Absence Of Laminin Research Articles

Merosin-deficient muscular dystrophy: pathogenesis, clinical manifestations and therapeutic strategies

Merosin-deficient muscular dystrophy is the most common form of congenital muscular dystrophies (СMD), characterized by genetic heterogeneity and a severe course in most cases. CMD pathogenesis is associated with a partial or complete absence of laminin 2 chains in the basal membrane of muscle fiber caused by a mutation in theLAMA2gene. The clinical manifestations of LAMA2-associated muscular dystrophy vary from severe СMD (СMD 1A) with an early onset to a relatively mild course with a late onset and phenotype of limb-girdle muscular dystrophy. CMD type 1A is characterized by a delay in motor development from the first months of the life (while the vast majority of children do not master the ability to walk independently), facial muscles weakness, ptosis, ophthalmoplegia, spine rigidity, early occurrence of contractures in the shoulder, elbow, hip and knee joints, restrictive respiratory disorders, nutritional problems, such as disorders of swallowing and chewing, gastroesophageal reflux, low body weight; some children suffer from mental retardation and epilepsy.

ADAP1 promotes invasive squamous cell carcinoma progression and predicts patient survival.

Invasive squamous cell carcinoma (SCC) is aggressive cancer with a high risk of recurrence and metastasis, but the critical determinants of its progression remain elusive. Here, we identify ADAP1, a GTPase-activating protein (GAP) for ARF6 up-regulated in TGF-β-responding invasive tumor cells, as a strong predictor of poor survival in early-stage SCC patients. Using a mouse model of SCC, we show that ADAP1 overexpression promotes invasive tumor progression by facilitating cell migration and breakdown of the basement membrane. We found that ADAP1-rich, TGF-β-responding tumor cells exhibit cytoplasmic laminin localization, which correlated with the absence of laminin and type IV collagen from the pericellular basement membrane. Interestingly, although tumors overexpressing a GAP activity-deficient mutant of ADAP1 resulted in morphologically complex tumors, those tumor cells failed to breach the basement membrane. Moreover, Adap1 deletion in tumor cells ameliorated the basement membrane breakdown and had less invading cells in the stroma. Our study demonstrates that ADAP1 is a critical mediator of TGF-β-induced cancer invasion and might be exploited for the treatment of high-risk SCC.

Immortalized myogenic cells from congenital muscular dystrophy type1A patients recapitulate aberrant caspase activation in pathogenesis: a new tool for MDC1A research

BackgroundCongenital muscular dystrophy Type 1A (MDC1A) is a severe, recessive disease of childhood onset that is caused by mutations in the LAMA2 gene encoding laminin-α2. Studies with both mouse models and primary cultures of human MDC1A myogenic cells suggest that aberrant activation of cell death is a significant contributor to pathogenesis in laminin-α2-deficiency.MethodsTo overcome the limited population doublings of primary cultures, we generated immortalized, clonal lines of human MDC1A myogenic cells via overexpression of both CDK4 and the telomerase catalytic component (human telomerase reverse transcriptase (hTERT)). ResultsThe immortalized MDC1A myogenic cells proliferated indefinitely when cultured at low density in high serum growth medium, but retained the capacity to form multinucleate myotubes and express muscle-specific proteins when switched to low serum medium. When cultured in the absence of laminin, myotubes formed from immortalized MDC1A myoblasts, but not those formed from immortalized healthy or disease control human myoblasts, showed significantly increased activation of caspase-3. This pattern of aberrant caspase-3 activation in the immortalized cultures was similar to that found previously in primary MDC1A cultures and laminin-α2-deficient mice.ConclusionsImmortalized MDC1A myogenic cells provide a new resource for studies of pathogenetic mechanisms and for screening possible therapeutic approaches in laminin-α2-deficiency.

