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Efficient, biocompatible, stereospecific strategies were developed to prepare eight probes to assay transketolase (TK) variants with new substrate specificities. The structure of these probes combines a sugar moiety ( D- threo or L- erythro ketose, or D- threo aldose) with the side chain of an amino acid (Ala, Leu, Val, Met, Thr) for in vivo detection of new TK activities using amino acid auxotrophs. To obtain D- threo ketose probes, biocatalysts, such as transketolase and fructose-6-phosphate aldolase Ala129Ser, were used whereas L- erythro ketoses and D- threo aldose probes were synthesized by the way of organocatalysis or Sharpless dihydroxylation as sustainable alternative key steps to biocatalysis. [ABSTRACT FROM AUTHOR]

Microcin B17 (MccB17) is a ribosomally synthesized peptide antibiotic of 43 amino acids that induces double-strand breaking of DNA in a DNA gyrase-dependent reaction. As a consequence, the SOS regulon is induced and massive DNA degradation occurs. In this work we have characterized an Escherichia coli gene, sbmC, that in high copy number determines high cell resistance to MccB17. sbmC encodes a cytoplasmic polypeptide of 157 amino acids (Mr, 18095) that has been visualized in SDS-polyacrylamide gels. The gene is located at min 44 of the E. coli genetic map, close to the sbcB gene. sbmC expression is induced by DNA-damaging agents and, also, by the entry of cells into the stationary growth phase. A G → T transversion at the fifth nucleotide of the quasicanonical LexA-box preceding the gene makes recA cells 16-fold more resistant to exogenous MccB17. The gene product, SbmC, also blocks MccB17 export from producing cells. Altogether, our results suggest that SbmC recognizes and sequesters MccB17 in a reversible way. [ABSTRACT FROM AUTHOR]

Effective freight transportation planning through different modes drives countries economic growth by ensuring the delivery of goods safely and sustainably. This article provides a systematic literature review focused on intermodal transport tactical planning, addressing logistical technical factors for efficient transportation modes management in cargo delivery. The methodology is based on the research questions definition, search process, and article selection criteria, exploring the network structures evolution to verify technical factors influencing network structures, including mode combinations, geographical distribution, and mathematical models' decision variables and constraints. According to the research question proposed, the most relevant technical factors for intermodal planning network structures are flow cargo, transport modal activation, flow conservation, delivery time, and vehicle management, generally found in countries with extensive road infrastructure and a high modal integration capacity, as China, Europe, and USA. Considering the decision variables and constraints, flow conservation is a common element observed in intermodal transport mathematical models. Thus, a balance between transportation costs and transportation modes utilization was verified, and insights into the relation between technical logistics factors application and network structures for cargo delivery efficiency were revealed. Finally, the systematic literature review contributes to understanding the main intermodal transportation planning evolving landscape, offering valuable insights for future research and practical implementations.

Atmospheric CO 2 poses a major threat to life on Earth by causing global warming and climate change. On the other hand, it can be considered as a resource that is scalable enough to establish a circular carbon economy. Accordingly, technologies to capture and convert CO 2 into reduced one-carbon (C 1 ) compounds (e.g. formic acid) are developing and improving fast. Driven by the idea of creating sustainable bioproduction platforms, natural and synthetic C 1 -utilization pathways are engineered into industrially relevant microbes. The realization of synthetic C 1 -assimilation cycles in living organisms is a promising but challenging endeavour. Here, we engineer the Serine Threonine Cycle, a synthetic C 1 -assimilation cycle in Escherichia coli to achieve growth on formic acid. Our stepwise engineering approach in tailored selection strains combined with adaptive laboratory evolution experiments enabled formatotrophic growth of the organism. Whole genome sequencing and reverse engineering allowed us to determine the key mutations linked to pathway activity. The Serine Threonine Cycle strains created in this work use formic acid as a carbon and energy source and can grow at ambient CO 2 cultivation conditions. This work sets an example for the engineering of complex C 1 -assimilation cycles in heterotrophic microbes., Competing Interests: Declaration of Competing Interest The authors declare no competing interest, (Copyright © 2024. Published by Elsevier Inc.)

