control the temperature of small sample volumes when using high- and super-resolution microscopes. Various effects can cause a deviation of the sample temperature from its expected value, specifically in the observed volume or field-of-view. These effects include direct thermal contact of the objective lens and the coverslip via immersion medium, illumination-induced heating, evaporative cooling, or triggered phase transitions. In this paper, we shed light on these phenomena and introduce our newly developed device VAHEAT, which follows a microscopic approach for controlling and measuring the sample temperature. Our system overcomes traditional limitations of temperature sensing and controlling in high-sensitivity microscopy applications, leading to more reproducible experimental results and conquering new experimental parameter ranges.

Hyphal cells of filamentous fungi grow at their tips in a method analogous to pollen tube and root hair elongation. This process, generally referred to as tip growth, requires precise regulation of the actin cytoskeleton, and characterizing the various actin structures in these cell types is currently an active area of research. Here, the actin marker Lifeact was used to document actin dynamics in the filamentous fungus Aspergillus nidulans. Contractile double rings were observed at septa, and annular clusters of puncta were seen subtending growing hyphal tips, corresponding to the well-characterized subapical endocytic collar. However, Lifeact also revealed two additional structures. One, an apical array, was dynamic on the face opposite the tip, while a subapical web was dynamic on the apical face and was located several microns behind the growth site. Each was observed turning into the other over time, implying that they could represent different localizations of the same structure, although hyphae with a subapical web grew faster than those exhibiting an apical array. The subapical web has not been documented in any filamentous fungus to date, and is separate from the networks of F-actin seen in other tip-growing organisms surrounding septa or stationary along the plasmalemma.

Polo-like kinase 1 (PLK1) is involved in essential events of cell cycle including mitosis in which it participates in centrosomal microtubule nucleation, spindle bipolarity establishment and cytokinesis. Although PLK1 function has been studied in cycling cancer cells, only limited data are known about its role in the first mitosis of mammalian zygotes. During the 1-cell stage of mouse embryo development, the acentriolar spindle is formed and the shift from acentriolar to centrosomal spindle formation progresses gradually throughout the preimplantation stage, thus providing a unique possibility to study acentriolar spindle formation. We have shown previously that PLK1 activity is not essential for entry into first mitosis, but is required for correct spindle formation and anaphase onset in 1-cell mouse embryos. In the present study, we extend this knowledge by employing quantitative confocal live cell imaging to determine spindle formation kinetics in the absence of PLK1 activity and answer the question whether metaphase arrest at PLK1-inhibited embryos is associated with low anaphase-promoting complex/cyclosome (APC/C) activity and consequently high securin level. We have shown that inhibition of PLK1 activity induces a delay in onset of acentriolar spindle formation during first mitosis. Although these PLK1-inhibited 1-cell embryos were finally able to form a bipolar spindle, not all chromosomes were aligned at the metaphase equator. PLK1-inhibited embryos were arrested in metaphase without any sign of APC/C activation with high securin levels. Our results document that PLK1 controls the onset of spindle assembly and spindle formation, and is essential for APC/C activation before anaphase onset in mouse zygotes.

Oocyte maturation is known to affect the chances for successful fertilization, embryonic development, establishment of pregnancy and delivery of a live, healthy, and viable offspring. Two-photon laser scanning microscopy (TPLSM) has previously been used to evaluate early embryonic development without a detectable impairment of subsequent development, but has never been applied to assess mammalian oocytes throughout in vitro maturation (IVM). Visualization of structures within live oocytes during IVM, followed by fertilization and embryo culture, may improve the understanding of oocyte maturation. To visualize structures within bovine oocytes using TPLSM, it is necessary to remove the cumulus cells that normally surround the oocyte during maturation. Repeated visualization of structures within the same oocyte is possible, if movement of the oocyte can be avoided. In this article, we describe the development of a method for repeated intravital imaging of denuded bovine oocytes using an upright TPLSM equipped with a specially constructed incubator. Oocytes were stained with Hoechst 33258, and the nuclear structures were evaluated. Oocyte fertilization rate was not affected by TPLSM exposure, but the developmental capacity of the denuded oocytes was significantly reduced. This is, to our knowledge, the first article describing repeated intravital imaging during mammalian oocyte maturation using TPLSM.