Motor Endplate-Anatomical, Functional, and Molecular Concepts in the Historical Perspective.

While understanding macromolecular structural elements and their roles in dictating cellular function is critical to grasp basic concepts in biology, it can be challenging for students to master this content—these elements naturally exist at the nanoscale and are not observable with the naked eye. Oftentimes this understanding is catalyzed by impactful illustrations and animations found online and in textbooks. In recent years, 3D printing technology has become readily accessible as an additional way to generate models and visualize entities of interest. In this report, we describe and discuss the efficacy of an approach using 3D-printed models in combination with online open-source molecular modeling analyses of the macromolecular structure of p53 to engage students with molecular concepts in cancer cell biology and human health. This pedagogy strategy has been successfully integrated into an upper-level undergraduate course at a primarily undergraduate institution and a graduate biology course at a public research university. We describe the potential benefits while providing tools for others to integrate this strategy into their teaching.

An essential principle of competency-based education (CBE) is use of observable outcomes with assessments as judgments of competence based on defined criteria. Faculty are accustomed to using learning objectives as the defining criteria for knowledge, assessing students using written exams. Faculty are less familiar with how the principles of CBE are applied to other competencies. We recently adopted school-wide goals and objectives, modeled after the ACGME Outcomes Project. The present objective was to give faculty first-hand experience in CBE within a basic science course, including both cognitive and non-cognitive outcomes. The format for the learner-centered, first-year Cell and Molecular Biology course was previously described.(1) Course goals were that students: (1) gain an understanding of the principles and concepts of cell and molecular biology, (2) develop an appreciation for how these principles and concepts are important to medicine, (3) demonstrate an ability to think critically using these principles and concepts. Goal 1 was measured by written exams. We assumed goals 2 and 3 were met through small-group problem-solving sessions, and outcomes were not assessed. The revised 2001 course goals were to prepare students for medical knowledge and lifelong learning and communication and professionalism. The goals for medical knowledge and lifelong learning were to: (1) demonstrate ability to use principles and concepts of cell biology, molecular biology, and genetics to analyze medically relevant data, solve problems, make predictions, and determine a course of action; (2) effectively use information technology to search, evaluate, and critically review scientific evidence related to principles and concepts covered in the course; (3) use appropriate techniques to teach peers in a conference setting. The goals for communication and professionalism were to: (1) use appropriate skills and attitudes to collaborate effectively with peers and faculty to accomplish learning goals; (2) maintain a personal learning portfolio to develop habits of reflective learning, broaden understanding of content beyond recall, and enhance communication with faculty; (3) demonstrate personal integrity in meeting course requirements and in interactions with peers and faculty throughout the course. Goals for medical knowledge and lifelong learning were assessed by written exams and by separate tools utilizing four-point Likert scales (novice, advanced beginner, proficient, distinguished) with specific observable criteria for a written research paper and a group PowerPoint presentation. Faculty and student assessments generated a number that was combined with exam grades for a lettered competency grade. A 19-item, five-point Likert scale was used by students to self- and peer-assess goals for communication and professionalism. Small-group faculty facilitators used the tool to give formative feedback midcourse, summative feedback at course conclusion, and competency grades. The tools may be viewed at: <www.meddean.luc.edu/lumen/meded/cellbio/index.htm>. Faculty achieved enhanced understanding of students, assisted by descriptive criteria, while suggesting improvements in forms. Better agreement on criteria definitions and consistency in form use is needed. Students developed understanding and improved communication/professionalism skills, based on repeated exposures to criteria and feedback. It remains to be seen whether the skills are used/developed in other courses. A majority of students did not use the learning portfolio as envisioned. Better design and implementation of school-wide rather than course-specific reflective portfolios may increase use and integrate learning in all courses with all six competencies.

A Presynaptic Giant Ankyrin Stabilizes the NMJ through Regulation of Presynaptic Microtubules and Transsynaptic Cell Adhesion

This paper reports on a study which investigated whether the addition of haptics (virtual touch) to a three-dimensional (3D) virtual reality (VR) simulation promotes learning of key concepts in biology for students aged 12 to 13 years. We developed a virtual model of a section of the cell membrane and a haptic-enabled interface that allows students to interact with the model and to manipulate objects in the model. Students, in two schools in England, worked collaboratively on activities, in pairs, designed to support learning of key difficult concepts. These concepts included the dynamic nature of the cell membrane, passive diffusion and facilitated diffusion. Findings from observation of the activities and student interviews revealed that students were very positive about using the system and believed that being able to feel structures and movements within the model assisted their learning. Results of pre- and post-tests of conceptual knowledge showed significant knowledge gains but there were no significant differences between the haptic and non-haptic condition.

