Immunochemical Analysis of Discoidins I and II at the Cell Surface in Wild Type and Aggregation-Defective Mutants of Dictyostelium discoideum

The endogenous lectins discoidins I and II are believed to be primary components of the morphogenetic cell cohesion system of D discoideum. We have developed two immunochemical methods to analyze the association of the discoidins with the cell surface. One method is a two-stage specific antibody binding assay in which intact cells are incubated on ice with rabbit serum (either control serum or antidiscoidin I and II), washed, then incubated with ¹²⁵I-Protein A. Specific antibody binding is defined as the difference between percent radioactivity bound with antidiscoidin versus control serum during the first stage. Substantial specific binding was observed with developed A3 cells but not with vegetative cells, and nearly all of the activity could be removed by pread-sorption of the antiserum with discoidin-Sepharose. As a complementary method, quantitative immunoadsorption analysis was performed in which we tested the ability of intact cells to remove antibodies reactive with purified ¹²⁵I-discoidin I or II. Developed cells, but not vegetative cells, were capable of adsorbing antibodies reactive with discoidin I as well as those reactive with discoidin II. This represents the first demonstration that both lectins are present on the surface of cohesive cells. These procedures, coupled with other methods to analyze soluble discoidin in cell extracts, were used to study discoidin expression in wild type cells and in two newly isolated aggregation-defective mutants. Strain EB-32 fails to aggregate and displays little or no discoidin in cell extracts or at the cell surface. On the other hand, strain EB-18 forms loose amorphous mounds, and expresses substantial quantities of the discoidins, both in cell extracts and at the cell surface. These mutants should prove valuable in studying the organization and regulation of discoidins I and II at the surface of aggregating cells.     

The Cellular Phenotype of Roberts Syndrome Fibroblasts as Revealed by Ectopic Expression of ESCO2

Cohesion between sister chromatids is essential for faithful chromosome segregation. In budding yeast, the acetyltransferase Eco1/Ctf7 establishes cohesion during DNA replication in S phase and in response to DNA double strand breaks in G2/M phase. In humans two Eco1 orthologs exist: ESCO1 and ESCO2. Both proteins are required for proper sister chromatid cohesion, but their exact function is unclear at present. Since ESCO2 has been identified as the gene defective in the rare autosomal recessive cohesinopathy Roberts syndrome (RBS), cells from RBS patients can be used to elucidate the role of ESCO2. We investigated for the first time RBS cells in comparison to isogenic controls that stably express V5- or GFP-tagged ESCO2. We show that the sister chromatid cohesion defect in the transfected cell lines is rescued and suggest that ESCO2 is regulated by proteasomal degradation in a cell cycle-dependent manner. In comparison to the corrected cells RBS cells were hypersensitive to the DNA-damaging agents mitomycin C, camptothecin and etoposide, while no particular sensitivity to UV, ionizing radiation, hydroxyurea or aphidicolin was found. The cohesion defect of RBS cells and their hypersensitivity to DNA-damaging agents were not corrected by a patient-derived ESCO2 acetyltransferase mutant (W539G), indicating that the acetyltransferase activity of ESCO2 is essential for its function. In contrast to a previous study on cells from patients with Cornelia de Lange syndrome, another cohesinopathy, RBS cells failed to exhibit excessive chromosome aberrations after irradiation in G2 phase of the cell cycle. Our results point at an S phase-specific role for ESCO2 in the maintenance of genome stability.     

Filopodia are enriched in a cell cohesion molecule of Mr 80,000 and participate in cell-cell contact formation in Dictyostelium discoideum