Laminin and collagen modulate expression of the small leucine-rich proteoglycan fibromodulin in rat anterior pituitary gland

The anterior pituitary is a complex organ consisting of five types of hormone-producing cells, non–hormone-producing cells such as folliculostellate (FS) cells and vascular cells (endothelial cells and pericytes). We have previously shown that FS cells and pericytes produce fibromodulin, a small leucine-rich proteoglycan (SLRP). SLRPs are major proteoglycans of the extracellular matrix (ECM) and are important in regulating cell signaling pathways and ECM assembly. However, the mechanism regulating fibromodulin expression in the anterior pituitary has not been elucidated. Here, we investigate whether fibromodulin expression is modulated by major anterior pituitary ECM components such as laminin and type I collagen. Using transgenic rats expressing green fluorescent protein (GFP) specifically in FS cells, we examine fibromodulin expression in GFP-positive (FS cells) and GFP-negative cells (e.g., pericytes, endocrine cells and endothelial cells). Immunostaining and Western blot analysis were used to assess protein expression in the presence and absence of laminin or type I collagen. We confirmed fibromodulin expression in the pituitary and observed the up-regulation of fibromodulin in FS cells in the presence of ECM components. However, neither laminin nor type I collagen affected expression in GFP-negative cells. This suggests that laminin and type I collagen support the function of FS cells by increasing fibromodulin protein expression in the anterior pituitary.

Pluripotency Network in Porcine Embryos and Derived Cell Lines

Huge amounts of work have been dedicated to the establishment of embryonic stem cell lines from farm animal species since the successful isolation of embryonic stem cells from the mouse and from the human. However, no conclusive results have been obtained so far, and validated lines have yet to be established in domestic animals. Many limiting factors have been suggested and need to be studied further to isolate truly pluripotent cell lines from livestock. In this review, we will discuss the difficulties in deriving and maintaining embryonic stem cell lines from farm animal embryos and how can this lack of success be explained. We will summarize results obtained in our laboratory regarding derivation of pluripotent cells in the pigs. Problems related to the identification of standard methods for derivation, maintenance and characterization of cell lines will also be examined. We will focus our attention on the need for appropriate stemness-related marker molecules that can be used to reliably investigate pluripotency in domestic species. Finally, we will review data presently available on functional key pluripotency-maintaining pathways in farm animals.

Laminin Expression Is Necessary for Maintenance of the Vascular Hematopoietic Niche

Abstract 217Laminins are heterotrimeric proteins that are the backbone for all basal laminas. They provide structural support, points for focal adhesion, and biochemical feedback for nearby cells. Laminins are found in many tissues, including bone marrow and lymphoid organs, and are detected in the discontinuous basal lamina of sinusoidal blood vessels. Laminin g1 is the predominant gamma subunit found in the bone marrow and lymphoid tissues. Here we show that the continuous expression of laminin in adulthood is necessary for viability of the vascular hematopoietic niche (sinusoidal endothelial cells). In the absence of laminin, this niche fails and hematopoiesis is impaired.To determine if sinusoidal laminin expression correlates with bone marrow failure and recovery, we used a well-established model of bone marrow failure. Wild type mice were treated with a sublethal dose of 5-fluoruracil (5-FU), and bone marrow was analyzed for disruption of sinusoid architecture and endothelial cell receptor and laminin expression. As described in other studies, shortly after treatment with 5-FU, bone marrow sinusoids became disorganized and hemorrhagic, and endothelial cells lost expression of VEGFR2. In addition, sinusoids lost their laminin-containing basal lamina. These changes correlated with bone marrow failure and onset of peripheral blood pancytopenia. As the sinusoids regained normal cytoarchitecture and VEGFR2 expression was normalized, sinusoid expression of laminin also returned to basal levels. With 5-FU-induced bone marrow failure and recovery, hematopoiesis correlated with laminin expression in the vascular hematopoietic stem cell niche.To test whether laminin expression directly affects the quality of the niche and hematopoiesis, a mutant mouse line was generated in which laminin g1 expression could be deleted by brief exposure to tamoxifen (TM). Control mice were littermates that lacked the TM-responsive Cre transgene. Prior to TM treatment, adult mutant and control mice had normal peripheral blood cell counts, and there was no evidence of gene recombination in genomic DNA samples. Adult mutant and control mice were then injected with either TM or vehicle, and the peripheral blood, bone marrow, spleen, and liver were collected after three weeks. Laminin g1 genes were deleted and laminin expression was lost in bone marrow, spleen, and liver sinusoids of only the TM-treated mutant mice. These mice also became thrombocytopenic and leukopenic. Flow cytometry showed early arrest of B-lymphocyte development within the bone marrow, bone marrow sinusoids became hemorrhagic, and the normal cytoarchitecture of the sinusoids and endothelial expression of VEGFR2 were lost. All other mouse groups were unaffected.These results suggest that continuous laminin expression is necessary for support of sinusoidal endothelial cells and the vascular hematopoietic niche. Disruption of the hematopoietic microenvironment is sufficient to alter blood cell development and release into peripheral circulation. These studies support the critical role of the microenvironment and stroma in supporting hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Differential expression of microRNA-1 in dorsal root ganglion neurons