The regulated release of neurotransmitter occurs via the fusion of synaptic vesicles (SVs) at specialized regions of the presynaptic membrane called active zones (AZs). These regions are defined by a cytoskeletal matrix assembled at AZs (CAZ), which functions to direct SVs toward docking and fusion sites and supports their maturation into the readily releasable pool. In addition, CAZ proteins localize voltage-gated Ca(2+) channels at SV release sites, bringing the fusion machinery in close proximity to the calcium source. Proteins of the CAZ therefore ensure that vesicle fusion is temporally and spatially organized, allowing for the precise and reliable release of neurotransmitter. Importantly, AZs are highly dynamic structures, supporting presynaptic remodeling, changes in neurotransmitter release efficacy, and thus presynaptic forms of plasticity. In this review, we discuss recent advances in the study of active zones, highlighting how the CAZ molecularly defines sites of neurotransmitter release, endocytic zones, and the integrity of synapses.

Although acetylcholine is the major neurotransmitter operating at the skeletal neuromuscular junction of many invertebrates and of vertebrates, glutamate participates in modulating cholinergic transmission and plastic changes in the last. Presynaptic terminals of neuromuscular junctions contain and release glutamate that contribute to the regulation of synaptic neurotransmission through its interaction with pre- and post-synaptic receptors activating downstream signaling pathways that tune synaptic efficacy and plasticity. During vertebrate development, the chemical nature of the neurotransmitter at the vertebrate neuromuscular junction can be experimentally shifted from acetylcholine to other mediators (including glutamate) through the modulation of calcium dynamics in motoneurons and, when the neurotransmitter changes, the muscle fiber expresses and assembles new receptors to match the nature of the new mediator. Finally, in adult rodents, by diverting descending spinal glutamatergic axons to a denervated muscle, a functional reinnervation can be achieved with the formation of new neuromuscular junctions that use glutamate as neurotransmitter and express ionotropic glutamate receptors and other markers of central glutamatergic synapses. Here, we summarize the past and recent experimental evidences in support of a role of glutamate as a mediator at the synapse between the motor nerve ending and the skeletal muscle fiber, focusing on the molecules and signaling pathways that are present and activated by glutamate at the vertebrate neuromuscular junction.

Concepts in Cell Biology - History and Evolution

The neuromuscular junction (NMJ) has been studied for over a century, yet we still do not have a complete picture of all its structural and functional components, knowledge of which is paramount in devising treatment strategies for neuromuscular diseases. Previous microarray-based approaches aimed at elucidating novel NMJ components were hindered by technological limitations. Recent technological advancements propelled next-generation RNA-sequencing with its wider dynamic range to the forefront of transcriptome-level gene expression profiling. We utilized laser-capture microdissection to isolate myonuclei underlying the NMJ combined with RNA-sequencing and successfully generated NMJ gene expression profiles of fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus (SOL) muscles and identified a large number of potential novel NMJ genes. The expression levels of canonical NMJ genes were nearly identical between the EDL and SOL, which suggests that the core NMJ gene program might be well conserved between different skeletal muscle types. We used in vivo muscle electroporation to overexpress one of our candidate genes, the transcription factor T-box 21 (TBX21), in the tibialis anterior (TA) muscle and observed an increased density of postsynaptic acetylcholine receptors. TBX21 may thus represent a novel transcription factor contributing to the regulation of the NMJ gene program, with a role in postsynaptic sensitivity. We also generated NMJ gene expression profiles of the TA muscle of 10-month-old (“young”) and 30-month-old (“old”) mice to investigate the effect of aging on the NMJ gene program. Strikingly, the NMJ gene program was remarkably stable, with nearly identical expression levels of canonical NMJ genes between young and old mice. This implies that age-related perturbations of the NMJ are likely caused by external factors, such as accumulated myofiber damage and changes in nerve input, rather than by gradual dysregulation of the NMJ gene program with increasing age. Our findings argue against the hypothesis that aging leads to a broad deterioration of the NMJ gene program that would contribute to perturbations of NMJ structure and function. Furthermore, functional annotation analysis of our different NMJ gene expression datasets strongly indicates the importance of an extensive number of hitherto unknown glycoproteins, as well as of posttranslational modifications, especially glycosylations, at the synaptic basal lamina. We highlight a set of candidate genes that encode for enzymes putatively involved in these processes at the NMJ, and which are potentially involved in the pathophysiology of neuromuscular diseases such as congenital myasthenic syndromes. This thesis expands our understanding of the complexity of the NMJ and lays the foundation for further research that will functionally characterize novel synaptic components and provide the basis for novel therapeutic treatment strategies.

Subcellular localization of the glutamate transporters GLAST and GLT at the neuromuscular junction in rodents

The Drosophila larval neuromuscular system is relatively simple, containing only 32 motor neurons in each abdominal hemisegment, and its neuromuscular junctions (NMJs) have been studied extensively. NMJ synapses exhibit developmental and functional plasticity while displaying stereotyped connectivity. Drosophila Type I NMJ synapses are glutamatergic, while the vertebrate NMJ uses acetylcholine as its primary neurotransmitter. The larval NMJ synapses use ionotropic glutamate receptors (GluRs) that are homologous to AMPA-type GluRs in the mammalian brain, and they have postsynaptic scaffolds that resemble those found in mammalian postsynaptic densities. These features make the Drosophila neuromuscular system an excellent genetic model for the study of excitatory synapses in the mammalian central nervous system. The first section of the review presents an overview of NMJ development. The second section describes genes that regulate NMJ development, including: (1) genes that positively and negatively regulate growth of the NMJ, (2) genes required for maintenance of NMJ bouton structure, (3) genes that modulate neuronal activity and alter NMJ growth, (4) genes involved in transsynaptic signaling at the NMJ. The third section describes genes that regulate acute plasticity, focusing on translational regulatory mechanisms. As this review is intended for a developmental biology audience, it does not cover NMJ electrophysiology in detail, and does not review genes for which mutations produce only electrophysiological but no structural phenotypes.

