Nuclei migrate through constricted spaces using microtubule motors and actin networks in C. elegans hypodermal cells.

Cellular migrations through constricted spaces are a crucial aspect of many developmental and disease processes including hematopoiesis, inflammation and metastasis. A limiting factor in these events is nuclear deformation. Here, we establish an in vivo model in which nuclei can be visualized while moving through constrictions and use it to elucidate mechanisms for nuclear migration. C. elegans hypodermal P-cell larval nuclei traverse a narrow space that is about 5% their width. This constriction is blocked by fibrous organelles, structures that pass through P cells to connect the muscles to cuticle. Fibrous organelles are removed just prior to nuclear migration, when nuclei and lamins undergo extreme morphological changes to squeeze through the space. Both actin and microtubule networks are organized to mediate nuclear migration. The LINC complex, consisting of the SUN protein UNC-84 and the KASH protein UNC-83, recruits dynein and kinesin-1 to the nuclear surface. Both motors function in P-cell nuclear migration, but dynein, functioning through UNC-83, plays a more central role as nuclei migrate towards minus ends of polarized microtubule networks. Thus, the nucleoskeleton and cytoskeleton are coordinated to move nuclei through constricted spaces.

A dynamic meiotic SUN belt includes the zygotene-stage telomere bouquet and is disrupted in chromosome segregation mutants of maize (Zea mays L.).

The nuclear envelope (NE) plays an essential role in meiotic telomere behavior and links the cytoplasm and nucleoplasm during homologous chromosome pairing and recombination in many eukaryotic species. Resident NE proteins including SUN (Sad-1/UNC-84) and KASH (Klarsicht/ANC-1/Syne-homology) domain proteins are known to interact forming the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex that connects chromatin to the cytoskeleton. To investigate the possible cross-kingdom conservation of SUN protein functions in plant meiosis, we immunolocalized maize SUN2 using 3D microscopy of pollen mother cells from maize (Zea mays L.), a large-genome plant model with a canonical NE zygotene-stage telomere bouquet. We detected SUN2 at the nuclear periphery and found that it exhibited a distinct belt-like structure that transitioned to a half-belt during the zygotene stage and back to a full belt during and beyond the pachytene stage. The zygotene-stage half-belt SUN structure was shown by 3D immuno-FISH to include the NE-associated telomere cluster that defines the bouquet stage and coincides with homologous chromosome synapsis. Microtubule and filamentous actin staining patterns did not show any obvious belt or a retracted-like structure other than a general enrichment of tubulin staining distributed widely around the nucleus and throughout the cytoplasm. Genetic disruption of the meiotic SUN belt staining patterns with three different meiosis-specific mutants, desynaptic (dy1), asynaptic1 (as1), and divergent spindle1 (dv1) provides additional evidence for the role of the nuclear envelope in meiotic chromosome behavior. Taking into account all of the observations from this study, we propose that the maize SUN belt is directly or indirectly involved in meiotic telomere dynamics, chromosome synapsis, and possibly integration of signals and forces across the meiotic prophase nuclear envelope.

Three-dimensional acrylamide fluorescence in situ hybridization for plant cells.

Plant meiosis involves complex and dynamic processes that occur within the space inside the nucleus. Direct inspection of meiotic chromosomes by fluorescence microscopy has been used to investigate many of these processes. In particular, optical sectioning microscopy of fluorescence in situ hybridization (FISH)-stained nuclei provides three-dimensional spatial information about the organization and distribution of specific sequences and chromosomal loci within the nucleus. Here we provide a fully detailed three-dimensional (3D) acrylamide FISH method for the analysis of plant meiotic nuclei. Several examples illustrate the versatility of this technique for the investigation of meiotic telomere dynamics in maize, Arabidopsis, and oat. Additional examples of 3D FISH include chromosome painting in a maize chromosome-addition line of oat and telomere FISH with maize nuclei from plants expressing a fluorescently tagged fusion protein, histone H2B-mCherry.

The maize (Zea mays) desynaptic (dy) mutation defines a pathway for meiotic chromosome segregation, linking nuclear morphology, telomere distribution and synapsis.