Damage to sensory neurons induces neural repair, regrowth and hyperexcitability. The regulation of such responses to injury must be organized in some way by the neurons. Regulation can occur at the post-transcriptional level via microRNAs (miRNAs). miRNAs are small non-coding RNAs that influence the stability or translation of mRNAs and thereby regulate gene expression. Although nociceptive neurons show transcriptional and post-transcriptional regulatory mechanisms at many levels, miRNAs have not yet been systematically investigated in these neurons. Based on our preliminary array data we investigated the presence of miR-1 in dorsal root ganglion (DRG) neurons of mice and humans. We detected miR-1 in total RNA from human and mouse DRG and localised miR-1 in human and murine sensory neurons in situ. In Situ Hybridization detected miR-1 expression by nearly all DRG neurons. In vitro studies of enriched sensory neuron subpopulations from mouse DRG showed higher miR-1 expression levels in I-B4 negative neurons compared with I-B4 positive cells. Culturing of primary sensory neurons reduced the relative miR-1 expression levels independent of the presence or absence of laminin on the culture substrate. Transfection with a miR-1 mimic induced a massive increase in neuronal miR-1 associated with attenuated neurite outgrowth. This first description of miR-1 in sensory neurons including nociceptors suggests that miR-1 has a role in modulating neurite outgrowth.

Immunocytochemical Studies of Adhesion Molecules on Mouse UNK Cells and Their Extracellular Matrix Ligands During Mouse Pregnancy

Uterine natural killer (uNK) cells are the dominant lymphocytes of pregnant mammals' uterus. Studies have identified four differentiation stage of mouse uNK cells based on Dolichos biflorus lectin cytochemistry, and their distribution showed preferential domain in the uterus through out the pregnancy. This work was done to investigate the expression of alpha5, alpha6, and beta7 integrins on uNK cells and their ligands distribution. Section of mouse uterus from sixth to seventeenth gestational days were submitted to immunocytochemistry and positive reactions for alpha5, alpha6, and beta7 integrins were found on uNK from eighth to tenth gestational days but not after twelfth gestational days. Fibronectin reactions were seemed from sixth to tenth gestational days around uNK from the myometrium and endometrium close to the myometrium. No reaction for fibronectin was seen in the decidualized and nondecidualized endometrium near the placenta. Laminin reaction was seen just in the antimesometrial side. beta7 integrin seems to be the active receptor to bind with VCAM-1 or MAdCAM-1 of endothelial cells, promoting the uNK cross through the vessels. The absence of laminin in an uNK domain suggests these cells are not dependent of laminin and alpha6 integrin for their establishment. However, fibronectin seems to support uNK migration, proliferation, differentiation, and survival in the uterus by binding with alpha5 integrin. The loss of alpha5 integrin ligation by the down regulation of fibronectin could inhibits these events and further studies are need to investigate whether unligated alpha5 can actively and initiate apoptosis, maybe in a caspase 8-dependent way that has been called integrin-mediated death.