Biosourced and biodegradable plastics offer a promising solution to reduce environmental impacts of plastics for specific applications. Here, we report a novel bacterium named Alteromonas plasticoclasticus MED1 isolated from the marine plastisphere that forms biofilms on foils of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV). Experiments of degradation halo, plastic matrix weight loss, bacterial oxygen consumption and heterotrophic biosynthetic activity showed that the bacterial isolate MED1 is able to degrade PHBV and to use it as carbon and energy source. The likely entire metabolic pathway specifically expressed by this bacterium grown on PHBV matrices was shown by further genomic and transcriptomic analysis. In addition to a gene coding for a probable secreted depolymerase, a gene cluster was located that encodes characteristic enzymes involved in the complete depolymerization of PHBV, the transport of oligomers, and in the conversion of the monomers into intermediates of central carbon metabolism. The transcriptomic experiments showed the activation of the glyoxylate shunt during PHBV degradation, setting the isocitrate dehydrogenase activity as regulated branching point of the carbon flow entering the tricarboxylic acid cycle. Our study also shows the potential of exploring the natural plastisphere to discover new bacteria with promising metabolic capabilities., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The reductive glycine pathway was described as the most energetically favorable synthetic route of aerobic formate assimilation. Here we report the successful implementation of formatotrophy in Escherichia coli by means of a stepwise adaptive evolution strategy. Medium swap and turbidostat regimes of continuous culture were applied to force the channeling of carbon flux through the synthetic pathway to pyruvate establishing growth on formate and CO 2 as sole carbon sources. Labeling with 13 C-formate proved the assimilation of the C1 substrate via the pathway metabolites. Genetic analysis of intermediate isolates revealed a mutational path followed throughout the adaptation process. Mutations were detected affecting the copy number (gene ftfL) or the coding sequence (genes folD and lpd) of genes which specify enzymes implicated in the three steps forming glycine from formate and CO 2 , the central metabolite of the synthetic pathway. The mutation R191S present in methylene-tetrahydrofolate dehydrogenase/cyclohydrolase (FolD) abolishes the inhibition of cyclohydrolase activity by the substrate formyl-tetrahydrofolate. The mutation R273H in lipoamide dehydrogenase (Lpd) alters substrate affinities as well as kinetics at physiological substrate concentrations likely favoring a reactional shift towards lipoamide reduction. In addition, genetic reconstructions proved the necessity of all three mutations for formate assimilation by the adapted cells. The largely unpredictable nature of these changes demonstrates the usefulness of the evolutionary approach enabling the selection of adaptive mutations crucial for pathway engineering of biotechnological model organisms., (Copyright © 2022 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.)

All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in E. coli , we complementarily employed in silico design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal and the other via serine biosynthesis and degradation. Then, we implemented both pathways in E. coli strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the "serine shunt" which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution study using an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism., Competing Interests: The authors declare no competing interests., (Copyright © 2022 Camillo Iacometti et al.)

