Overlapping roles for PLK1 and Aurora A during meiotic centrosome biogenesis in mouse spermatocytes

The establishment of bipolar spindles during meiotic divisions ensures faithful chromosome segregation to prevent gamete aneuploidy. We analyzed centriole duplication, as well as centrosome maturation and separation during meiosis I and II using mouse spermatocytes. The first round of centriole duplication occurs during early prophase I, and then, centrosomes mature and begin to separate by the end of prophase I to prime formation of bipolar metaphase I spindles. The second round of centriole duplication occurs at late anaphase I, and subsequently, centrosome separation coordinates bipolar segregation of sister chromatids during meiosis II. Using a germ cell‐specific conditional knockout strategy, we show that Polo‐like kinase 1 and Aurora A kinase are required for centrosome maturation and separation prior to metaphase I, leading to the formation of bipolar metaphase I spindles. Furthermore, we show that PLK1 is required to block the second round of centriole duplication and maturation until anaphase I. Our findings emphasize the importance of maintaining strict spatiotemporal control of cell cycle kinases during meiosis to ensure proficient centrosome biogenesis and, thus, accurate chromosome segregation during spermatogenesis.     

Heat shock protein 25 plays multiple roles during mouse skin development

Heat shock protein (Hsp) 25 is a member of the small Hsp family. High levels of Hsp25 can be detected in skin. During adult epidermis differentiation, the concentration of Hsp25 increases as the distance of keratinocytes from the basal layer increases, in parallel with the extent of keratinization. We previously showed that Hsp25, mouse keratin (MK) 5, and MK14 participated in the formation of characteristic ring-shaped aggregates during the differentiation of the PAM212 keratinocyte cell line. We suggested that Hsp25 was involved in the disorganization of the MK5-MK14 keratin network before the establishment of the MK1-MK10 keratin network at the beginning of epidermis stratification. In this study, we have investigated the distribution of Hsp25 and keratins throughout skin development. We demonstrate that the distribution of Hsp25 and MK5 in the epidermis at the beginning of stratification and before keratinization is similar to that observed in PAM212 keratinocytes. These results indicate that there is a strong correlation between the mechanism we described ex vivo and the events taking place in vivo. Moreover, we show that Hsp25 is produced in different cell types in the epidermis and in the hair follicle at different stages of their development. Thus, our results suggest that Hsp25 is involved in more than one process during skin development.     

Distinct roles for neuropilin1 and neuropilin2 during mouse corneal innervation

Trigeminal sensory innervation of the cornea is critical for protection and synthesis of neuropeptides required for normal vision. Little is known about axon guidance during mammalian corneal innervation. In contrast to the chick where a pericorneal nerve ring forms via Npn/Sema signaling, mouse corneal axons project directly into the presumptive cornea without initial formation of an analogous nerve ring. Here we show that during development of the mouse cornea, Npn1 is strongly expressed by the trigeminal ganglion whereas Npn2 is expressed at low levels. At the same time Sema3A and Sema3F are expressed in distinct patterns in the ocular tissues. Npn1(sema-/-) mutant corneas become precociously and aberrantly innervated by nerve bundles that project further into the corneal stroma. In contrast, stromal innervation was not affected in Npn2(-/-) mutants. The corneal epithelium was prematurely innervated in both Npn1(sema-/-) and Npn2(-/-) mutants. These defects were exacerbated in Npn1(sema-/-);Npn2(-/-) double mutants, which in addition showed ectopic innervation of the region between the optic cup and lens vesicle. Collectively, our data show that Sema3A/Npn1 and Sema3F/Npn2 signaling play distinct roles and both are required for proper innervation of the mouse cornea.     

Epstein-Barr virus nuclear antigen 3C (EBNA3C) interacts with the metabolism sensing C-terminal binding protein (CtBP) repressor to upregulate host genes

Epstein-Barr virus (EBV) infection is associated with the development of specific types of lymphoma and some epithelial cancers. EBV infection of resting B-lymphocytes in vitro drives them to proliferate as lymphoblastoid cell lines (LCLs) and serves as a model for studying EBV lymphomagenesis. EBV nuclear antigen 3C (EBNA3C) is one of the genes required for LCL growth and previous work has suggested that suppression of the CDKN2A encoded tumor suppressor p16^INK4A and possibly p14^ARF is central to EBNA3C’s role in this growth transformation. To directly assess whether loss of p16 and/or p14 was sufficient to explain EBNA3C growth effects, we used CRISPR/Cas9 to disrupt specific CDKN2A exons in EBV transformed LCLs. Disruption of p16 specific exon 1α and the p16/p14 shared exon 2 were each sufficient to restore growth in the absence of EBNA3C. Using EBNA3C conditional LCLs knocked out for either exon 1α or 2, we identified EBNA3C induced and repressed genes. By trans-complementing with EBNA3C mutants, we determined specific genes that require EBNA3C interaction with RBPJ or CtBP for their regulation. Unexpectedly, interaction with the CtBP repressor was required not only for repression, but also for EBNA3C induction of many host genes. Contrary to previously proposed models, we found that EBNA3C does not recruit CtBP to the promoters of these genes. Instead, our results suggest that CtBP is bound to these promoters in the absence of EBNA3C and that EBNA3C interaction with CtBP interferes with the repressive function of CtBP, leading to EBNA3C mediated upregulation.     