During the early phase of Dictyostelium discoideum development, cells undergo chemotactic migration to form tight aggregates. A developmentally regulated surface glycoprotein of Mr 80,000 (gp80) has been implicated in mediating the EDTA-resistant type of cell cohesion at this stage. We have used a monoclonal antibody directed against gp80 to study the topographical distribution of gp80 on the cell surface. Indirect immunofluorescence studies showed that gp80 was primarily localized on the cell surface, with a higher concentration at contact areas. Immunoelectron microscopy was carried out by indirect labeling using protein A-gold, and a nonrandom distribution of gp80 was revealed. In addition to contact regions, gold particles were found preferentially localized on filopodia. Quantitative analysis using transmission electron microscopy (TEM) showed that approximately 60% more gold particles were localized in contact regions in comparison with the noncontact regions, and the filopodial surfaces had a twofold higher gold density. Both TEM and scanning electron microscopy showed that contact areas were enriched in filopodial structures. Filopodia often appeared to adhere to either smooth surfaces or similar filopodial structures of an adjacent cell. These observations suggest that the formation of stable cell-cell contacts involves at least four sequential steps in which filopodia and gp80 probably play an important role in the initial stages of recognition and cohesion among cells.     

A proteomic approach to analysing spheroid formation of two human thyroid cell lines cultured on a random positioning machine

The human cell lines FTC-133 and CGTH W-1, both derived from patients with thyroid cancer, assemble to form different types of spheroids when cultured on a random positioning machine. In order to obtain a possible explanation for their distinguishable aggregation behaviour under equal culturing conditions, we evaluated a proteomic analysis emphasising cytoskeletal and membrane-associated proteins. For this analysis, we treated the cells by ultrasound, which freed up some of the proteins into the supernatant but left some attached to the cell fragments. Both types of proteins were further separated by free-flow IEF and SDS gel electrophoresis until their identity was determined by MS. The MS data revealed differences between the two cell lines with regard to various structural proteins such as vimentin, tubulins and actin. Interestingly, integrin α-5 chains, myosin-10 and filamin B were only found in FTC-133 cells, while collagen was only detected in CGTH W-1 cells. These analyses suggest that FTC-133 cells express surface proteins that bind fibronectin, strengthening the three-dimensional cell cohesion.     

C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus types 1 and 2.

Glycoprotein C from herpes simplex virus type 1 (gC-1 from HSV-1) acts as a receptor for the C3b fragment of the third component of complement on HSV-1-infected cell surfaces. Direct binding assays with purified gC-1 and C3b demonstrate that other viral and cellular proteins are not required for this interaction. Although C3b receptor activity is not expressed on HSV-2-infected cell surfaces, purified gC-2 specifically binds C3b in direct binding assays, suggesting that gC-1 and gC-2 are functionally similar. Here, we used a transient transfection system to further characterize the role of gC-1 and gC-2 as C3b receptors and to localize the site(s) on gC involved in C3b binding. The genes for gC-1 and gC-2 were each cloned into a eucaryotic expression vector containing the Rous sarcoma virus long terminal repeat as the promoter and transfected into NIH 3T3 cells. The expressed proteins were similar in molecular size, extent of carbohydrate processing, and antigenic properties to gC-1 and gC-2 purified from infected cells. Using a double-label immunofluorescence assay, we found that both gC-1 and gC-2 were expressed on the surfaces of transfected cells and bound C3b. These results suggest that other proteins expressed during HSV-2 infection prevent receptor activity. We constructed three in-frame deletion mutants of gC-2 to identify domains on the protein important for C3b receptor activity. These mutants lacked amino acids 26 to 73, 219 to 244, or 318 to 346. The mutant protein lacking residues 26 to 73 was reactive with two monoclonal antibodies recognizing distinct epitopes, showed a wild-type pattern of carbohydrate processing, and bound C3b on the transfected cell surface. These results suggest that residues 26 to 73 are not involved in C3b binding. The other two mutant proteins were present on the cell surface, but did not bind C3b. In addition, these mutant proteins showed altered patterns of carbohydrate processing, formed aggregates, and were no longer recognized by the monoclonal antibodies. These properties indicate that removal of residues 219 to 244 or 318 to 346 disrupted the native conformation of gC-2, possibly owing to an alteration in the spacing between critical cysteine residues.     

