Multiple roles for Cdc42 in cell regulation

The Rho family member Cdc42 can signal through a number of cellular pathways fundamental to growth, differentiation and apoptosis. Recently, information has come at an impressive pace, both with regard to previously identified targets for Cdc42 that regulate the actin cytoskeleton (e.g. WASP) and cellular stress pathways (e.g. PAK) and with regard to newly identified targets such as the coatomer protein complex and PAR6. Recent results hint at a previously unappreciated link between these various cellular processes.     

Multiple roles of integrins in cell motility

Motility is essential for many important biological events, including embryonic development, inflammatory responses, wound healing, and tumor metastasis. During these events cells are in dynamic contact with the extracellular matrix through integrins. Integrins are the primary receptors for extracellular matrix proteins and consequently are required for cell motility. Cells have evolved multiple mechanisms to modulate integrin adhesive functions, which impact cell migration. In addition to providing a mechanism that allows cells to contact the extracellular matrix, integrins also promote intracellular signals that stimulate and regulate cell movement. Here we discuss the role of integrins during the multiple steps of cell migration.     

Multiple essential roles for EzrA in cell division of Staphylococcus aureus

In Bacillus subtilis, EzrA is involved in preventing aberrant formation of FtsZ rings and has also been implicated in the localization cycle of Pbp1. We have identified the orthologue of EzrA in Staphylococcus aureus to be essential for growth and cell division in this organism. Phenotypic analyses following titration of EzrA levels in S. aureus have shown that the protein is required for peptidoglycan synthesis as well as for assembly of the divisome at the midcell and cytokinesis. Protein interaction studies revealed that EzrA forms a complex with both the cytoplasmic components of the division machinery and those with periplasmic domains, suggesting that EzrA may be a scaffold molecule permitting the assembly of the division complex and forming an interface between the cytoplasmic cytoskeletal element FtsZ and the peptidoglycan biosynthetic apparatus active in the periplasm.     

Mutation of a Src phosphorylation site in the PDGF beta-receptor leads to increased PDGF-stimulated chemotaxis but decreased mitogenesis.

Ligand induced activation of the beta-receptor for platelet-derived growth factor (PDGF) leads to activation of Src family tyrosine kinases. We have explored the possibility that the receptor itself is a substrate for Src. We show that Tyr934 in the kinase domain of the PDGF receptor is phosphorylated by Src. Cell lines expressing a beta-receptor mutant, in which Tyr934 was replaced with a phenyalanine residue, showed reduced mitogenic signaling in response to PDGF-BB. In contrast, the mutant receptor mediated increased signals for chemotaxis and actin reorganization. Whereas the motility responses of cells expressing wild-type beta-receptors were attenuated by inhibition of phosphatidylinositol 3'-kinase, those of cells expressing the mutant receptor were only slightly influenced. In contrast, PDGF-BB-induced chemotaxis of the cells with the mutant receptor was attenuated by inhibition of protein kinase C, whereas the chemotaxis of cells expressing the wild-type beta-receptor was less affected. Moreover, the PDGF-BB-stimulated tyrosine phosphorylation of phospholipase C-gamma was increased in the mutant receptor cells compared with wild-type receptor cells. In conclusion, the characteristics of the Y934F mutant suggest that the phosphorylation of Tyr934 by Src negatively modulates a signal transduction pathway leading to motility responses which involves phospholipase C-gamma, and shifts the response to increased mitogenicity.     

Multiple roles of cadmium in cell death and survival

Cadmium is a toxic metal with no known biological function. It is increasingly important as an environmental hazard to both humans and wildlife, and it exemplifies the double edged nature of many toxic substances. Thus, on the one hand cadmium can act as a mitogen, stimulate cell proliferation, inhibit apoptosis, inhibit DNA repair, and promote cancer in a number of tissues. On the other hand, it causes tissue damage, notably in the kidney, by inducing cell death. At low and moderate concentrations in cell culture systems (e.g., 0.1–10 μM) cadmium primarily causes apoptosis, and at higher concentrations (>50 μM) necrosis becomes evident. This generalization appears to hold in vivo. There is also evidence of cadmium-induced autophagy, although whether this is a direct cause of cell death remains uncertain. After discussing these generalities, this review considers the details of apoptotic death, and its inhibition, in renal mesangial cells. We also present evidence for the effect of environmental exposure to cadmium in affecting renal function, and in particular review the evidence for the role of the mesangial cell in cadmium nephrotoxicity.     

