Ingrid C. McCall

Microtubules regulate disassembly of epithelial apical junctions

BackgroundEpithelial tight junction (TJ) and adherens junction (AJ) form the apical junctional complex (AJC) which regulates cell-cell adhesion, paracellular permeability and cell polarity. The AJC is anchored on cytoskeletal structures including actin microfilaments and microtubules. Such cytoskeletal interactions are thought to be important for the assembly and remodeling of apical junctions. In the present study, we investigated the role of microtubules in disassembly of the AJC in intestinal epithelial cells using a model of extracellular calcium depletion.ResultsCalcium depletion resulted in disruption and internalization of epithelial TJs and AJs along with reorganization of perijunctional F-actin into contractile rings. Microtubules reorganized into dense plaques positioned inside such F-actin rings. Depolymerization of microtubules with nocodazole prevented junctional disassembly and F-actin ring formation. Stabilization of microtubules with either docetaxel or pacitaxel blocked contraction of F-actin rings and attenuated internalization of junctional proteins into a subapical cytosolic compartment. Likewise, pharmacological inhibition of microtubule motors, kinesins, prevented contraction of F-actin rings and attenuated disassembly of apical junctions. Kinesin-1 was enriched at the AJC in cultured epithelial cells and it also accumulated at epithelial cell-cell contacts in normal human colonic mucosa. Furthermore, immunoprecipitation experiments demonstrated association of kinesin-1 with the E-cadherin-catenin complex.ConclusionOur data suggest that microtubules play a role in disassembly of the AJC during calcium depletion by regulating formation of contractile F-actin rings and internalization of AJ/TJ proteins.

Effects of phenol on barrier function of a human intestinal epithelial cell line correlate with altered tight junction protein localization.

Phenol contamination of soil and water has raised concerns among people living near phenol-producing factories and hazardous waste sites containing the chemical. Phenol, particularly in high concentrations, is an irritating and corrosive substance, making mucosal membranes targets of toxicity in humans. However, few data on the effects of phenol after oral exposure exist. We used an in vitro model employing human intestinal epithelial cells (SK-CO15) cultured on permeable supports to examine effects of phenol on epithelial barrier function. We hypothesized that phenol disrupts epithelial barrier by altering tight junction (TJ) protein expression. The dose-response effect of phenol on epithelial barrier function was determined using transepithelial electrical resistance (TER) and FITC-dextran permeability measurements. We studied phenol-induced changes in cell morphology and expression of several tight junction proteins by immunofluorescence and Western blot analysis. Effects on cell viability were assessed by MTT, Trypan blue, propidium iodide and TUNEL staining. Exposure to phenol resulted in decreased TER and increased paracellular flux of FITC-dextran in a dose-dependent manner. Delocalization of claudin-1 and ZO-1 from TJs to cytosol correlated with the observed increase in permeability after phenol treatment. Additionally, the decrease in TER correlated with changes in the distribution of a membrane raft marker, suggesting phenol-mediated effects on membrane fluidity. Such observations were independent of effects of phenol on cell viability as enhanced permeability occurred at doses of phenol that did not cause cell death. Overall, these findings suggest that phenol may affect transiently the lipid bilayer of the cell membrane, thus destabilizing TJ-containing microdomains.

Novel structural determinants on SIRPα that mediate binding to CD47.