The cytoplasmic domain of L-selectin interacts with cytoskeletal proteins via alpha-actinin: receptor positioning in microvilli does not require interaction with alpha-actinin

The leukocyte adhesion molecule L-selectin mediates binding to lymph node high endothelial venules (HEV) and contributes to leukocyte rolling on endothelium at sites of inflammation. Previously, it was shown that truncation of the L-selectin cytoplasmic tail by 11 amino acids abolished binding to lymph node HEV and leukocyte rolling in vivo, but the molecular basis for that observation was not determined. This study examined potential interactions between L-selectin and cytoskeletal proteins. We found that the cytoplasmic domain of L- selectin interacts directly with the cytoplasmic actin-binding protein alpha-actinin and forms a complex with vinculin and possibly talin. Solid phase binding assays using the full-length L-selectin cytoplasmic domain bound to microtiter wells demonstrated direct, specific, and saturable binding of purified alpha-actinin to L-selectin (Kd = 550 nM), but no direct binding of purified talin or vinculin. Interestingly, talin potentiated binding of alpha-actinin to the L- selectin cytoplasmic domain peptide despite the fact that direct binding of talin to L-selectin could not be measured. Vinculin binding to the L-selectin cytoplasmic domain peptide was detectable only in the presence of alpha-actinin. L-selectin coprecipitated with a complex of cytoskeletal proteins including alpha-actinin and vinculin from cells transfected with L-selectin, consistent with the possibility that alpha- actinin binds directly to L-selectin and that vinculin associates by binding to alpha-actinin in vivo to link actin filaments to the L- selectin cytoplasmic domain. In contrast, a deletion mutant of L- selectin lacking the COOH-terminal 11 amino acids of the cytoplasmic domain failed to coprecipitate with alpha-actinin or vinculin. Surprisingly, this mutant L-selectin localized normally to the microvillar projections on the cell surface. These data suggest that the COOH-terminal 11 amino acids of the L-selectin cytoplasmic domain are required for mediating interactions with the actin cytoskeleton via a complex of alpha-actinin and vinculin, but that this portion of the cytoplasmic domain is not necessary for proper localization of L- selectin on the cell surface. Correct L-selectin receptor positioning is therefore insufficient for leukocyte adhesion mediated by L- selectin, suggesting that this adhesion may also require direct interactions with the cytoskeleton.     

Perturbation of HP1 localization and chromatin binding ability causes defects in sister-chromatid cohesion

Sister-chromatid cohesion, the machinery used in eukaryote organisms to prevent aneuploidy, tethers sister chromatids together after their replication in S phase until mitosis. Previous studies in fission yeast, Drosophila and mammals have demonstrated the requirement for the heterochromatin formation pathway for proper centromeric cohesion. However, the exact role of heterochromatin protein 1 (HP1) in sister-chromatid cohesion in mammals is still unknown. In this study, we disrupted endogenous HP1 expression in HeLa cells using a dominant-negative mutant of HP1beta and wild-type or mutant forms of HP1alpha. We then examined their effects on chromosome alignment, segregation and cohesion. Enforced expression of these constructs leads to frequent chromosome misalignment and missegregation. Mitotic chromosomes from these cells also exhibit a loosened primary constriction and separated sister chromatids. We further demonstrate that alignment of the cohesin proteins around kinetochores was also aberrant and that cohesin complexes bound less tightly in these cells. Unexpectedly, we observed a "wavy" chromosome morphology resembling that seen upon depletion of condensin proteins in cells with over-expression of HP1alpha, but not in cells expressing the HP1beta mutant. These results indicate that proper HP1 status is required for sister-chromatid cohesion in mammalian cells, and suggest that HP1alpha might be required for chromosome condensation.     

Intracellular localization of the P21rho proteins

We have surveyed the proteins expressed at the surface of different primary neurons as a first step in elucidating how axons regulate their ensheathment by glial cells. We characterized the surface proteins of dorsal root ganglion neurons, superior cervical ganglion neurons, and cerebellar granule cells which are myelinated, ensheathed but unmyelinated, and unensheathed, respectively. We found that the most abundant proteins are common to all three types of neurons. Reproducible differences in the composition of the integral membrane proteins (enriched by partitioning into a Triton X-114 detergent phase) were detected. These differences were most striking when the expression of glycosylphosphatidyl-inositol (GPI)-anchored membrane proteins by these different neurons was compared. Variations in the relative abundance and degree of glycosylation of several well known GPI-anchored proteins, including Thy-1, F3/F11, and the 120-kD form of the neural cell adhesion molecule (N-CAM), and an abundant 60-kD GPI-linked protein were observed. In addition, we have identified several potentially novel GPI-anchored glycoproteins on each class of neurons. These include a protein that is present only on superior cervical ganglion neurons and is 90 kD; an abundant protein of 69 kD that is essentially restricted in its expression to dorsal root ganglion neurons; and proteins of 38 and 31 kD that are expressed only on granule cell neurons. Finally, the relative abundance of the three major isoforms of N-CAM was found to vary significantly between these different primary neurons. These results are the first demonstration that nerve fibers with diverse ensheathment fates differ significantly in the composition of their surface proteins and suggest an important role for GPI-anchored proteins in generating diversity of the neuronal cell surface.     

