Big Lessons from Little Yeast: Budding and Fission Yeast Centrosome Structure, Duplication, and Function.

Centrosomes are a functionally conserved feature of eukaryotic cells that play an important role in cell division. The conserved γ-tubulin complex organizes spindle and astral microtubules, which, in turn, separate replicated chromosomes accurately into daughter cells. Like DNA, centrosomes are duplicated once each cell cycle. Although in some cell types it is possible for cell division to occur in the absence of centrosomes, these divisions typically result in defects in chromosome number and stability. In single-celled organisms such as fungi, centrosomes [known as spindle pole bodies (SPBs)] are essential for cell division. SPBs also must be inserted into the membrane because fungi undergo a closed mitosis in which the nuclear envelope (NE) remains intact. This poorly understood process involves events similar or identical to those needed for de novo nuclear pore complex assembly. Here, we review how analysis of fungal SPBs has advanced our understanding of centrosomes and NE events.

Fungal microtubule organizing centers are evolutionarily unstable structures.

For most Eukaryotic species the requirements of cilia formation dictate the structure of microtubule organizing centers (MTOCs). In this study we find that loss of cilia corresponds to loss of evolutionary stability for fungal MTOCs. We used iterative search algorithms to identify proteins homologous to those found in Saccharomyces cerevisiae, and Schizosaccharomyces pombe MTOCs, and calculated site-specific rates of change for those proteins that were broadly phylogenetically distributed. Our results indicate that both the protein composition of MTOCs as well as the sequence of MTOC proteins are poorly conserved throughout the fungal kingdom. To begin to reconcile this rapid evolutionary change with the rigid structure and essential function of the S. cerevisiae MTOC we further analyzed how structural interfaces among proteins influence the rates of change for specific residues within a protein. We find that a more stable protein may stabilize portions of an interacting partner where the two proteins are in contact. In summary, while the protein composition and sequences of the MTOC may be rapidly changing the proteins within the structure have a stabilizing effect on one another. Further exploration of fungal MTOCs will expand our understanding of how changes in the functional needs of a cell have affected physical structures, proteomes, and protein sequences throughout fungal evolution.

The role of gene dosage in budding yeast centrosome scaling and spontaneous diploidization.

Ploidy is the number of whole sets of chromosomes in a species. Ploidy is typically a stable cellular feature that is critical for survival. Polyploidization is a route recognized to increase gene dosage, improve fitness under stressful conditions and promote evolutionary diversity. However, the mechanism of regulation and maintenance of ploidy is not well characterized. Here, we examine the spontaneous diploidization associated with mutations in components of the Saccharomyces cerevisiae centrosome, known as the spindle pole body (SPB). Although SPB mutants are associated with defects in spindle formation, we show that two copies of the mutant in a haploid yeast favors diploidization in some cases, leading us to speculate that the increased gene dosage in diploids 'rescues' SPB duplication defects, allowing cells to successfully propagate with a stable diploid karyotype. This copy number-based rescue is linked to SPB scaling: certain SPB subcomplexes do not scale or only minimally scale with ploidy. We hypothesize that lesions in structures with incompatible allometries such as the centrosome may drive changes such as whole genome duplication, which have shaped the evolutionary landscape of many eukaryotes.

Two developmentally distinct populations of neural crest cells contribute to the zebrafish heart.

Cardiac neural crest cells are essential for outflow tract remodeling in animals with divided systemic and pulmonary circulatory systems, but their contributions to cardiac development in animals with a single-loop circulatory system are less clear. Here we genetically labeled neural crest cells and examined their contribution to the developing zebrafish heart. We identified two populations of neural crest cells that contribute to distinct compartments of zebrafish cardiovascular system at different developmental stages. A stream of neural crest cells migrating through pharyngeal arches 1 and 2 integrates into the myocardium of the primitive heart tube between 24 and 30 h post fertilization and gives rise to cardiomyocytes. A second wave of neural crest cells migrating along aortic arch 6 envelops the endothelium of the ventral aorta and invades the bulbus arteriosus after three days of development. Interestingly, while inhibition of FGF signaling has no effect on the integration of neural crest cells to the primitive heart tube, it prevents these cells from contributing to the outflow tract, demonstrating disparate responses of neural crest cells to FGF signaling. Furthermore, neural crest ablation in zebrafish leads to multiple cardiac defects, including reduced heart rate, defective myocardial maturation and a failure to recruit progenitor cells from the second heart field. These findings add to our understanding of the contribution of neural crest cells to the developing heart and provide insights into the requirement for these cells in cardiac maturation.