Synergistic roles of acyl‐CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. ¹³C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.     

Isoform-specific roles of the Drosophila filamin-type protein Jitterbug (Jbug) during development

Filamins are highly conserved actin-crosslinking proteins that regulate organization of the actin cytoskeleton. As key components of versatile signaling scaffolds, filamins are implicated in developmental anomalies and cancer. Multiple isoforms of filamins exist, raising the possibility of distinct functions for each isoform during development and in disease. Here, we provide an initial characterization of jitterbug (jbug), which encodes one of the two filamin-type proteins in Drosophila. We generate Jbug antiserum that recognizes all of the spliced forms and reveals differential expression of different Jbug isoforms during development, and a significant maternal contribution of Jbug protein. To reveal the function of Jbug isoforms, we create new genetic tools, including a null allele that deletes all isoforms, hypomorphic alleles that affect only a subset, and UAS lines for Gal4-driven expression of the major isoforms. Using these tools, we demonstrate that Jbug is required for viability and that specific isoforms are required in the formation of actin-rich protrusions including thoracic bristles in adults and ventral denticles in the embryo. We also show that specific isoforms of Jbug show differential localization within epithelia and that maternal and zygotic loss of jbug disrupts Crumbs (Crb) localization in several epithelial cell types.     

Overlapping and divergent localization of Frem1 and Fras1 and its functional implications during mouse embryonic development

Frem1 belongs to a family of structurally related extracellular matrix proteins of which Fras1 is the founding member. Mutations in Fras1 and Frem1 have been identified in mouse models for Fraser syndrome, which display a strikingly similar embryonic skin blistering phenotype due to impaired dermal-epidermal adhesion. Here we show that Frem1 originates from both epithelial and mesenchymal cells, in contrast to Fras1 that is exclusively derived from epithelia. However, both proteins are localized in an absolutely overlapping fashion in diverse epithelial basement membranes. At the ultrastructural level, Frem1 exhibits a clustered arrangement in the sublamina densa coinciding with fibrillar structures reminiscent of anchoring fibrils. Furthermore, in addition to its extracellular deposition, around E16, Frem1 displays an intracellular distribution in distinct epidermal cell types such as the periderm layer and basal keratinocytes. Since periderm cells are known to participate in temporary epithelial fusions like embryonic eyelid closure, defective function of Frem1 in these cells could provide a molecular explanation for the "eyes open at birth" phenotype, a feature unique for Frem1 deficient mouse mutants. Finally, we demonstrate loss of Frem1 localization in the basement membrane but not in periderm cells in the skin of Fras1(-/-) embryos. Taken together, our findings indicate that besides a cooperative function with Fras1 in embryonic basement membranes, Frem1 can also act independently in processes related to epidermal differentiation.     

Overlapping and selective roles of endothelial intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 in lymphocyte trafficking

The integrin LFA-1 interacts with a variety of ligands termed ICAMs. ICAM-1 and ICAM-2 are both expressed on endothelium and serve as counterreceptors during lymphocyte trafficking. In this study, we analyzed their relative contribution to lymphocyte recirculation through lymph nodes and to recruitment into lung and inflamed skin by blocking mAbs against ICAM-1 and ICAM-2 and mice deficient for ICAM-1. The entry of lymphocytes into peripheral and mesenteric lymph nodes was found to be unaffected by the functional deletion of either ICAM-1 or ICAM-2. However, when both pathways were blocked, recirculation through lymph nodes was strongly reduced. Trapping of lymphocytes in the lung after i.v. injection is partly mediated by LFA-1/ICAM interactions; the data presented in this study show an exclusive role of ICAM-1 in LFA-1-dependent lung trapping. Similarly, ICAM-1, but not ICAM-2, was required for the migration of T effector cells into the inflamed skin. These results indicate that ICAM-1 and ICAM-2 have redundant functions in lymphocyte recirculation through lymph nodes, but ICAM-1 is unique in supporting migration into inflamed sites and trapping within the lung.     

Distinct roles for visceral endoderm during embryonic mouse development.

The murine visceral endoderm is an extraembryonic cell layer that appears prior to gastrulation and performs critical functions during embryogenesis. The traditional role ascribed to the visceral endoderm entails nutrient uptake and transport. Besides synthesizing a number of specialized proteins that facilitate uptake, digestion, and secretion of nutrients, the extraembryonic visceral endoderm coordinates blood cell differentiation and vessel formation in the adjoining mesoderm, thereby facilitating efficient exchange of nutrients and gases between the mother and embryo. Recent studies suggest that in addition to this nutrient exchange function the visceral endoderm overlying the egg cylinder stage embryo plays an active role in guiding early development. Cells in the anterior visceral endoderm function as an early organizer. Prior to formation of the primitive streak, these cells express specific gene products that specify the fate of underlying embryonic tissues. In this review we highlight recent investigations demonstrating this dual role for visceral endoderm as a provider of both nutrients and developmental cues for the early embryo.