Signal regulatory proteins (SIRP-α, -β, and -γ) are important regulators of several innate immune functions that include leukocyte migration. Membrane distal (D1) domains of SIRPα and SIRPγ, but not SIRPβ, mediate binding to a cellular ligand termed CD47. Because the extracellular domains of all SIRPs are highly homologous, we hypothesized that some of the 16 residues unique to SIRPα.D1 mediate binding to CD47. By site-directed mutagenesis, we determined that SIRPα binding to CD47 is independent of N-glycosylation. We also identified three residues critical for CD47 binding by exchanging residues on SIRPα with corresponding residues from SIRPβ. Cumulative substitutions of the critical residues into SIRPβ resulted in de novo binding of the mutant protein to CD47. Homology modeling of SIRPα.D1 revealed topological relationships among critical residues and allowed the identification of critical residues common to SIRPα and SIRPβ. Mapping these critical residues onto the recently reported crystal structure of SIRPα.D1 revealed a novel region that is required for CD47 binding and is distinct and lateral to another putative CD47 binding site described on that crystal structure. The importance of this lateral region in mediating SIRPα.D1 binding to CD47 was confirmed by epitope mapping analyses of anti-SIRP Abs. These observations highlight a complex nature of the ligand binding requirements for SIRPα that appear to be dependent on two distinct but adjacent regions on the membrane distal Ig loop. A better understanding of the structural basis of SIRPα/CD47 interactions may provide insights into therapeutics targeting pathologic inflammation.

Neutrophil-derived JAML Inhibits Repair of Intestinal Epithelial Injury During Acute Inflammation

Neutrophil transepithelial migration (TEM) during acute inflammation is associated with mucosal injury. Using models of acute mucosal injury in vitro and in vivo, we describe a new mechanism by which neutrophils infiltrating the intestinal mucosa disrupt epithelial homeostasis. We report that junctional adhesion molecule-like protein (JAML) is cleaved from neutrophil surface by zinc metalloproteases during TEM. Neutrophil-derived soluble JAML binds to the epithelial tight junction protein coxsackie-adenovirus receptor (CAR) resulting in compromised barrier and inhibition of wound repair, through decreased epithelial proliferation. The deleterious effects of JAML on barrier and wound repair are reversed with an anti-JAML monoclonal antibody that inhibits JAML–CAR binding. JAML released from transmigrating neutrophils across inflamed epithelia may thus promote recruitment of leukocytes and aid in clearance of invading microorganisms. However, sustained release of JAML under pathologic conditions associated with persistence of large numbers of infiltrated neutrophils would compromise intestinal barrier and inhibit mucosal healing. Thus, targeting JAML–CAR interactions may improve mucosal healing responses under conditions of dysregulated neutrophil recruitment.

The role of Cis dimerization of signal regulatory protein α (SIRPα) in binding to CD47

Interaction of SIRPα with its ligand, CD47, regulates leukocyte functions, including transmigration, phagocytosis, oxidative burst, and cytokine secretion. Recent progress has provided significant insights into the structural details of the distal IgV domain (D1) of SIRPα. However, the structural roles of proximal IgC domains (D2 and D3) have been largely unstudied. The high degree of conservation of D2 and D3 among members of the SIRP family as well as the propensity of known IgC domains to assemble in cis has led others to hypothesize that SIRPα forms higher order structures on the cell surface. Here we report that SIRPα forms noncovalently linked cis homodimers. Treatment of SIRPα-expressing cells with a membrane-impermeable cross-linker resulted in the formation of SDS-stable SIRPα dimers and oligomers. Biochemical analyses of soluble recombinant extracellular regions of SIRPα, including domain truncation mutants, revealed that each of the three extracellular immunoglobulin loops of SIRPα formed dimers in solution. Co-immunoprecipitation experiments using cells transfected with different affinity-tagged SIRPα molecules revealed that SIRPα forms cis dimers. Interestingly, in cells treated with tunicamycin, SIRPα dimerization but not CD47 binding was inhibited, suggesting that a SIRPα dimer is probably bivalent. Last, we demonstrate robust dimerization of SIRPa in adherent, stimulated human neutrophils. Collectively, these data are consistent with SIRPα being expressed on the cell surface as a functional cis-linked dimer.