A proteomics approach to the cell-surface interactome using the enzyme-mediated activation of radical sources reaction

We previously reported a simple method to analyze the interaction of cell-surface molecules in living cells. This method termed enzyme-mediated activation of radical sources (EMARS) is featured by radical formation of the labeling reagent by horseradish peroxidase (HRP). Herein, we propose an approach to the cell-surface molecular interactome by using combination of this EMARS reaction and MS-based proteomics techniques. In the current study, we employed a novel labeling reagent, fluorescein-conjugated arylazide. The fluorescein-tagged proteins resulting from the EMARS reaction were directly detected in the electrophoresis gels with a fluorescence image analyzer. These products were also purified and concentrated by immunoaffinity chromatography with anti-fluorescein antibody-immobilized resins. The purified fluorescein-tagged proteins were subsequently subjected to an MS-based proteomics analysis. Analysis using HRP-conjugated cholera toxin subunit B, which recognizes a lipid raft marker, ganglioside GM1, revealed 30 membrane and secreted proteins that were candidates for the cell-surface molecules coclustering with GM1. The proposed approach will provide a clue to study functional molecular interactions in a variety of biological events on the cell surface.     

Morphology and dynamics of protruding spirochete periplasmic flagella.

We recently characterized the three-dimensional shape of Treponema phagedenis periplasmic flagella (PFs). In the course of these studies, we observed protrusions on swimming cells that resembled PFs. Here we present a detailed characterization of the shape, structure, and motion of these protrusions. Although protrusion formation occurred primarily in wild-type cells during the stationary phase, a large fraction of exponential-phase cells of cell cylinder helicity mutants (greater than 90% of mutant T-52) had protrusions. These results suggest that cells bearing protrusions can still participate in cell division. T. phagedenis protrusions had the identical helix handedness, pitch, and diameter to those of purified PFs. Protrusions were not present on mutants unable to synthesize PFs, but were present in all motile revertants which regained PFs. These results, taken together with electron microscope observations, suggest that protrusions consist of PFs surrounded by an outer membrane sheath. To analyze protrusion movements, we held cells against a coverglass surface with optical tweezers and observed the motion of protrusions by video-enhanced differential interference contrast light microscopy. Protrusions were found to gyrate in both clockwise and counterclockwise directions, and direct evidence was obtained that protrusions rotate. Protrusions were also observed on Treponema denticola and Borrelia burgdorferi. These were also left-handed and had the same helix handedness, pitch, and diameter as purified PFs from their respective species. The PFs from T. denticola had a helix diameter of 0.26 microns and a helix pitch of 0.78 micron; PFs from B. burgdorferi had a helix diameter of 0.28 micron and a helix pitch of 1.48 microns. Protrusions from these spirochete species had similar structures and motion to those of T. phagedenis. Our results present direct evidence that PFs rotate and support previously proposed models of spirochete motility.     

