Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy.

Salmonella is a gram-negative facultative intracellular pathogen that is capable of infecting a variety of hosts. Inside host cells, most Salmonella bacteria reside and replicate within Salmonella-containing vacuoles. They use virulence proteins to manipulate the host cell machinery for their own benefit and hijack the host cytoskeleton to travel toward the perinuclear area. However, a fraction of bacteria escapes into the cytosol where they get decorated with a dense layer of polyubiquitin, which labels the bacteria for clearance by autophagy. More specifically, autophagy receptor proteins recognize the ubiquitinated bacteria and deliver them to autophagosomes, which subsequently fuse to lysosomes. Here, we describe methods used to infect HeLa cells with Salmonella bacteria and to detect their ubiquitination via immunofluorescence and laser scanning confocal microscopy.

ARVCF depletion cooperates with Tbx1 deficiency in the development of 22q11.2DS-like phenotypes in Xenopus.

The 22q11.2 deletion syndrome is a common dominant genetic disorder characterized by a heterozygous deletion of a cluster of genes on chromosome 22q11.2. TBX1, a transcription factor belonging to the T-box gene family, is a key player in the syndrome. However, heterozygosity of Tbx1 in mouse models does not fully recapitulate the phenotypes characteristic of the disease, which may point to the involvement of other genes in the deleted chromosomal region. Hence, we investigated the contribution of the catenin ARVCF, another gene that is deleted in 22q11.2DS. During Xenopus development, ARVCF mRNA is expressed in the pharyngeal arches and depleting either ARVCF or Tbx1 results in delayed migration of the cranial neural crest cells and in defects in the craniofacial skeleton and aortic arches. Moreover, double depletion of ARVCF and Tbx1 revealed that they act cooperatively, indicating that decreased ARVCF levels may also contribute to 22q11.2DS-associated phenotypes.

Thrombomodulin mutations in atypical hemolytic-uremic syndrome.

BACKGROUND: The hemolytic-uremic syndrome consists of the triad of microangiopathic hemolytic anemia, thrombocytopenia, and renal failure. The common form of the syndrome is triggered by infection with Shiga toxin-producing bacteria and has a favorable outcome. The less common form of the syndrome, called atypical hemolytic-uremic syndrome, accounts for about 10% of cases, and patients with this form of the syndrome have a poor prognosis. Approximately half of the patients with atypical hemolytic-uremic syndrome have mutations in genes that regulate the complement system. Genetic factors in the remaining cases are unknown. We studied the role of thrombomodulin, an endothelial glycoprotein with anticoagulant, antiinflammatory, and cytoprotective properties, in atypical hemolytic-uremic syndrome. METHODS: We sequenced the entire thrombomodulin gene (THBD) in 152 patients with atypical hemolytic-uremic syndrome and in 380 controls. Using purified proteins and cell-expression systems, we investigated whether thrombomodulin regulates the complement system, and we characterized the mechanisms. We evaluated the effects of thrombomodulin missense mutations associated with atypical hemolytic-uremic syndrome on complement activation by expressing thrombomodulin variants in cultured cells. RESULTS: Of 152 patients with atypical hemolytic-uremic syndrome, 7 unrelated patients had six different heterozygous missense THBD mutations. In vitro, thrombomodulin binds to C3b and factor H (CFH) and negatively regulates complement by accelerating factor I-mediated inactivation of C3b in the presence of cofactors, CFH or C4b binding protein. By promoting activation of the plasma procarboxypeptidase B, thrombomodulin also accelerates the inactivation of anaphylatoxins C3a and C5a. Cultured cells expressing thrombomodulin variants associated with atypical hemolytic-uremic syndrome had diminished capacity to inactivate C3b and to activate procarboxypeptidase B and were thus less protected from activated complement. CONCLUSIONS: Mutations that impair the function of thrombomodulin occur in about 5% of patients with atypical hemolytic-uremic syndrome.