Meiosis involves a dramatic reorganization of the genetic material, along with changes in the architecture of the nucleoplasm and cytoplasm. In the opisthokonts, nuclear envelope and meiotic chromosome behavior are coordinated by forces generated in the cytoplasm and transferred to the nucleus by the nuclear-envelope protein linkers SUN and KASH. During meiotic prophase I, the telomere bouquet arrangement has roles in interhomolog recognition, pairing, synapsis, interlock resolution and homologous chromosome recombination. The maize desynaptic (dy) mutant is defective in homologous chromosome synapsis, recombination, telomere-nuclear envelope interactions and chromosome segregation. A detailed three-dimensional cytological analysis of dy revealed telomere misplacement during the bouquet stage, synaptic irregularities, nuclear envelope distortion and chromosome bridges at anaphase I. Using linkage and B-A translocation mapping, we placed dy on the long arm of chromosome 3, genetic bin 3.06. SSR marker analysis narrowed the mapping interval to 9 cM. Candidate genes in this region include a PM3-type SUN domain protein, ZmSUN3. No obvious genetic lesions were found in the ZmSUN3 allele of dy, but a conspicuous splice variant, ZmSUN3-sv1, was observed in mRNA from dy. The variant message is predicted to result in the synthesis of a truncated ZmSUN3 protein lacking two C-terminal transmembrane domains. Other potential candidate genes relevant to the documented phenotypes were also considered. In summary, this study reveals that dy causes disruption of a central meiotic pathway connecting nuclear envelope integrity to telomere localization and synapsis during meiotic prophase.

Innate immunity in transplant tolerance and rejection.

Historically, research on transplant rejection and tolerance has focused on cells of the adaptive immune system, especially T cells. Anti-graft effector T cells are necessary and sufficient for the rejection of most allografts, while regulatory T cells, either arising naturally or as a result of a specific treatment regimen, are crucial to long-term graft tolerance. Although the role of T cells in transplant rejection and tolerance is well-established, the role that the innate immune system plays in these processes is only recently being appreciated. Cells of the innate immune system, such as dendritic cells (DCs) and natural killer cells, can become activated by microbial products or endogenous pro-inflammatory ligands released during the mechanical and ischemia-reperfusion injury associated with transplantation surgery, promoting the initiation of T-cell responses against the graft. In addition, innate immune cells are required for acute and chronic rejection in certain animal transplant models and by extension perhaps in clinical transplantation. However, innate immune cells are also implicated in the establishment of transplant tolerance through mechanisms including elimination or inhibition of activated host effector T cells and killing of donor DCs. A deeper understanding of the functions of the innate immune system during transplantation may lead to more successful tolerance strategies.

Structure and expression of the maize (Zea mays L.) SUN-domain protein gene family: evidence for the existence of two divergent classes of SUN proteins in plants.

BACKGROUND: The nuclear envelope that separates the contents of the nucleus from the cytoplasm provides a surface for chromatin attachment and organization of the cortical nucleoplasm. Proteins associated with it have been well characterized in many eukaryotes but not in plants. SUN (Sad1p/Unc-84) domain proteins reside in the inner nuclear membrane and function with other proteins to form a physical link between the nucleoskeleton and the cytoskeleton. These bridges transfer forces across the nuclear envelope and are increasingly recognized to play roles in nuclear positioning, nuclear migration, cell cycle-dependent breakdown and reformation of the nuclear envelope, telomere-led nuclear reorganization during meiosis, and karyogamy. RESULTS: We found and characterized a family of maize SUN-domain proteins, starting with a screen of maize genomic sequence data. We characterized five different maize ZmSUN genes (ZmSUN1-5), which fell into two classes (probably of ancient origin, as they are also found in other monocots, eudicots, and even mosses). The first (ZmSUN1, 2), here designated canonical C-terminal SUN-domain (CCSD), includes structural homologs of the animal and fungal SUN-domain protein genes. The second (ZmSUN3, 4, 5), here designated plant-prevalent mid-SUN 3 transmembrane (PM3), includes a novel but conserved structural variant SUN-domain protein gene class. Mircroarray-based expression analyses revealed an intriguing pollen-preferred expression for ZmSUN5 mRNA but low-level expression (50-200 parts per ten million) in multiple tissues for all the others. Cloning and characterization of a full-length cDNA for a PM3-type maize gene, ZmSUN4, is described. Peptide antibodies to ZmSUN3, 4 were used in western-blot and cell-staining assays to show that they are expressed and show concentrated staining at the nuclear periphery. CONCLUSIONS: The maize genome encodes and expresses at least five different SUN-domain proteins, of which the PM3 subfamily may represent a novel class of proteins with possible new and intriguing roles within the plant nuclear envelope. Expression levels for ZmSUN1-4 are consistent with basic cellular functions, whereas ZmSUN5 expression levels indicate a role in pollen. Models for possible topological arrangements of the CCSD-type and PM3-type SUN-domain proteins are presented.