Internalization and re-expression of antigens of human melanoma cells following exposure to monoclonal antibody

Modulation of the surface membrane of human Sk-Mel-28 melanoma cells by monoclonal antibody (MoAb) 96.5 recognizing p97 determinants was examined using direct radioimmunoassay and indirect fluorescent antibody-staining techniques. It was determined that the majority of 111In-labeled antibody that remained associated with cells after a 24-hr incubation at 37 degrees C had been internalized because MoAb 96.5 was no longer visible on the cell surface. A second treatment of these cells with the same antibody 24 hr later not only increased the cell-associated radioactivity, reflecting an increase of total antibody bound, but also rendered these cells membrane immunofluorescent again, indicating the re-expression of surface antigens. Autoradiographs of the electrophoretically analyzed membrane components of Sk-Mel-28 cells further demonstrated the appearance of newly synthesized 97-kDa proteins that were immunoprecipitable with MoAb 96.5. Taken together, the present findings suggest that p97 antigens undergo endocytosis in Sk-Mel-28 cells following exposure to MoAb 96.5. However, the same antigens were regenerated and expressed on the cell surface within a period of 24 hr. The re-expression of tumor cell surface antigen following initial internalization of the MoAb-antigen complex may have implications for diagnosis and therapy.     

Influence of growth phase on adhesion kinetics of Escherichia coli D21g

The influence of bacterial growth stage and the evolution of surface macromolecules on cell adhesion have been examined by using a mutant of Escherichia coli K-12. To better understand the adhesion kinetics of bacteria in the mid-exponential and stationary growth phases under flow conditions, deposition experiments were conducted in a well-controlled radial stagnation point flow (RSPF) system. Complementary cell characterization techniques were conducted in combination with the RSPF experiments to evaluate the hydrophobicity, electrophoretic mobility, size, and titratable surface charge of the cells in the two growth phases considered. It was observed that cells in stationary phase were notably more adhesive than those in mid-exponential phase. This behavior is attributed to the high degree of local charge heterogeneity on the outer membranes of stationary-phase cells, which results in decreased electrostatic repulsion between the cells and a quartz surface. The mid-exponential-phase cells, on the other hand, have a more uniform charge distribution on the outer membrane, resulting in greater electrostatic repulsion and, subsequently, less adhesion. Our results suggest that the macromolecules responsible for this phenomenon are outer membrane-bound proteins and lipopolysaccharide-associated functional groups.     

The inducible blood--brain barrier specific molecule HT7 is a novel immunoglobulin-like cell surface glycoprotein.

The unique properties of brain endothelial cells, which form the blood-brain barrier, are reflected by the expression of specific cell surface molecules. We report here the purification, cloning and expression of one such molecule which is recognized by HT7 monoclonal antibodies. The HT7 antigen is a highly glycosylated 45-52 kd protein localized in brain endothelial cells, kidney epithelial cells and erythroblasts. The protein was purified to homogeneity from plasma membrane proteins isolated from all three sources using immunoaffinity chromatography and reverse phase HPLC. The amino-terminal amino acid sequences of the proteins were found to be identical. Based on amino acid sequence information, specific primers were designed and the polymerase chain reaction was used to obtain a full length cDNA clone. The nucleotide sequence encoded a novel glycoprotein with two C2-like immunoglobulin related domains, one transmembrane domain and a cytoplasmic tail. Expression of the transfected cDNA in COS cells resulted in the appearance of the HT7 antigen on the surface of these cells. On the basis of our results we propose that the protein may be a receptor involved in cell surface recognition at the blood-brain barrier.     

Saccharomyces cerevisiae CTF18 and CTF4 are required for sister chromatid cohesion

CTF4 and CTF18 are required for high-fidelity chromosome segregation. Both exhibit genetic and physical ties to replication fork constituents. We find that absence of either CTF4 or CTF18 causes sister chromatid cohesion failure and leads to a preanaphase accumulation of cells that depends on the spindle assembly checkpoint. The physical and genetic interactions between CTF4, CTF18, and core components of replication fork complexes observed in this study and others suggest that both gene products act in association with the replication fork to facilitate sister chromatid cohesion. We find that Ctf18p, an RFC1-like protein, directly interacts with Rfc2p, Rfc3p, Rfc4p, and Rfc5p. However, Ctf18p is not a component of biochemically purified proliferating cell nuclear antigen loading RF-C, suggesting the presence of a discrete complex containing Ctf18p, Rfc2p, Rfc3p, Rfc4p, and Rfc5p. Recent identification and characterization of the budding yeast polymerase kappa, encoded by TRF4, strongly supports a hypothesis that the DNA replication machinery is required for proper sister chromatid cohesion. Analogous to the polymerase switching role of the bacterial and human RF-C complexes, we propose that budding yeast RF-C(CTF18) may be involved in a polymerase switch event that facilities sister chromatid cohesion. The requirement for CTF4 and CTF18 in robust cohesion identifies novel roles for replication accessory proteins in this process.     