Neuregulin 1β/erbB signalling in cardiac myocytes enhances integrin based myocyte‐matrix interactions via src

The Neuregulin‐1β (Nrg)‐erbB2/ErbB4 (E2/E4) system is critical for the heart and it is needed for myocyte survival in stress conditions. Nrg activates Src and FAK, both part of integrin signaling. Hyp.: Nrg signaling regulates myocyte matrix interactions, critical for coupling force generation to the extracellular matrix.Immunostaining of mouse hearts showed E4 and E2 are at intercalated discs and have a t‐tubule like striated pattern. Isolated myocytes were incubated 30 min or 24h with an E2 monoclonal antibody (Ab) or an E2 tyrosine kinase inhibitor (TKI). Western‐blot showed E2 TKI blocks activation of FAK, Erk1/2, and Akt by Nrg; Ab is a weak inhibitor. ErbB activation in myocyte‐matrix coupling was tested by plating myocytes onto untreated culture dishes spot‐coated with laminin, with or without Nrg (10ng/ml). After 30 minutes unattached cells were stained and counted. Attachment was enhanced 4‐fold by Nrg vs. laminin alone. Cells did not attach to Nrg coated plates in absence of laminin. Pre‐treatment of myocytes with E2 inhibitors did not prevent Nrg‐enhanced attachment; pre‐treated with Src inhibitor PP2 (10 μM) Nrg effect was inhibited.Nrg‐1β treatment of cardiac myocytes enhances integrin mediated myocyte‐matrix coupling via Src dependent signalling. This appears to be E2 independent pathway, involving E4 signaling, supported by e4 localization to the intercalated disks in vivo.Support by SNSF, NIH.

Adhesion Receptors Mediate Efficient Non-viral Gene Delivery

For a variety of reasons, including production limitations, potential unanticipated side effects, and an immunological response upon repeated systemic administration, virus-based vectors are as yet not ideal gene delivery vehicles, justifying further research into alternatives. Unlike viral vectors, non-viral vectors pose minimal health risks, but to meet therapeutic requirements their efficacy needs major improvement. This goal may be accomplished by better defining the mechanism of non-viral gene delivery and exploiting specific cellular properties. Here we demonstrate that transfection of epithelial cells with lipoplexes is almost exclusively mediated by the beta1 integrin cell surface receptor. More important, we show that in general, adhesion receptors can be exploited by lipoplexes to gain access to cells, including difficult-to-transfect primary neural stem cells and suspension cells, thereby leading to productive transfection. We propose that adhesion receptors serve as "natural" receptors for lipoplexes. As no natural cellular receptors for lipoplexes have previously been identified, our results are an important step forward in understanding the mechanisms of non-viral gene delivery. Moreover, the finding that adhesion receptors mediate efficient non-viral gene delivery paves the way for the optimization of (standard) transfection procedures as well as ex vivo gene therapy protocols using non-viral vectors.

Extracellular Muscle Myosin II Promotes Sensory Axon Formation

Myosin II is an intracellular force-generating enzyme with no known extracellular action. In the course of experiments involving trituration loading of skeletal myosin II into embryonic sensory neurons we observed that extracellular application of myosin II to neurons resulted in a robust increase in the number of axons initiated by each neuron, but did not alter the rate of axon extension. Substratum bound myosin II in the presence of laminin was sufficient to elicit increases in axon formation. However, in the absence of laminin, extracellular myosin II alone was not sufficient to promote axon formation, although it allowed neuron survival in the presence of neurotrophin. Myosin II promoted the attachment of neurons to the substratum in the absence or presence of laminin. In addition to promoting the initiation of axons, extracellular myosin II also increased the frequency of axon collateral branching. Finally, extracellular myosin II did not affect growth cone collapse in response to semaphorin-IIIA, but attenuated the inhibitory action of chondroitin sulfate proteoglycans on axon extension. Surprisingly, these results demonstrate that extracellular myosin II promotes attachment of neurons and increases axon formation and branching. The potential significance of these observations is discussed in the context of myosin II release from injured muscle and a previous demonstration of extracellular myosin II association with the extracellular matrix.