Evidence for participation of uterine natural killer cells in the mechanisms responsible for spontaneous preterm labor and delivery.

OBJECTIVE: The purpose of this study was to determine in a mouse model whether uterine natural killer (uNK) cell cytotoxic activation induces infection/inflammation-associated preterm labor and delivery. STUDY DESIGN: Wild type or interleukin (IL)-10(-/-) mice were injected intraperitoneally with lipopolysaccharide on gestational day 14. Mice were either killed for collection of uteroplacental tissue, spleen, and serum or allowed to deliver. Uteroplacental tissue was used for histology and characterization of uNK cells. RESULTS: Low-dose lipopolysaccharide treatment triggered preterm labor and delivery in IL-10(-/-), but not wild type mice, in a manner independent of progesterone levels. Preterm labor and delivery in IL-10(-/-) mice was associated with an increased number and placental infiltration of cytotoxic uNK cells and placental cell death. Depletion of NK cells or tumor necrosis factor (TNF)-alpha neutralization in these mice restored term delivery. Furthermore, TNF-alpha neutralization prevented uNK cell infiltration and placental cell apoptosis. CONCLUSION: The uNK cell-TNF-alpha-IL-10 axis plays an important role in the genesis of infection/inflammation-induced preterm labor/delivery.

TLR6 modulates first trimester trophoblast responses to peptidoglycan.

Intrauterine bacterial infections are a well-established cause of pregnancy complications. One key observation in a number of abnormal pregnancies is that placental apoptosis is significantly elevated. First trimester trophoblast cells are known to express TLR1 and TLR2 and to undergo apoptosis following exposure to Gram-positive bacterial peptidoglycan (PDG). Thus, the objectives of this study were to determine whether PDG-induced pregnancy complications are associated with placental apoptosis and to characterize the cellular mechanisms involved. We have demonstrated, using an animal model, that delivery of PDG to pregnant mice early in gestation resulted in highly elevated placental apoptosis, evidenced by trophoblast M-30 and active caspase 3 immunostaining. Using an in vitro model of human first trimester trophoblasts, apoptosis induced by PDG was found to be mediated by both TLR1 and TLR2 and that this could be blocked by the presence of TLR6. Furthermore, in the presence of TLR6, exposure to PDG resulted in trophoblast NF-kappaB activation and triggered these cells to secrete IL-8 and IL-6. The findings of this study suggest that a Gram-positive bacterial infection, through TLR2 and TLR1, may directly promote the elevated trophoblast cell death and that this may be the underlying mechanism of pregnancy complications, such as preterm delivery. Furthermore, the expression of TLR6 may be a key factor in determining whether the response to PDG would be apoptosis or inflammation.

Genes regulating implantation and fetal development: a focus on mouse knockout models.

Timely and efficient regulation of blastocyst implantation and fetal growth are essential for the successful reproduction of viviparous mammals. Disruptions in this regulation can result in a wide variety of human gestational complications including infertility, spontaneous abortion, fetal growth restriction, and premature delivery. The role of several groups of factors, including cytokines, hormones, transcription factors, extra-cellular proteinases, and angiogenic factors has been suggested in both implantation and regulation of fetal growth. Due to the inherent difficulties of studying implantation and fetal development in humans, much of our knowledge of the genes involved in these processes has been derived from animal models. The critical genetic loci involved in blastocyst implantation and fetal growth will be discussed with a focus on those genes with available mouse knockout models.

Uterine NK cells mediate inflammation-induced fetal demise in IL-10-null mice.

Specialized NK cells are recruited in high numbers to the mammalian embryo implantation sites, yet remain pregnancy compatible. It is not well understood whether uterine NK (uNK) cells become adversely activated and mediate fetal demise, a common complication of early pregnancy. In this study we show that mating of IL-10(-/-) mice resulted in fetal resorption or intrauterine growth restriction in response to very low doses of LPS. Pregnancy in congenic wild-type mice was normal even at 10-fold higher LPS doses. Fetal resorption in IL-10(-/-) mice was associated with a significant increase in uNK cell cytotoxic activation and invasion into the placenta. Depletion of uNK cells, TNF-alpha neutralization, or IL-10 administration rescued pregnancy in LPS-treated IL-10(-/-) animals. Our results identify an immune mechanism of fetal demise involving IL-10 deficiency, NK cells, and inflammation. These results may provide insight into adverse pregnancy outcomes in humans.