Splenic B cells and antigen-specific B cells process anti-Ig in a similar manner

B lymphocytes can process and present antigen to T cells. However, the fate of native antigen after its binding to specific B cells, i.e., the intracellular events involved in the processing and recycling of the antigenic fragments to the cell surface for antigen presentation, are not well understood. In the present study, we demonstrate that murine B cells degrade anti-Ig molecules bound to their surface and release acid soluble fragments into the supernatant. We also demonstrate that the kinetics of this process are identical for anti-mu, anti-delta, and anti-light chain antibodies, indicating that both surface IgM and surface IgD are equally effective in binding antigen and directing its processing. We also describe the effects of azide, chloroquine, and irradiation on this process. To extend these studies to the processing of specifically bound antigen, we demonstrate that highly purified trinitrophenyl antigen-binding cells degrade anti-Ig molecules with the same kinetics as unpurified splenic B cells. Thus, this purified population provides a suitable model system for the analysis of antigen degradation by antigen-specific cells.     

High affinity nanobodies against human epidermal growth factor receptor selected on cells by E. coli display

Most therapeutic antibodies (Abs) target cell surface proteins on tumor and immune cells. Cloning of Ab gene libraries in E. coli and their display on bacteriophages is commonly used to select novel therapeutic Abs binding target antigens, either purified or expressed on cells. However, the sticky nature of bacteriophages renders phage display selections on cells challenging. We previously reported an E. coli display system for expression of VHHs (i.e., nanobodies, Nbs) on the surface of bacteria and selection of high-affinity clones by magnetic cell sorting (MACS). Here, we demonstrate that E. coli display is also an attractive method for isolation of Nbs against cell surface antigens, such as the epidermal growth factor receptor (EGFR), upon direct selection and screening of Ab libraries on live cells. We employ a whole cell-based strategy using a VHH library obtained by immunization with human tumor cells over-expressing EGFR (i.e., A431), and selection of bacterial clones bound to murine fibroblast NIH-3T3 cells transfected with human EGFR, after depletion of non-specific clones on untransfected cells. This strategy resulted in the isolation of high-affinity Nbs binding distinct epitopes of EGFR, including Nbs competing with the ligand, EGF, as characterized by flow cytometry of bacteria displaying the Nbs and binding assays with purified Nbs using surface plasmon resonance. Hence, our study demonstrates that E. coli display of VHH libraries and selection on cells enables efficient isolation and characterization of high-affinity Nbs against cell surface antigens.     

Role of outer-membrane cytochromes MtrC and OmcA in the biomineralization of ferrihydrite by Shewanella oneidensis MR-1

In an effort to improve the understanding of electron transfer mechanisms at the microbe–mineral interface, Shewanella oneidensis MR-1 mutants with in-frame deletions of outer-membrane cytochromes (OMCs), MtrC and OmcA, were characterized for the ability to reduce ferrihydrite (FH) using a suite of microscopic, spectroscopic, and biochemical techniques. Analysis of purified recombinant proteins demonstrated that both cytochromes undergo rapid electron exchange with FH in vitro with MtrC displaying faster transfer rates than OmcA. Immunomicroscopy with cytochrome-specific antibodies revealed that MtrC co-localizes with iron solids on the cell surface while OmcA exhibits a more diffuse distribution over the cell surface. After 3-day incubation of MR-1 with FH, pronounced reductive transformation mineral products were visible by electron microscopy. Upon further incubation, the predominant phases identified were ferrous phosphates including vivianite [Fe₃(PO₄)₂·8H₂O] and a switzerite-like phase [Mn₃,Fe₃(PO₄)₂·7H₂O] that were heavily colonized by MR-1 cells with surface-exposed outer-membrane cytochromes. In the absence of both MtrC and OmcA, the cells ability to reduce FH was significantly hindered and no mineral transformation products were detected. Collectively, these results highlight the importance of the outer-membrane cytochromes in the reductive transformation of FH and support a role for direct electron transfer from the OMCs at the cell surface to the mineral.     