The nicotinamide cofactor specificity of enzymes plays a key role in regulating metabolic processes and attaining cellular homeostasis. Multiple studies have used enzyme engineering tools or a directed evolution approach to switch the cofactor preference of specific oxidoreductases. However, whole-cell adaptation toward the emergence of novel cofactor regeneration routes has not been previously explored. To address this challenge, we used an Escherichia coli NADPH-auxotrophic strain. We continuously cultivated this strain under selective conditions. After 500 to 1,100 generations of adaptive evolution using different carbon sources, we isolated several strains capable of growing without an external NADPH source. Most isolated strains were found to harbor a mutated NAD + -dependent malic enzyme (MaeA). A single mutation in MaeA was found to switch cofactor specificity while lowering enzyme activity. Most mutated MaeA variants also harbored a second mutation that restored the catalytic efficiency of the enzyme. Remarkably, the best MaeA variants identified this way displayed overall superior kinetics relative to the wild-type variant with NAD + . In other evolved strains, the dihydrolipoamide dehydrogenase (Lpd) was mutated to accept NADP + , thus enabling the pyruvate dehydrogenase and 2-ketoglutarate dehydrogenase complexes to regenerate NADPH. Interestingly, no other central metabolism oxidoreductase seems to evolve toward reducing NADP + , which we attribute to several biochemical constraints, including unfavorable thermodynamics. This study demonstrates the potential and biochemical limits of evolving oxidoreductases within the cellular context toward changing cofactor specificity, further showing that long-term adaptive evolution can optimize enzyme activity beyond what is achievable via rational design or directed evolution using small libraries. IMPORTANCE In the cell, NAD(H) and NADP(H) cofactors have different functions. The former mainly accepts electrons from catabolic reactions and carries them to respiration, while the latter provides reducing power for anabolism. Correspondingly, the ratio of the reduced to the oxidized form differs for NAD + (low) and NADP + (high), reflecting their distinct roles. We challenged the flexibility of E. coli 's central metabolism in multiple adaptive evolution experiments using an NADPH-auxotrophic strain. We found several mutations in two enzymes, changing the cofactor preference of malic enzyme and dihydrolipoamide dehydrogenase. Upon deletion of their corresponding genes we performed additional evolution experiments which did not lead to the emergence of any additional mutants. We attribute this restricted number of mutational targets to intrinsic thermodynamic barriers; the high ratio of NADPH to NADP + limits metabolic redox reactions that can regenerate NADPH, mainly by mass action constraints.

There is an increasing interest in product recovery, closed-loop supply chains, and reverse logistics (RL) for mitigating environmental impairment. Although RL is becoming a mandatory policy in developed countries, it is still in an embryonic stage in some industrial sectors of emerging economies. The purpose of this study is twofold: (1) identify the critical factors to the successful implementation of RL in the Brazilian pharmaceutical care process (PCP) and (2) determine the cause-and-effect relationships among them. We use snowball sampling to select the relevant RL studies and deductive reasoning and classification to identify the critical factors and a grey decision-making trial and evaluation laboratory (DEMATEL) to evaluate the cause-and-effect relationships among them. The study revealed management, collaboration, information technology, infrastructure, policy, financial and economic, end-of-life management practices, and logistic performance factors as the most relevant factors to the successful implementation of RL in the Brazilian PCP. The end-of-life management practices were identified as the most critical factor, and information technology was identified as the least critical factor. We further determined the end-of-life management practices and policy have the strongest casual relationship. The municipal PCP coordinators can use the findings of this study to formulate mitigating strategies to identify and eliminate barriers to the successful implementation of RL in the Brazilian PCP.

Small- and medium-sized enterprises primarily focus on their operations and rarely pay attention to issues related to sustainable solid waste management that originate from their production processes. A suitable strategy to support sustainable solid waste management is reverse logistics. Through the use of maturity models, it is possible to determine the grade to which small- and medium-sized enterprises are prepared to perform this strategy. This study proposes an adapted maturity model to measure maturity levels of reverse logistics aspects at small- and medium-sized enterprises in regions from Colombia in order to contribute to sustainable solid waste management. The maturity model was applied to seven small- and medium-sized enterprises in the plastics sector in the central and southern regions of Colombia by adapting a maturity model that was previously correlated to suggested drivers and barriers in this sector. Results show that maturity levels range from naïve to immature owing to the incipient development of reverse logistics in Colombia. Therefore, it is necessary to establish a holistic vision of the organisation to improve the reverse logistics decision-making process to achieve sustainable solid waste management.