Multiple roles for kinases in DNA replication

DNA replication is carried out by the replisome, which includes several proteins that are targets of cell-cycle-regulated kinases. The phosphorylation of proteins such as replication protein A, DNA polymerase-α and -δ, replication factor C, flap endonuclease 1 and DNA ligase I leads to their inactivation, suggesting that phosphorylation is important in the prevention of re-replication. Moreover, the phosphorylation of several of these replication proteins has been shown to block their association with the 'moving platform'—proliferating cell nuclear antigen. Therefore, phosphorylation seems to be a crucial regulator of replisome assembly and DNA replication, although its precise role in these processes remains to be clarified.     

Multiple roles for protein palmitoylation in plants

Palmitoylation, more correctly known as S-acylation, aids in the regulation of cellular functions including stress response, disease resistance, hormone signalling, cell polarisation, cell expansion and cytoskeletal organization. S-acylation is the reversible addition of fatty acids to proteins, which increases their membrane affinity. Membrane-protein interactions are important for signalling complex formation and signal propagation, protein sequestration and segregation, protein stability, and maintaining polarity within the cell. S-acylation is a dynamic modification that modulates the activity and membrane association of many signalling molecules, including ROP GTPases, heterotrimeric G-proteins and calcium-sensing kinases. Recent advances in methods to study S-acylation are permitting an in-depth examination of its function in plants.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

Multiple roles for integrins during development

Summry— Interactions between cells and extracellular matrix play a crucial role during development by controlling tissue remodelling and cell migration. Integrins are the main family of cell surface receptors for extracellular matrix. The knockout of integrin genes in mouse embryos has provided new insights into the function of these receptors during embryonic development and morphogenesis. The lethality observed either during embryonic life or after birth suggests that many integrins are essential.     

Multiple roles for protein phosphatase 1 in regulating the Xenopus early embryonic cell cycle.

Using cytostatic factor metaphase II-arrested extracts as a model system, we show that protein phosphatase 1 is regulated during early embryonic cell cycles in Xenopus. Phosphatase 1 activity peaks during interphase and decreases shortly before the onset of mitosis. A second peak of activity appears in mitosis at about the same time that cdc2 becomes active. If extracts are inhibited in S-phase with aphidicolin, then phosphatase 1 activity remains high. The activity of phosphatase 1 appears to determine the timing of exit from S-phase and entry into M-phase; inhibition of phosphatase 1 by the specific inhibitor, inhibitor 2 (Inh-2), causes premature entry into mitosis, whereas exogenously added phosphatase 1 lengthens the interphase period. Analysis of DNA synthesis in extracts treated with Inh-2, but lacking the A- and B-type cyclins, shows that phosphatase 1 is also required for the process of DNA replication. These data indicate that phosphatase 1 is a component of the signaling pathway that ensures that M-phase is not initiated until DNA synthesis is complete.     

Conflicting roles for a cell surface modification in Salmonella

Chemical modifications of components of the bacterial cell envelope can enhance resistance to antimicrobial agents. Why then are such modifications produced only under specific conditions? Here, we address this question by examining the role of regulated variations in O‐antigen length in the lipopolysaccharide (LPS), a glycolipid that forms most of the outer leaflet of the outer membrane in Gram‐negative bacteria. We determined that activation of the PmrA/PmrB two‐component system, which is the major regulator of LPS alterations in Salmonella enterica serovar Typhimurium, impaired growth of Salmonella in bile. This growth defect required the PmrA‐activated gene wzz_st, which encodes the protein that determines long O‐antigen chain length and confers resistance to complement‐mediated killing. By contrast, this growth defect did not require the wzz_fepE gene, which controls production of very long O‐antigen, or other PmrA‐activated genes that mediate modifications of lipid A or core regions of the LPS. Additionally, we establish that long O‐antigen inhibits growth in bile only in the presence of enterobacterial common antigen, an outer‐membrane glycolipid that contributes to bile resistance. Our results suggest that Salmonella regulates the proportion of long O‐antigen in its LPS to respond to the different conditions it faces during infection.     

