Paul A. Sims

Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia

The function of epithelial cell sheets depends on the integrity of specialized cell–cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR–HMP (cadherin–catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (α-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.

Three-dimensional extracellular matrix textured biomaterials

Clinical and experimental investigations have reported that manufactured surface topographies have significant effects on cell adhesion and tissue integration. However, essentially all previously examined topographies bear little relation to cell adhesion substrates found in biological tissues. In vivo, many cells are adherent to extracellular matrices (ECM), which have an extremely complex 3-D topography in the micrometre to nanometre range. In addition, many studies indicate that micro- and nano-scale mechanical stresses generated by cell-matrix adhesion have significant effects on cellular phenotypic behaviour. In this report we describe methodology for the fabrication of topographic replicas of the subendothelial ECM topography with a biomedical polyurethane. Using three-dimensional high resolution scanning electron microscopy, accurate replication of subendothelial ECM topography from the macroscopic to the macromolecular scale is demonstrated. Bovine aortic endothelial cells cultured on the ECM replicas spread more rapidly and had a three-dimensional appearance and spread areas at confluence which appeared more like endothelial cells in native arteries, compared with cells cultured on untextured control surfaces. Since the fabrication process may be used with many different types of materials, including polymers of synthetic and biological origin, these biomimetic ECM-textured surfaces may find both research and clinical applications.

The cell junction protein VAB-9 regulates adhesion and epidermal morphology in C. elegans

Epithelial cell junctions are essential for cell polarity, adhesion and morphogenesis. We have analysed VAB-9, a cell junction protein in Caenorhabditis elegans. VAB-9 is a predicted four-pass integral membrane protein that has greatest similarity to BCMP1 (brain cell membrane protein 1, a member of the PMP22/EMP/Claudin family of cell junction proteins) and localizes to the adherens junction domain of C. elegans apical junctions. Here, we show that VAB-9 requires HMR-1/cadherin for localization to the cell membrane, and both HMP-1/α-catenin and HMP-2/β-catenin for maintaining its distribution at the cell junction. In vab-9 mutants, morphological defects correlate with disorganization of F-actin at the adherens junction; however, localization of the cadherin–catenin complex and epithelial polarity is normal. These results suggest that VAB-9 regulates interactions between the cytoskeleton and the adherens junction downstream of or parallel to α-catenin and/or β-catenin. Mutations in vab-9 enhance adhesion defects through functional loss of the cell junction genes apical junction molecule 1 (ajm-1) and discs large 1 (dlg-1), suggesting that VAB-9 is involved in cell adhesion. Thus, VAB-9 represents the first characterized tetraspan adherens junction protein in C. elegans and defines a new family of such proteins in higher eukaryotes.

Fluorescence-Integrated Transmission Electron Microscopy Images

This chapter describes high-pressure freezing (HPF) techniques for correlative light and electron microscopy on the same sample. Laser scanning confocal microscopy (LSCM) is exploited for its ability to collect fluorescent, as well as transmitted and back scattered light (BSL) images at the same time. Fluorescent information from a whole mount (preembedding) or from thin sections (post-embedding) can be displayed as a color overlay on transmission electron microscopy (TEM) images. Fluorescence-integrated TEM (F-TEM) images provide a fluorescent perspective to TEM images. The pre-embedding method uses a thin two-part agarose pad to immobilize live Caenorhabditis elegans embryos for LSCM, HPF, and TEM. Pre-embedding F-TEM images display fluorescent information collected from a whole mount of live embryos onto all thin sections collected from that sample. In contrast, the postembedding method uses HPF and freeze substitution with 1% paraformaldehyde in 95% ethanol followed by low-temperature embedding in methacrylate resin. This procedure preserves the structure and function of green fluorescent protein (GFP) as determined by immunogold labeling of GFP, when compared with GFP expression, both demonstrated in the same thin section.

The Inositol 1,4,5-Trisphosphate Receptor Regulates Epidermal Cell Migration in Caenorhabditis elegans

Polarized migration and spreading of epithelial sheets is important during many processes in vivo, including embryogenesis and wound healing. However, the signaling pathways that regulate epithelial migrations are poorly understood. To identify molecular components that regulate the spreading of epithelial sheets, we performed a screen for mutations that perturb epidermal cell migration during embryogenesis in Caenorhabditis elegans. We identified one mutant (jc5) as a weak mutation in itr-1, which encodes the single inositol 1,4,5-trisphosphate receptor (ITR) in C. elegans. During the migration of the embryonic epidermis, jc5 embryos display defects including misdirected migration or premature cessation of migration. Cells that halt their migration have disorganized F-actin and display reduced filopodial protrusive activity at their leading edge. Furthermore, some filopodia formed by epidermal cells in itr-1(jc5) embryos exhibit abnormally long lifetimes. Pharmacological studies with the inositol 1,4,5-trisphosphate antagonist xestospongin C phenocopy these defects, confirming that ITR function is important for proper epidermal migration. Our results provide the first molecular evidence that movements of embryonic epithelial cell sheets can be controlled by ITRs and suggest that such regulation may be a widespread mechanism for coordinating epithelial cell movements during embryogenesis.