The bio-economy relies on microbial strains optimized for efficient large scale production of chemicals and fuels from inexpensive and renewable feedstocks under industrial conditions. The reduced one carbon compound methanol, whose production does not involve carbohydrates needed for the feed and food sector, can be used as sole carbon and energy source by methylotrophic bacteria like Methylobacterium extorquens AM1. This strain has already been engineered to produce various commodity and high value chemicals from methanol. The toxic effect of methanol limits its concentration as feedstock to 1% v/v. We obtained M. extorquens chassis strains tolerant to high methanol via adaptive directed evolution using the GM3 technology of automated continuous culture. Turbidostat and conditional medium swap regimes were employed for the parallel evolution of the recently characterized strain TK 0001 and the reference strain AM1 and enabled the isolation of derivatives of both strains capable of stable growth with 10% methanol. The isolates produced more biomass at 1% methanol than the ancestor strains. Genome sequencing identified the gene metY coding for an O -acetyl-L-homoserine sulfhydrylase as common target of mutation. We showed that the wildtype enzyme uses methanol as substrate at elevated concentrations. This side reaction produces methoxine, a toxic homolog of methionine incorporated in polypeptides during translation. All mutated metY alleles isolated from the evolved populations coded for inactive enzymes, designating O -acetyl-L-homoserine sulfhydrylase as a major vector of methanol toxicity. A whole cell transcriptomic analysis revealed that genes coding for chaperones and proteases were upregulated in the evolved cells as compared with the wildtype, suggesting that the cells had to cope with aberrant proteins formed during the adaptation to increasing methanol exposure. In addition, the expression of ribosomal proteins and enzymes related to energy production from methanol like formate dehydrogenases and ATP synthases was boosted in the evolved cells upon a short-term methanol stress. D-lactate production from methanol by adapted cells overexpressing the native D-lactate dehydrogenase was quantified. A significant higher lactate yield was obtained compared with control cells, indicating an enhanced capacity of the cells resistant to high methanol to assimilate this one carbon feedstock more efficiently.

Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.

Endowing biotechnological platform organisms with new carbon assimilation pathways is a key challenge for industrial biotechnology. Here we report progress toward the construction of formatotrophic Escherichia coli strains. Glycine and serine, universal precursors of one-carbon compounds oxidized during heterotrophic growth, are produced from formate and CO 2 through a reductive route. An adaptive evolution strategy was applied to optimize the enzymatic steps of this route in appropriate selection strains. Metabolic labeling experiments with 13 C-formate confirm the redirected carbon-flow. These results demonstrate the high plasticity of the central carbon metabolism of E. coli and the applicative potential of directed evolution for implementing synthetic pathways in microorganisms.

Assimilation of one-carbon compounds presents a key biochemical challenge that limits their use as sustainable feedstocks for microbial growth and production. The reductive glycine pathway is a synthetic metabolic route that could provide an optimal way for the aerobic assimilation of reduced C1 compounds. Here, we show that a rational integration of native and foreign enzymes enables the tetrahydrofolate and glycine cleavage/synthase systems to operate in the reductive direction, such that Escherichia coli satisfies all of its glycine and serine requirements from the assimilation of formate and CO 2 . Importantly, the biosynthesis of serine from formate and CO 2 does not lower the growth rate, indicating high flux that is able to provide 10% of cellular carbon. Our findings assert that the reductive glycine pathway could support highly efficient aerobic assimilation of C1-feedstocks.

Methylobacterium extorquens TK 0001 (DSM 1337, ATCC 43645) is an aerobic pink-pigmented facultative methylotrophic alphaproteobacterium isolated from soil in Poland. Here, we report the whole-genome sequence and annotation of this organism, which consists of a single 5.71-Mb chromosome., (Copyright © 2018 Belkhelfa et al.)

One-carbon metabolism is an ubiquitous metabolic pathway that encompasses the reactions transferring formyl-, hydroxymethyl- and methyl-groups bound to tetrahydrofolate for the synthesis of purine nucleotides, thymidylate, methionine and dehydropantoate, the precursor of coenzyme A. An alternative cyclic pathway was designed that substitutes 4-hydroxy-2-oxobutanoic acid (HOB), a compound absent from known metabolism, for the amino acids serine and glycine as one-carbon donors. It involves two novel reactions, the transamination of l-homoserine and the transfer of a one-carbon unit from HOB to tetrahydrofolate releasing pyruvate as coproduct. Since canonical reactions regenerate l-homoserine from pyruvate by carboxylation and subsequent reduction, every one-carbon moiety made available for anabolic reactions originates from CO 2 . The HOB-dependent pathway was established in an Escherichia coli auxotroph selected for prototrophy using long-term cultivation protocols. Genetic, metabolic and biochemical evidence support the emergence of a functional HOB-dependent one-carbon pathway achieved with the recruitment of the two enzymes l-homoserine transaminase and HOB-hydroxymethyltransferase and of HOB as an essential metabolic intermediate. Escherichia coli biochemical reprogramming was achieved by minimally altering canonical metabolism and leveraging on natural selection mechanisms, thereby launching the resulting strain on an evolutionary trajectory diverging from all known extant species.

