The centrosome - diverse functions in fertilization and development across species.

The centrosome is a non-membrane-bound organelle that is conserved across most animal cells and serves various functions throughout the cell cycle. In dividing cells, the centrosome is known as the spindle pole and nucleates a robust microtubule spindle to separate genetic material equally into two daughter cells. In non-dividing cells, the mother centriole, a substructure of the centrosome, matures into a basal body and nucleates cilia, which acts as a signal-transducing antenna. The functions of centrosomes and their substructures are important for embryonic development and have been studied extensively using in vitro mammalian cell culture or in vivo using invertebrate models. However, there are considerable differences in the composition and functions of centrosomes during different aspects of vertebrate development, and these are less studied. In this Review, we discuss the roles played by centrosomes, highlighting conserved and divergent features across species, particularly during fertilization and embryonic development.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during zebrafish left-right organizer development.

An essential process during Danio rerio's left-right organizer (Kupffer's Vesicle, KV) formation is the formation of a motile cilium by developing KV cells which extends into the KV lumen. Beating of motile cilia within the KV lumen directs fluid flow to establish the embryo's left-right axis. However, the timepoint at which KV cells start to form cilia and how cilia formation is coordinated with KV lumen formation have not been examined. We identified that nascent KV cells form cilia at their centrosomes at random intracellular positions that then move towards a forming apical membrane containing cystic fibrosis transmembrane conductance regulator (CFTR). Using optogenetic clustering approaches, we found that Rab35 positive membranes recruit Rab11 to modulate CFTR delivery to the apical membrane, which is required for lumen opening, and subsequent cilia extension into the lumen. Once the intracellular cilia reach the CFTR positive apical membrane, Arl13b-positive cilia extend and elongate in a Rab8 dependent manner into the forming lumen once the lumen reaches an area of 300 µm2. These studies demonstrate the need to acutely coordinate Rab8, Rab11, and Rab35-mediated membrane trafficking events to ensure appropriate timing in lumen and cilia formation during KV development.

Mating and fitness consequences of variation in male allocation in a wind-pollinated plant.

In hermaphrodites, the allocation of resources to each sex function can influence fitness through mating success. A prediction that arises from sex allocation theory is that in wind-pollinated plants, male fitness should increase linearly with investment of resources into male function but there have been few empirical tests of this prediction. In a field experiment, we experimentally manipulated allocation to male function in Ambrosia artemisiifolia (common ragweed) and measured mating success in contrasting phenotypes using genetic markers. We investigated the effects of morphological traits and flowering phenology on male siring success, and on the diversity of mates. Our results provide evidence for a linear relation between allocation to male function, mating, and fitness. We find earlier onset of male flowering time increases reproductive success, whereas later flowering increases the probability of mating with diverse individuals. Our study is among the first empirical tests of the prediction of linear male fitness returns in wind-pollinated plants and emphasizes the importance of a large investment into male function by wind-pollinated plants and mating consequences of temporal variation in sex allocation.

Pericentriolar matrix (PCM) integrity relies on cenexin and polo-like kinase (PLK)1.

Polo-like-kinase (PLK) 1 activity is associated with maintaining the functional and physical properties of the centrosome's pericentriolar matrix (PCM). In this study, we use a multimodal approach of human cells (HeLa), zebrafish embryos, and phylogenic analysis to test the role of a PLK1 binding protein, cenexin, in regulating the PCM. Our studies identify that cenexin is required for tempering microtubule nucleation by maintaining PCM cohesion in a PLK1-dependent manner. PCM architecture in cenexin-depleted zebrafish embryos was rescued with wild-type human cenexin, but not with a C-terminal cenexin mutant (S796A) deficient in PLK1 binding. We propose a model where cenexin's C terminus acts in a conserved manner in eukaryotes, excluding nematodes and arthropods, to sequester PLK1 that limits PCM substrate phosphorylation events required for PCM cohesion.

Imaging the early zebrafish embryo centrosomes following injection of small-molecule inhibitors to understand spindle formation.

During the earliest division stages, zebrafish embryos have large cells that divide rapidly and synchronously to create a cellular layer on top of the yolk. Here, we describe a protocol for monitoring spindle dynamics during these early embryonic divisions. We outline techniques for injecting zebrafish embryos with small-molecule inhibitors toward polo-like kinases, preparing and mounting embryos for three-dimensional imaging using confocal microscopy. These techniques are used to understand how the early zebrafish embryo's centrosome constructs the mitotic spindle. For complete details on the use and execution of this protocol, please refer to Rathbun et al. (2020).

PLK1- and PLK4-Mediated Asymmetric Mitotic Centrosome Size and Positioning in the Early Zebrafish Embryo.

Factors that regulate mitotic spindle positioning remain unclear within the confines of extremely large embryonic cells, such as the early divisions of the vertebrate embryo, Danio rerio (zebrafish). We find that the mitotic centrosome, a structure that assembles the mitotic spindle [1], is notably large in the zebrafish embryo (246.44 ± 11.93 µm2 in a 126.86 ± 0.35 µm diameter cell) compared to a C. elegans embryo (5.78 ± 0.18 µm2 in a 55.83 ± 1.04 µm diameter cell). During embryonic cell divisions, cell size changes rapidly in both C. elegans and zebrafish [2, 3], where mitotic centrosome area scales more closely with changes in cell size compared to changes in spindle length. Embryonic zebrafish spindles contain asymmetrically sized mitotic centrosomes (2.14 ± 0.13-fold difference between the two), with the larger mitotic centrosome placed toward the embryo center in a polo-like kinase (PLK) 1- and PLK4-dependent manner. We propose a model in which uniquely large zebrafish embryonic centrosomes direct spindle placement within disproportionately large cells.