Laminin-induced aggregation of the inwardly rectifying potassium channel, Kir4.1, and the water-permeable channel, AQP4, via a dystroglycan-containing complex in astrocytes.

Dystroglycan (DG) is part of a multiprotein complex that links the extracellular matrix to the actin cytoskeleton of muscle fibers and that is involved in aggregating acetylcholine receptors at the neuromuscular junction. This complex is also expressed in regions of the central nervous system where it is localized to both neuronal and glial cells. DG and the inwardly rectifying potassium channels, Kir4.1, are concentrated at the interface of astroglia and small blood vessels. These channels are involved in siphoning potassium released into the extracellular space after neuronal excitation. This raises the possibility that DG may be involved in targeting Kir4.1 channels to specific domains of astroglia. To address this question, we used mixed hippocampal cultures to investigate the distribution of DG, syntrophin, dystrobrevin, and Kir4.1 channels, as well as aquaporin-permeable water channels, AQP4. These proteins exhibit a similar distribution pattern and form aggregates in astrocytes cultured on laminin. Both DG and syntrophin colocalize with Kir4.1 channel aggregates in astrocytes. Similarly, DG colocalizes with AQP4 channel aggregates. Quantitative studies show a significant increase of Kir4.1 and AQP4 channel aggregates in astrocytes cultured in the presence of laminin when compared with those in the absence of laminin. These findings show that laminin has a role in Kir4.1 and AQP4 channel aggregation and suggest that this may be mediated via a dystroglycan-containing complex. This study reveals a novel functional role for DG in brain including K+ buffering and water homeostasis.

Microtubule and Rac 1-dependent F-actin in growth cones.

Extracellular cues control the rate and direction of growth of neuronal processes in large part by regulating the cytoskeleton of the growth cone. The actin filament network of the peripheral region is thought to be the primary target for these cues, with consequences for the advance and organization of microtubules. Binding of laminin to integrin receptors is a cue that accelerates the growth of processes from many types of neurons. It was applied acutely to sympathetic neurons in culture to study its effects on the cytoskeleton of the growth cone. Microtubules advance to the edge of the growth cone and bundle in response to laminin, and it was found that small veils of membrane appear near the ends of some of those microtubules. To examine more clearly the relationship between the microtubules and the appearance of actin-rich structures at the periphery, a low dose of cytochalasin D was used to deplete the peripheral region of the growth cone of pre-existing F-actin. The subsequent addition of laminin resulted in the bundling of ends of dynamic (tyrosinated) microtubules at the distal edge of the growth cone, most of which were associated with foci of F-actin. Observations of labeled actin within living growth cones confirmed that these foci formed in response to laminin. Suppression of microtubule dynamics with drugs eliminated the actin foci; washout of drug restored them. Rac 1 did not co-concentrate with F-actin in the peripheral region of the growth cone in the absence of laminin, but did co-concentrate with the foci of F-actin that formed in response to laminin. Inhibition of Rac 1 functioning prevented the formation of the foci and also inhibited laminin-induced neurite growth with or without cytochalasin. These results indicate that extracellular cues can affect actin in the growth cone via microtubules, as well as affect microtubules via actin. They also point to the mediation of microtubule-dependent accumulation of F-actin at the front of the growth cone as a role of Rac 1 in neurite growth.

Animal Models for Skin Blistering Conditions: Absence of Laminin 5 Causes Hereditary Junctional Mechanobullous Disease in the Belgian Horse