Streptomyces cattleya, a producer of the antibiotics thienamycin and cephamycin C, is one of the rare bacteria known to synthesize fluorinated metabolites. The genome consists of two linear replicons. The genes involved in fluorine metabolism and in the biosynthesis of the antibiotic thienamycin were mapped on both replicons., (Copyright © 2011, American Society for Microbiology. All Rights Reserved.)

Probes were developed for the in vivo detection of transketolase activity by the use of a complementation assay in Escherichia coli auxotrophs They combine the d-threo ketose moiety recognised by transketolase and the side chain of leucine or methionine. These compounds were donor substrates of yeast transketolase leading to the release of the corresponding alpha-hydroxyaldehydes which could be converted in E. coli by a cascade of reactions into leucine or methionine required for cellular growth.

We identified in the genome of Salmonella enterica serovar Typhi the gene encoding deoxyribokinase, deoK. Two other genes, vicinal to deoK, were determined to encode the putative deoxyribose transporter (deoP) and a repressor protein (deoQ). This locus, located between the uhpA and ilvN genes, is absent in Escherichia coli. The deoK gene inserted on a plasmid provides a selectable marker in E. coli for growth on deoxyribose-containing medium. Deoxyribokinase is a 306-amino-acid protein which exhibits about 35% identity with ribokinase from serovar Typhi, S. enterica serovar Typhimurium, or E. coli. The catalytic properties of the recombinant deoxyribokinase overproduced in E. coli correspond to those previously described for the enzyme isolated from serovar Typhimurium. From a sequence comparison between serovar Typhi deoxyribokinase and E. coli ribokinase, whose crystal structure was recently solved, we deduced that a key residue differentiating ribose and deoxyribose is Met10, which in ribokinase is replaced by Asn14. Replacement by site-directed mutagenesis of Met10 with Asn decreased the V(max) of deoxyribokinase by a factor of 2.5 and increased the K(m) for deoxyribose by a factor of 70, compared to the parent enzyme.

The chemical diversification of DNA precursors was undertaken in Escherichia coil by expressing the human gene for deoxycytidine kinase, and supplying such recombinant strains with nucleoside analogues bearing an altered base or sugar. Arabinocytidine and dideoxycytidine thus became highly toxic to E. coli in the sub-millimolar range. Deoxynucleosides bearing isoadenine (2-aminopurine) and isoguanine (2-hydroxy-6-aminopurine) showed a high mutagenic potency towards the recombinant strains, to an extent comparable to that of the most efficient mutator alleles (dnaQ). These findings open the way to the propagation of chemically remodelled nucleic acids and to the controlled hypermutagenesis of plasmids in vivo.

In the course of a study of genes located at min 44 of the Escherichia coli genome, we identified an open reading frame with the capacity to encode a 43-kDa polypeptide whose predicted amino acid sequence is strikingly similar to those of the well-known DD-carboxipeptidases penicillin-binding proteins PBP5 and PBP6. The gene product was shown to bind [3H]benzylpenicillin and to have DD-carboxypeptidase activity on pentapeptide muropeptides in vivo. Therefore, we called the protein PBP6b and the gene dacD. As with other E. coli DD-carboxypeptidases, PBP6b is not essential for cell growth. A quadruple dacA dacB dacC dacD mutant was constructed and shown to grow as well as its isogenic wild-type strain, indicating that the loss of any known PBP-associated DD-carboxypeptidase activity is not deleterious for E. coli. We also identified the homologous gene of dacD in Salmonella typhimurium as one of the components of the previously described phsBCDEF gene cluster.