A Numbers Game: Ribosome Densities, Bacterial Growth, and Antibiotic-Mediated Stasis and Death

ABSTRACT We postulate that the inhibition of growth and low rates of mortality of bacteria exposed to ribosome-binding antibiotics deemed bacteriostatic can be attributed almost uniquely to these drugs reducing the number of ribosomes contributing to protein synthesis, i.e., the number of effective ribosomes. We tested this hypothesis with Escherichia coli K-12 MG1655 and constructs that had been deleted for 1 to 6 of the 7 rRNA (rrn) operons. In the absence of antibiotics, constructs with fewer rrn operons have lower maximum growth rates and longer lag phases than those with more ribosomal operons. In the presence of the ribosome-binding “bacteriostatic” antibiotics tetracycline, chloramphenicol, and azithromycin, E. coli strains with 1 and 2 rrn operons are killed at a substantially higher rate than those with more rrn operons. This increase in the susceptibility of E. coli with fewer rrn operons to killing by ribosome-targeting bacteriostatic antibiotics is not reflected in their greater sensitivity to killing by the bactericidal antibiotic ciprofloxacin, which does not target ribosomes, but also to killing by gentamicin, which does. Finally, when such strains are exposed to these ribosome-targeting bacteriostatic antibiotics, the time before these bacteria start to grow again when the drugs are removed, referred to as the post-antibiotic effect (PAE), is markedly greater for constructs with fewer rrn operons than for those with more rrn operons. We interpret the results of these other experiments reported here as support for the hypothesis that the reduction in the effective number of ribosomes due to binding to these structures provides a sufficient explanation for the action of bacteriostatic antibiotics that target these structures. IMPORTANCE Chemotherapeutic agents, including antibiotics, have been used for more than a century; nevertheless, there are still major gaps in our understanding of how these drugs operate which limit future advances in antibacterial chemotherapy. Although the molecular mechanisms by which antibiotics bind to their target structures are largely known, fundamental questions about how these drugs actually kill and/or inhibit the replication of bacteria remain unanswered and subjects of controversy. We postulate that for the broad class of ribosome-binding bacteriostatic antibiotics, their reducing the number of active (functional) ribosomes per cell provides a sufficient explanation for the abatement of replication and the low rate of decline in densities of viable cells of bacteria exposed to these drugs. Using E. coli K-12 constructs with deletions of from one to six of the seven ribosome-RNA operons and the ribosome-binding bacteriostatic antibiotics tetracycline, chloramphenicol, and azithromycin, we tested this hypothesis. The results of our experiments are consistent with this “numbers game” hypothesis. Chemotherapeutic agents, including antibiotics, have been used for more than a century; nevertheless, there are still major gaps in our understanding of how these drugs operate which limit future advances in antibacterial chemotherapy. Although the molecular mechanisms by which antibiotics bind to their target structures are largely known, fundamental questions about how these drugs actually kill and/or inhibit the replication of bacteria remain unanswered and subjects of controversy. We postulate that for the broad class of ribosome-binding bacteriostatic antibiotics, their reducing the number of active (functional) ribosomes per cell provides a sufficient explanation for the abatement of replication and the low rate of decline in densities of viable cells of bacteria exposed to these drugs. Using E. coli K-12 constructs with deletions of from one to six of the seven ribosome-RNA operons and the ribosome-binding bacteriostatic antibiotics tetracycline, chloramphenicol, and azithromycin, we tested this hypothesis. The results of our experiments are consistent with this “numbers game” hypothesis.

Phagocytes, Antibiotics, and Self-Limiting Bacterial Infections

Most antibiotic use in humans is to reduce the magnitude and term of morbidity of acute, community-acquired infections in immune competent patients, rather than to save lives. Thanks to phagocytic leucocytes and other host defenses, the vast majority of these infections are self-limiting. Nevertheless, there has been a negligible amount of consideration of the contribution of phagocytosis and other host defenses in the research for, and the design of, antibiotic treatment regimens, which hyper-emphasizes antibiotics as if they were the sole mechanism responsible for the clearance of infections. Here, we critically review this approach and its limitations. With the aid of a heuristic mathematical model, we postulate that if the rate of phagocytosis is great enough, for acute, normally self-limiting infections, then (i) antibiotics with different pharmacodynamic properties would be similarly effective, (ii) low doses of antibiotics can be as effective as high doses, and (iii) neither phenotypic nor inherited antibiotic resistance generated during therapy are likely to lead to treatment failure.