Recent achievements in the genetic correction of keratinocytes isolated from patients with junctional epidermolysis bullosa have paved the way to a gene therapy approach for the disease. Because gene therapy protocols require preclinical validation in animals, we have characterized spontaneous animal models of junctional epidermolysis bullosa. In this study we have elucidated the genetic basis of the hereditary junctional mechanobullous disease in the Belgian horse, a condition characterized by blistering of the skin and mouth epithelia, and exungulation (loss of the hoof). Immunofluorescence analysis associated the condition to the absent expression of the gamma2 chain of laminin 5 and designated Lamc2 as the candidate gene. Comparative analysis of the nucleotide sequence of the full-length gamma2 cDNA isolated by reverse transcription polymerase chain reaction amplification of total RNA purified from the epithelium of a junctional epidermolysis bullosa foal and a healthy control disclosed a homozygous basepair insertion (1368insC) in the affected animal. Mutation 1368insC results in a downstream premature termination codon and is predicted to cause absent expression of the laminin gamma2 polypeptide. Our results also show that: (i) the horse junctional epidermolysis bullosa genetically corresponds to the severe Herlitz form of junctional epidermolysis bullosa in man; (ii) the amino acid sequence and structure of the horse laminin gamma2 chain are virtually identical to the human counterpart; (iii) the moderate eruption of skin blisters in the affected animals with respect to the human Herlitz junctional epidermolysis bullosa patients correlates with the protection provided by hair. Our observations suggest that the affected foals are a convenient source of epithelial cells from tissues that cannot be obtained from human junctional epidermolysis bullosa patients, and imply that hairless strains of animals with recessive skin disorders would be the best models for in vivo gene therapy approaches to skin blistering diseases.

Puerperal involution in the mare – a comparison of clinical signs and pathology

A detailed morphological and functional description of equine endometrial involution is necessary for the understanding of alterations leading to a reduced fertility rate following peripartal disturbances. A complete involution of the microcaruncles, glands and stroma by the 9th day post parturition and the return to cyclical activity is essential for an early post partal pregnancy. After normal parturition, a loss of the glandular cytokeratin-expression, the absence of laminin in the glandular basal lamina and an intraepithelial expression of vimentin occurs especially on the 3rd and 6th day post partum (p.p.). The expression of estrogen receptors and progesterone receptors of intact glandular epithelial cells decreases from the 3rd to the 6th day p.p. and increases from the 6th to 9th day p.p.. Decreased fertility rate in the first post-partum heats in mares with dystocia or retained placental delivery may be due to a higher incidence of periglandular fibrosis and a disturbed glandular redifferentiation, which is characterized by a delayed inset of epithelial cytokeratin-expression and an unphysiological expression of the hormone receptors. Moreover, in mares with dystocia a disturbed microcaruncular involution may contribute to the decreased conception rate.

Chemotaxis of Rat Mast Cells Toward Adenine Nucleotides

Rat mucosal mast cells express P2 purinoceptors, occupation of which mobilizes cytosolic Ca2+ and activates a potassium conductance. The primary function of this P2 system in mast cell biology remains unknown. Here, we show that extracellular ADP causes morphological changes in rat bone marrow-cultured mast cells (BMMC) typical of those occurring in cells stimulated by chemotaxins, and that the nucleotides ADP, ATP, and UTP are effective chemoattractants for rat BMMC. ADP was also a chemotaxin for murine J774 monocytes. The nucleotide selectivity and pertussis toxin sensitivity of the rat BMMC migratory response suggest the involvement of P2U receptors. Poorly hydrolyzable derivatives of ADP and ATP were effective chemotaxins, obviating a role for adenosine receptors. Buffering of external Ca2+ at 100 nM or reduction of the electrical gradient driving Ca2+ entry (by elevating external K+) blocked ADP-driven chemotaxis, suggesting a role for Ca2+ influx in this process. Anaphylatoxin C5a was a potent chemotaxin (EC50 approximately 0.5 nM) for J774 monocytes, but it was inactive on rat BMMC in the presence or absence of laminin. Ca2+ removal or elevated [K+] had modest effects on C5a-driven chemotaxis of J774 cells, implicating markedly different requirements for Ca2+ signaling in C5a- vs ADP-mediated chemotaxis. This is supported by the observation that depletion of Ca2+ stores with thapsigargin completely blocked migration induced by ADP but not C5a. These findings suggest that adenine nucleotides liberated from parasite-infested tissue could participate in the recruitment of mast cells by intestinal mucosa.