The properties of EHS laminin and its proteolytic fragments E8 and P1 to promote spreading of B16 F1 murine melanoma cells were studied in short-term adhesion assays. The cells exhibited similar attachment rates but distinct spread morphologies on laminin, P1, and E8 fragments. The extent of spreading and the shape of the cells were quantitatively defined by two geometrical parameters: the surface and the form factor. These parameters were computed with an automatic image analyzer. Wheat germ agglutinin (WGA), applied to laminin-coated substrates, totally blocked cell spreading, but did not modify attachment percentages. Under similar conditions, WGA partially inhibited cell spreading on the E8 fragment and had no effect on the P1 fragment. In Western blot analysis, P1 fragment, contrary to laminin and E8, did not bind WGA. Laminin galactosylation and cell treatment with alpha-lactalbumin, which should prevent cell galactosyltransferase (GalTase) from binding to N-acetylglucosamine (GlcNAc) residues of the substrate, had no effect on the spreading ability of B16 F1 cells. The role of laminin N-linked carbohydrate chains in the induction of B16 F1 cell spreading was studied further after endoglycosidase F (Endo F) treatment of the substrates. The loss of carbohydrate chains was estimated by the reduction of iodinated lectin binding and by SDS-PAGE. Endo F treatment of laminin (85% of WGA binding inhibition) and E8 (40-50%) had no effect on cell spreading. In contrast, Endo F treatment of P1 fragment (85% of Con A binding inhibition) reduced both cell surface and form factor of B16 F1 cells. These results suggest that: (i) other spreading systems may act in concert with or in place of GalTase/GlcNAc interactions, (ii) the N-linked sugar chains of P1, which are not recognized by WGA, are involved in the spreading process of B16 F1 cells on this fragment, (iii) the epitopes of E8 fragment and E8 domain in laminin which are responsible for spreading are differently masked by WGA, (iv) the binding of WGA to laminin may impair cell spreading by steric hindrance.

The biosynthesis of the basement-membrane glycoprotein laminin in the mouse teratocarcinoma cell line PFHR9 was studied by immunoelectron microscopy and pulse-chase experiments using monoclonal and polyclonal antibodies. By immunoelectron microscopy, most of the protein was found to be aggregated on the outer cell surface. Cytoplasmic stainings were rare and were located next to the intracellular side of the plasma membrane. Sequential immunoprecipitations of cell extracts with a monoclonal antibody (4C12) sensitive to the laminin native conformation and with a polyclonal antibody enables laminin, the B1 subunit and a 410 kDa molecule to be distinguished. Most of the laminin is of the A(B1B2) type, and the 410 kDa molecule appears to be a B1B2 heterodimer. The assembly of laminin from subunits is completed in less than 1 h, and B chains are incorporated via the formation of the B heterodimers. The B2 and A chains are not found as free forms, so their levels appear to be the rate-limiting factors for the assembly of the dimers and laminin respectively. The formation of an uncross-linked A(B1B2) complex as a short-lived intermediate in the biosynthetic process is possible. Together with immunoelectron microscopy, the present study suggests that the protein is rapidly exported after assembly to accumulate on the outer side of the cell membrane. The biosynthesis of laminin in the PFHR9 cell line appears to be similar to that in other matrix-producing cell lines.

The present study reveals the dynamic distribution of membrane laminin receptors induced by laminin binding in a rat rhabdomyosarcoma cell line RMS S4. The treatment of the cells with soluble laminin did not modify cell adhesion to laminin-coated substrates in in vitro attachment assays. Fluorescent labeling of membrane-bound laminin revealed that occupied receptors were induced to cluster and cap. New free membrane binding sites were made evident after capping of bound laminin by a double labeling technique. Cytochalasin D (CD) treatment prevented the capping process. The adhesion of CD-treated cells to laminin-coated substrates was inhibited by cell preincubation with soluble laminin. Cycloheximide treatment had no effect on the ability of RMS S4 cells to adhere to adsorbed laminin after preincubation in the presence of soluble laminin. These results taken as a whole suggest that free receptors may arise from an intracellular pool that could be maintained by membrane receptor recycling. Since capping and motility seem related events, migration of RMS S4 cells on laminin was studied in the agarose drop assay. Immobilized laminin stimulated basic cell motility by more than 200%. E8 laminin fragment retained partially the motility stimulating property of laminin while P1 pepsinic fragment had no effect. The presence of constantly available receptors at the cell surface could be determinant in the ability of cells to migrate on laminin substrates.