Multiple roles for IL-12 in a model of acute septic peritonitis

The present study addressed the role of IL-12 in a murine model of septic peritonitis, induced by cecal ligation and puncture (CLP). Although CLP surgery induced IL-12 production at 6 and 24 h after surgery, IL-12 immunoneutralization was clearly deleterious in this model: 54% of CLP mice receiving preimmune serum survived, whereas mice administered IL-12 antisera prior to CLP experienced a 25% survival rate. IL-12 immunoneutralization not only led to increased mortality, but also appeared to promote a shift away from IL-12 and IFN-gamma, in favor of IL-10. This cytokine shift corresponded to changes in bacterial load, as CLP mice receiving IL-12 antiserum yielded more CFUs from the peritoneal cavity at 24 h after CLP. To address the role of bacterial infection in IL-12 antiserum-induced mortality following CLP, antibiotics were administered for 4 days after surgery. Despite regular antibiotic administration, IL-12 immunoneutralization still reduced survival in CLP mice. Furthermore, histology of the ceca revealed that mice administered IL-12 antisera failed to show typical organization of the damaged cecum wall. Accordingly, Gram staining revealed bacteria within peritoneal fluids from these mice, while peritoneal fluids from CLP mice that received preimmune serum and antibiotics were free of bacteria. Altogether, these data suggested multiple important roles for IL-12 in the evolution of murine septic peritonitis.     

Multiple roles for Hedgehog signaling in zebrafish pituitary development

The endocrine-secreting lobe of the pituitary gland, or adenohypophysis, forms from cells at the anterior margin of the neural plate through inductive interactions involving secreted morphogens of the Hedgehog (Hh), fibroblast growth factor (FGF), and bone morphogenetic protein (BMP) families. To better understand when and where Hh signaling influences pituitary development, we have analyzed the effects of blocking Hh signaling both pharmacologically (cyclopamine treatments) and genetically (zebrafish Hh pathway mutants). While current models state that Shh signaling from the oral ectoderm patterns the pituitary after placode induction, our data suggest that Shh plays a direct early role in both pituitary induction and patterning, and that early Hh signals comes from adjacent neural ectoderm. We report that Hh signaling is necessary between 10 and 15 h of development for induction of the zebrafish adenohypophysis, a time when shh is expressed only in neural tissue. We show that the Hh responsive genes ptc1 and nk2.2 are expressed in preplacodal cells at the anterior margin of the neural tube at this time, indicating that these cells are directly receiving Hh signals. Later (15-20 h) cyclopamine treatments disrupt anterior expression of nk2.2 and Prolactin, showing that early functional patterning requires Hh signals. Consistent with a direct role for Hh signaling in pituitary induction and patterning, overexpression of Shh results in expanded adenohypophyseal expression of lim3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in Prolactin- and Somatolactin-secreting cells. We also use the zebrafish Hh pathway mutants to document the range of pituitary defects that occur when different elements of the Hh signaling pathway are mutated. These defects, ranging from a complete loss of the adenohypophysis (smu/smo and yot/gli2 mutants) to more subtle patterning defects (dtr/gli1 mutants), may correlate to human Hh signaling mutant phenotypes seen in Holoprosencephaly and other congenital disorders. Our results reveal multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland, and suggest that Hh signaling from neural ectoderm is necessary for induction and functional patterning of the vertebrate pituitary gland.     

Multiple roles for elastic fibers in the skin.

Dermal elastic fibers are believed to have a primary role in providing elastic stretch and recoil to the skin. Here we compare the structural arrangement of dermal elastic fibers of chick skin and different animal species. Most elastic fibers in chick skin are derived from cells that line the feather follicle and/or smooth muscle that connects the pterial and apterial muscle bundles to feather follicles. Elastic fibers in the dermis of animals with single, primary hair follicles are derived from cells lining the hair follicle or from the ends of the pili muscle, which anchors the muscle to the matrix or to the hair follicle. Each follicle is interconnected with elastic fibers. Follicles of animals with primary and secondary (wool) hair follicles are also interconnected by elastic fibers, yet only the elastic fibers derived from the primary follicle are connected to each primary follicle. Only the primary hair follicles are connected to the pili muscle. Human skin, but not the skin of other primates, is significantly different from other animals with respect to elastic fiber organization and probably cell of origin. The data suggest that the primary role for elastic fibers in animals, with the possible exception of humans, is movement and/or placement of feathers or hair.