Targeted disruption of the LAMA3 gene in mice reveals abnormalities in survival and late stage differentiation of epithelial cells.

Laminin 5 regulates anchorage and motility of epithelial cells through integrins alpha6beta4 and alpha3beta1, respectively. We used targeted disruption of the LAMA3 gene, which encodes the alpha3 subunit of laminin 5 and other isoforms, to examine developmental functions that are regulated by adhesion to the basement membrane (BM). In homozygous null animals, profound epithelial abnormalities were detected that resulted in neonatal lethality, consistent with removal of all alpha3-laminin isoforms from epithelial BMs. Alterations in three different cellular functions were identified. First, using a novel tissue adhesion assay, we found that the mutant BM could not induce stable adhesion by integrin alpha6beta4, consistent with the presence of junctional blisters and abnormal hemidesmosomes. In the absence of laminin 5 function, we were able to detect a new ligand for integrin alpha3beta1 in the epidermal BM, suggesting that basal keratinocytes can utilize integrin alpha3beta1 to interact with an alternative ligand. Second, we identified a survival defect in mutant epithelial cells that could be rescued by exogenous laminin 5, collagen, or an antibody against integrin alpha6beta4, suggesting that signaling through beta1 or beta4 integrins is sufficient for survival. Third, we detected abnormalities in ameloblast differentiation in developing mutant incisors indicating that events downstream of adhesion are affected in mutant animals. These results indicate that laminin 5 has an important role in regulating tissue organization, gene expression, and survival of epithelium.

Absence of PS Integrins or Laminin A Affects Extracellular Adhesion, but Not Intracellular Assembly, of Hemiadherens and Neuromuscular Junctions inDrosophilaEmbryos

We have examined the role of integrins in the formation of the cell junctions that connect muscles to epidermis (muscle attachments) and muscles to neurons (neuromuscular junctions). To this end we have analyzed muscle attachments and neuromuscular junctions ultrastructurally in single or double mutantDrosophilaembryos lacking PS1 integrin (αPS1βPS), PS2 integrin (αPS2βPS), and/or their potential extracellular ligand laminin A. At the muscle attachments PS integrins are essential for the adhesion of hemiadherens junctions (HAJs) to extracellular matrix, but not for their intracellular link to the cytoskeleton. The PS2 integrin is only expressed in the muscles, but it is essential for the adhesion of muscleandepidermal HAJs to electron dense extracellular matrix. It is also required for adhesion of muscle HAJs to a less electron dense form of extracellular matrix, the basement membrane. The PS1 integrin is expressed in epidermal cells and can mediate adhesion of the epidermal HAJs to the basement membrane. The ligands involved in adhesion mediated by both PS integrins seem distinct because adhesion mediated by PS1 appears to require the extracellular matrix component laminin A, while adhesion mediated by PS2 integrin does not. At neuromuscular junctions the formation of functional synapses occurs normally in embryos lacking PS integrins and/or laminin A, but the extent of contact between neuronal and muscle surfaces is altered significantly. We suggest that neuromuscular contact in part requires basement membrane adhesion to the general muscle surface, and this form of adhesion is completely abolished in the absence of laminin A.

The functions of laminins: lessons from in vivo studies.