αIIbβ3 Redistribution Triggered by Receptor Cross-Linking

Abstract Fibrinogen binding to αIIbβ3 on adherent, spread platelets triggers active, cytoskeletally-directed redistribution of fibrinogen/αIIbβ3 complexes on the platelet surface. Gold-conjugated fibrinogen, unlabeled, soluble fibrinogen, and individual fibrinogen molecules have been demonstrated to trigger receptor redistribution. Here we examine the respective roles of receptor cross-linking and ligand occupancy of receptors in initiating this movement. Monovalent, αIIbβ3-binding fibrinogen fragments RGDS and HHLGGAKQAGDV did not trigger receptor redistribution, suggesting that ligand binding to a single receptor is an insufficient stimulus. Binding of monoclonal antibodies 10E5, AP2, and AP3 to the receptor did not trigger receptor movement. However, cross-linking these receptor-bound monoclonal antibodies by polyclonal anti-mouse IgG or by conjugation of the anti-receptor antibody to large colloidal gold particles triggered receptor redistribution identical in rate, pattern, and final distribution to ...

Corrosion Casting in the Reproduction of the Microsurface Topography of Fibrillar Collagen.

: We have used a two-step casting procedure to create replicas of fibrillar collagen. A negative replica or mold is first created using either Mercox casting resin or a prepolymerized methacrylate resin that was originally described by Murakami. In the second step, a positive replica is made from the first using a solution of polystyrene or polyurethane to coat the methacrylate cast. The resulting replica is removed from the methacrylate cast. Results indicate the highest level of spatial resolution is obtained using a modified Murakami resin for the negative cast and polystyrene for the positive replica. The 29-nm-wide repeat banding structure present on collagen fibrils could be clearly identified in both the methacrylate cast and in the polystyrene positive replica of the methacrylate cast. Thus high-resolution replicas can be obtained. Such replicas may be useful to imprint inplantable devices with biologically relevant textures to improve tissue integration or for the development of textured surfaces for the in vitro expansion of cultured cells.

When Light Microscope Resolution Is Not Enough:Correlational Light Microscopy and Electron Microscopy

Notwithstanding the many ways, amply documented elsewhere in this book, in which fluorescent light microscopy can elucidate biological structure and function, there are times when the available spatial resolution is just not sufficient to answer the biological question. Consequently, there is a need to follow up the initial light microscope (LM) findings by subsequently viewing the LM specimens in the transmission or scanning electron microscope (TEM or SEM). Correlative microscopy of this type has a long history. In 1973, shortly after LM stains had been developed that identified T- and B-lymphocytes, Wetzel viewed the same exact cells, first in the LM and then in the SEM to show that being a T or a B cell bears no relation to whether the cells appeared to be “rough” or “smooth” when viewed in the SEM (Wetzel et al., 1973.

Cytokine and Eicosanoid Production by Cultured Human Monocytes Exposed to Titanium Particulate Debris

: Phagocytosis of particulate wear debris from arthroplasties by macrophages induces an inflammatory response that has been linked to implant loosening and premature failure of artificial joints. Inflammatory mediators released by phagocytic macrophages such as tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), interleukin-6 (IL-6), and prostaglandin E(2) (PGE(2)) are believed to play a central role in the pathogenesis of aseptic loosening. The objective of this study was to characterize titanium alloy particulates that closely match wear debris found around joint arthroplasties and to study their effects on the biosynthesis of inflammatory mediators by cultured monocytes. Peripheral blood monocytes were isolated from healthy human volunteers. Monocytes were cultured in 96-well plates for 24 h, washed, and exposed to three concentrations of titanium particulates and controls from 18-24 h. Supernatants were assayed for TNF-alpha, IL-1beta, IL-6, and PGE(2) activity. Energy dispersive X-ray spectroscopy (EDX) verified the titanium alloy to be Ti6A14V. Scanning electron microscopy (SEM) analysis showed significant titanium particulate heterogeneity with approximately 95% of the particles <1 µm in diameter. SEM and EDX technology was useful in the characterization of the titanium particulates utilized for in vitro models of titanium-induced cytokine release by monocytes. Incubation of titanium particulates (in concentrations similar to those found around loosened prosthetic joints) with cultured monocytes significantly increased their production of TNF-alpha, IL-1beta, and PGE(2).