Coagulation and innate immune responses: can we view them separately?

The horseshoe crab is often referred to as a "living fossil," representative of the oldest classes of arthropods, almost identical to species in existence more than 500 million years ago. Comparative analyses of the defense mechanisms used by the horseshoe crab that allowed it to survive mostly unchanged throughout the millennia reveal a common ancestry of the coagulation and innate immune systems that are totally integrated-indeed, almost inseparable. In human biology, we traditionally view the hemostatic pathways and those regulating innate immune responses to infections and tissue damage as entirely separate entities. But are they? The last couple of decades have revealed a remarkable degree of interplay between these systems, and the linking cellular and molecular mechanisms are rapidly being delineated. In this review, we present some of the major points of intersection between coagulation and innate immunity. We attempt to highlight the potential impact of these findings by identifying recently established paradigms that will hopefully result in the emergence of new strategies to treat a range of inflammatory and hemostatic disorders.

Role of the 2 zebrafish survivin genes in vasculo-angiogenesis, neurogenesis, cardiogenesis and hematopoiesis.

BACKGROUND: Normal growth and development of organisms requires maintenance of a dynamic balance between systems that promote cell survival and those that induce apoptosis. The molecular mechanisms that regulate these processes remain poorly understood, and thus further in vivo study is required. Survivin is a member of the inhibitor of apoptosis protein (IAP) family, that uniquely also promotes mitosis and cell proliferation. Postnatally, survivin is hardly detected in most tissues, but is upregulated in all cancers, and as such, is a potential therapeutic target. Prenatally, survivin is also highly expressed in several tissues. Fully delineating the properties of survivin in vivo in mice has been confounded by early lethal phenotypes following survivin gene inactivation. RESULTS: To gain further insights into the properties of survivin, we used the zebrafish model. There are 2 zebrafish survivin genes (Birc5a and Birc5b) with overlapping expression patterns during early development, prominently in neural and vascular structures. Morpholino-induced depletion of Birc5a causes profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. Similar abnormalities, all less severe except for hematopoiesis, were evident with suppression of Birc5b. The phenotypes induced by morpholino knockdown of one survivin gene, were rescued by overexpression of the other, indicating that the Birc5 paralogs may compensate for each. The potent vascular endothelial growth factor (VEGF) also entirely rescues the phenotypes induced by depletion of either Birc5a and Birc5b, highlighting its multi-functional properties, as well as the power of the model in characterizing the activities of growth factors. CONCLUSION: Overall, with the zebrafish model, we identify survivin as a key regulator of neurogenesis, vasculo-angiogenesis, hematopoiesis and cardiogenesis. These properties of survivin, which are consistent with those identified in mice, indicate that its functions are highly conserved across species, and point to the value of the zebrafish model in understanding the role of this IAP in the pathogenesis of human disease, and for exploring its potential as a therapeutic target.

p120 catenin is required for morphogenetic movements involved in the formation of the eyes and the craniofacial skeleton in Xenopus.

During Xenopus development, p120 transcripts are enriched in highly morphogenetic tissues. We addressed the developmental function of p120 by knockdown experiments and by expressing E-cadherin mutants unable to bind p120. This resulted in defective eye formation and provoked malformations in the craniofacial cartilage structures, derivatives of the cranial neural crest cells. Closer inspection showed that p120 depletion impaired evagination of the optic vesicles and migration of cranial neural crest cells from the neural tube into the branchial arches. These morphogenetic processes were also affected by p120-uncoupled cadherins or E-cadherin containing a deletion of the juxtamembrane domain. Irrespective of the manipulation that caused the malformations, coexpression of dominant-negative forms of either Rac1 or LIM kinase rescued the phenotypes. Wild-type RhoA and constitutively active Rho kinase caused partial rescue. Our results indicate that, in contrast to invertebrates, p120 is an essential factor for vertebrate development and an adequate balance between cadherin activity and cytoskeletal condition is critical for correct morphogenetic movements.