PKCµ promotes keratinocyte cell migration through Cx43 phosphorylation-mediated suppression of intercellular communication.

Downregulation of intercellular communication through suppression of gap junctional conductance is necessary during wound healing. Connexin 43 (Cx43), a prominent gap junction protein in skin, is downregulated following wounding to restrict communication between keratinocytes. Previous studies found that PKCµ, a novel PKC isozyme, regulates efficient cutaneous wound healing. However, the molecular mechanism by which PKCµ regulates wound healing remains unknown. We have identified that PKCµ suppresses intercellular communication and enhances cell migration in an in vitro wound healing model by regulating Cx43 containing gap junctions. PKCµ can directly interact with and phosphorylate Cx43 at S368, which leads to Cx43 internalization and downregulation. Finally, utilizing phosphomimetic and non-phosphorylatable S368 substitutions and gap junction inhibitors, we confirmed that PKCµ regulates intercellular communication and in vitro wound healing by controlling Cx43-S368 phosphorylation. These results define PKCµ as a critical regulator of Cx43 phosphorylation to control cell migration and wound healing in keratinocytes.

The PAF1 complex differentially regulates cardiomyocyte specification.

The specification of an appropriate number of cardiomyocytes from the lateral plate mesoderm requires a careful balance of both positive and negative regulatory signals. To identify new regulators of cardiac specification, we performed a phenotype-driven ENU mutagenesis forward genetic screen in zebrafish. In our genetic screen we identified a zebrafish ctr9 mutant with a dramatic reduction in myocardial cell number as well as later defects in primitive heart tube elongation and atrioventricular boundary patterning. Ctr9, together with Paf1, Cdc73, Rtf1 and Leo1, constitute the RNA polymerase II associated protein complex, PAF1. We demonstrate that the PAF1 complex (PAF1C) is structurally conserved among zebrafish and other metazoans and that loss of any one of the components of the PAF1C results in abnormal development of the atrioventricular boundary of the heart. However, Ctr9, Cdc73, Paf1 and Rtf1, but not Leo1, are required for the specification of an appropriate number of cardiomyocytes and elongation of the heart tube. Interestingly, loss of Rtf1 function produced the most severe defects, resulting in a nearly complete absence of cardiac precursors. Based on gene expression analyses and transplantation studies, we found that the PAF1C regulates the developmental potential of the lateral plate mesoderm and is required cell autonomously for the specification of cardiac precursors. Our findings demonstrate critical but differential requirements for PAF1C components in zebrafish cardiac specification and heart morphogenesis.

A Forward Genetic Screen Targeting the Endothelium Reveals a Regulatory Role for the Lipid Kinase Pi4ka in Myelo- and Erythropoiesis.

Given its role as the source of definitive hematopoietic cells, we sought to determine whether mutations initiated in the hemogenic endothelium would yield hematopoietic abnormalities or malignancies. Here, we find that endothelium-specific transposon mutagenesis in mice promotes hematopoietic pathologies that are both myeloid and lymphoid in nature. Frequently mutated genes included previously recognized cancer drivers and additional candidates, such as Pi4ka, a lipid kinase whose mutation was found to promote myeloid and erythroid dysfunction. Subsequent validation experiments showed that targeted inactivation of the Pi4ka catalytic domain or reduction in mRNA expression inhibited myeloid and erythroid cell differentiation in vitro and promoted anemia in vivo through a mechanism involving deregulation of AKT, MAPK, SRC, and JAK-STAT signaling. Finally, we provide evidence linking PI4KAP2, previously considered a pseudogene, to human myeloid and erythroid leukemia.

MT2-MMP expression during early avian morphogenesis.

Membrane-type 2 matrix metalloproteinase (MT2-MMP; also called MMP15) is a membrane-bound protease that degrades extracellular matrix and activates proMMPs such as proMMP-2. MMP-2 expression in avian embryos is well documented, but it is not clear how proMMP-2 is activated during avian embryogenesis. Herein, we report that MT2-MMP mRNA is expressed in several tissues including the neural folds and epidermal ectoderm, intermediate mesoderm, pharyngeal arches, limb buds, and dermis. Several, but not all, of these tissues are known to express MMP-2. These observations suggest MT2-MMP may play a role during embryonic development not only through its own proteolytic activity but also by activating proMMP-2.

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples. Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter (1). We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.