Activity of plasma membrane β-galactosidase and β-glucosidase

Human fibroblasts produce ceramide from sialyllactosylceramide on the plasma membranes. Sialidase Neu3 is known to be plasma membrane associated, while only indirect data suggest the plasma membrane association of β-galactosidase and β-glucosidase. To determine the presence of β-galactosidase and β-glucosidase on plasma membrane, cells were submitted to cell surface biotinylation. Biotinylated proteins were purified by affinity column and analyzed for enzymatic activities on artificial substrates. Both enzyme activities were found associated with the cell surface and were up-regulated in Neu3 overexpressing cells. These enzymes were capable to act on both artificial and natural substrates without any addition of activator proteins or detergents and displayed a trans activity in living cells.     

An extracellular matrix-associated zinc metalloprotease is required for dilauroyl phosphatidylethanolamine chemotactic excitation in Myxococcus xanthus

An extracellular matrix connects bacteria that live in organized assemblages called biofilms. While the role of the matrix in the regulation of cell behavior has not been extensively examined in bacteria, we suggest that, like mammalian cells, the matrix facilitates cell-cell interactions involved with regulation of cohesion, motility, and sensory transduction. The extracellular matrix of the soil bacterium Myxococcus xanthus is essential for biofilm formation and fruiting body development. The matrix material is extruded as long, thin fibrils that mediate adhesion to surfaces, cohesion to other cells, and excitation by the chemoattractant dilauroyl phosphatidylethanolamine. We report the identification of a putative matrix-associated zinc metalloprotease called FibA (fibril protein A). Western blotting with FibA-specific monoclonal antibody 2105 suggests extensive proteolytic processing of FibA during assembly into fibrils, consistent with the autoprocessing observed with other members of the M4 metalloprotease family. Disruption of fibA had no obvious effect on the structure of the fibrils and did not inhibit cell cohesion, excitation by dioleoyl phosphatidylethanolamine, or activity of the A- or S-motility motors. However, the cells lost the ability to respond to dilauroyl phosphatidylethanolamine and to form well-spaced fruiting bodies, though substantial aggregation was observed. Chemotactic excitation of the fibA mutant was restored by incubation with purified wild-type fibrils. The results suggest that this metalloprotease is involved in sensory transduction.     

Radio-iodination of plasma membrane polypeptides from isolated mouse spermatogenic cells

Cell surface polypeptides of mouse pachytene spermatocytes and round spermatids (steps 1–8) have been iodinated using 1,2,3,6,tetracholoro-3α, 6α-diphenylglycouril (IODOGEN). Labeled proteins have been assayed using two-dimensional polyacrylamide electrophoresis and radioautography. Purified plasma membranes, prepared from both spermatocytes and spermatids after the iodination of intact cells, exhibit 25–30 polypeptides which label reproducibly. No significant qualitative differences are noted in the labeled polypeptide map obtained from each of the purified cell types. Iodinated proteins range in molecular weight from greater than 100k daltons to approximately 40k daltons. The isoelectric points of labeled constituents range from pI 5.7 to 7.2. Three polypeptides represent the major iodinated species: p 94/5.8, p 75/5.9, and p 53/7.1. Comparison with total plasma membrane constituents assayed using Coomassie brilliant blue indicates that many of the radioactively labeled proteins are not present in quantities sufficient to allow ready detection without isotopic techniques. As a result, many of the proteins identified autoradiographically represent newly described surface components of mouse pachytene spermatocytes and round spermatids. The preparation of purified plasma membrane fractions prior to electrophoresis ensures that all iodinated species are in fact cell surface components. Furthermore, experiments designed to assess the vectorial nature of the IODOGEN-catalyzed labeling procedure suggest that most, if not all, of the iodinated species are exposed on the external side of the cell plasma membrane. Therefore, these studies have (1) identified hitherto unrecognized plasma membrane components of mouse pachytene spermatocytes and round spermatids and (2) provided the first available biochemical data concerning the molecular orientation of particular proteins in the surface membranes of developing mouse spermatogenic cells.