This series of three short reviews is an attempt to summarize our current knowledge of the in vivo tests of hypotheses of laminin functions. The structures of the laminins have been thoroughly reviewed recently (P. Ekblom and R. Timpl, in press), and I will not attempt to repeat this information here. Instead, I will focus on the recent evidence gathered from gene knock out experiments in mice and from naturally occurring human and mouse gene mutations. The most obvious lesson from the above studies--other than demonstrating the importance of laminins in general--is that the structural diversity of the laminin family members makes highly specialized functions possible. While all laminins may share many functional properties, the individual chains are involved in interactions which cannot be substituted for by other laminins or by other basement membrane components. While this concept is not new, it is very satisfying to see its validity so dramatically confirmed. It is therefore predictable that additional gene ablation experiments using other known and yet undescribed laminin genes will be equally interesting and informative. To me, one of the most striking lessons from these studies is how strongly the induced mouse mutations mimic human disease. With all the concerns with genetic background differences and species specific effects, manipulation of the laminin genes appears to be a particularly good first approach to identifying the causes of human disease. There is an abundant literature accumulated from biochemical and, more recently, molecular structural analyses, and from in vitro systems, suggesting a role of laminins contributing directly to the stability of the basement membrane. There is an equally vast literature supporting an indirect role in mediating cellular behavior, through interactions with various receptors. It is interesting that the in vivo studies summarized above support both activities. In the case of laminin 5 mutations, the phenotypic consequence appears to be due primarily to the loss of an important structural link between the epithelial cytokeratins and the dermal anchoring fibrils. The ultrastructure of the epithelium appears normal, as does the architecture of the papillary dermis. Only the anchoring complex itself is aberrant. The absence of laminin 5 appears not to compromise the development or viability of the epidermis. The basement membrane appears normal-other than the anchoring complex itself. The pathology observed in the newborn is believed to be due to the frictional trauma of birth, with the expectation that the function of the fetal skin is normal in utero. The Herlitz epidermolysis bullosa phenotype is obvious immediately at birth, and it does not progress postnatally beyond the extent to which the affected individual experiences additional frictional trauma or secondary consequences such as infection or fluid loss. Since laminin 5 is only one of a series of structural links within the anchoring complex, one would predict that a loss of any of these links would result in the same phenotype. Current evidence supports this view, as the absence of integrin alpha 6 beta 4 (Vidal et al., 1995; Dowling et al., 1996; Georges-Labouesse et al., 1996; van der Neut et al., 1996) or of collagen VII (A. M. Christiano and J. Uitto, in press) also results in dramatic neonatal dermal-epidermal fragility. The differences in phenotype, such as the pyloric atresia in the case of loss of integrin alpha 6 beta 4, are presumably due to additional functions of the integrin in other tissues or in other developmental processes. Therefore, the laminin 5 mutations may be unique, in that the in vivo studies suggest that the primary role of the molecule is in the elaboration and stability of the anchoring complex, but not in the basement membrane itself. Of course, since the in vivo phenotype reflects only losses that cannot be compensated, this interpretation may be much too narrow. (ABSTRACT TRUNCATED)

Expression of synapsin I correlates with maturation of the neuromuscular synapse.

Synapsins are a family of neuron-specific phosphoproteins that are localized within the presynaptic terminals in adult brain. Previous work has demonstrated that introduction of exogenous synapsins I(a + b) or IIa into Xenopus spinal neurons promoted maturation of the neuromuscular synapse in a nerve-muscle co-culture system. We have now studied the expression of endogenous Xenopus synapsin I during synaptic maturation in vivo and in culture, using a polyclonal antibody raised against Xenopus synapsin I. Immunoprecipitation experiments indicated that synapsin I was not detectable during the early phase of synaptogenesis in vivo, and exhibited a marked increase during the period of synaptic maturation. In contrast, the expression of synaptophysin, another synaptic vesicle protein, was detected at the start of nervous system formation, and remained at a high level thereafter. Similar expression profiles for the two proteins were also observed in immunocytochemical studies of Xenopus spinal neurons in culture: intense staining of synaptophysin was found on the first day, while synapsin I was not detected until after three days in culture. The expression of synapsin I correlated very well with the appearance of a bell-shaped amplitude distribution of spontaneous synaptic currents, a physiological parameter which reflects functional maturation of the neuromuscular synapse. In one-day-old cultures grown in the absence of laminin, an extracellular matrix protein known to be present at the neuromuscular junction, the amplitude distribution of virtually all synapses was skewed towards smaller values. In contrast, when laminin was used as a culture substrate, many synapses exhibited a bell-shaped amplitude distribution. Laminin treatment also induced synapsin I expression in one-day-old cultures. These results suggest that the expression of endogenous synapsin I may regulate maturation at neuromuscular synapses.