dystrophin-glycoprotein complex The sarcolemma (muscle plasma membrane) plays a central role in skeletal muscle structure and function (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar). In addition to the housekeeping functions of a cell plasma membrane, the sarcolemma is directly involved in synaptic transmission, action potential propagation, and excitation-contraction coupling (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar). Besides these well established physiological functions, the sarcolemma, subsarcolemma cytoskeleton, and surrounding basement membrane (extracellular matrix) play an essential structural role in skeletal muscle (1Engel A.G. Franzini-Armstrong C. Myology. 2nd Ed. McGraw-Hill, New York1994Google Scholar, 2Clark K.A. McElhinny A.S. Beckerle M.C. Gregorio C.C. Annu. Rev. Cell Biol. 2002; 18: 637-706Crossref Scopus (474) Google Scholar, 3Colognato H. Yurchenco P.D. Dev. Dyn. 2000; 218: 213-234Crossref PubMed Scopus (1052) Google Scholar). The biological importance of the basement membrane-sarcolemma-cytoskeleton in skeletal muscle is underscored by the number of inherited muscle diseases caused by mutations in components of the basement membrane, or cytoskeleton, or the sarcolemma protein complexes that link the basement membrane to the cytoskelton (4Cohn R.D. Campbell K.P. Muscle Nerve. 2000; 23: 1456-1471Crossref PubMed Scopus (426) Google Scholar, 5Blake D.F. Weir A. Newey S.E. Davies K.E. Physiol. Rev. 2002; 82: 291-329Crossref PubMed Scopus (925) Google Scholar, 6Spence H.J. Chen Y.J. Winder S.J. Bioessays. 2002; 24: 542-552Crossref PubMed Scopus (57) Google Scholar, 7Brown S.C. Muntoni F. Sewer C.A. Brain Pathol. 2002; 11: 193-205Crossref Scopus (17) Google Scholar, 8Durbeej M. Campbell K.P. Curr. Opin. Genet. Dev. 2002; 9: 2019-2027Google Scholar). The minireview in this and the following issues updates our understanding of the structure and function of the basement membrane, cytoskeletal costameres, and the major trans-sarcolemma links (integrins and dystroglycan) in skeletal muscle. The basement membrane surrounds skeletal muscle fibers and is now known to be critical in muscle fiber structure and function. The skeletal muscle basement membrane is composed of the basal lamina and the reticular lamina. Basal lamina is directly linked to the sarcolemma. Genetic studies of muscular dystrophy patients and animal models of muscular dystrophy have demonstrated the importance of the basement membrane in maintenance of muscle integrity. In addition to maintenance of muscle integrity, the basement membrane is essential in the promotion of myogenesis and muscle development. Muscle regeneration is also a process that depends on the skeletal muscle basement membrane. Satellite cells (endogenous stem cells of skeletal muscle) reside between the muscle fiber and the basal lamina. Following injury, new muscle fibers regenerate within a basement membrane tube, which is believed to act as a mechanical barrier to limit migration of satellite cells, and a scaffold to orient myotube regeneration. Finally, the basement membrane is structurally and functionally specialized in areas of neuromuscular and myotendinous junctions and is required for the assembly of these structures. In the first minireview of this series entitled "The Basement Membrane/Basal Lamina of Skeletal Muscle," Joshua R. Sanes reviews the structure and function of the skeletal muscle basement membrane. In particular, he focuses on recent molecular studies that have led to a better understanding of the function of the basement membrane in skeletal muscle physiology and pathophysiology (9Sanes J.R. J. Biol. Chem. 2003; 278: 12601-12604Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). A major cytoskeletal structure in muscle that has the unique role of connecting the sarcomere to the basement membrane is the costamere. Costameres were originally described as subsarcolemma protein complexes that align in register with the Z-disk and are physically coupled to the sarcomeres. Costameres may be equivalent to focal adhesions that are expressed in non-muscle cells and are believed to be involved in the lateral transmission of contractile forces from sarcomeres across the sarcolemma to the basement membrane. In the second minireview, entitled "Costameres: the Achilles Heel of Herculean Muscle," James M. Ervasti reviews the structure and function of the striated muscle costamere (10Ervasti J.M. J. Biol. Chem. 2003; 278: 13591-13594Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar). As with the basement membrane, the importance of the costamere for normal muscle function has been revealed by genetic studies of muscular dystrophies and dilated cardiomyopathies. Dystrophin is known to be enriched in costameres, but dystrophin is not required for costamere assembly. In the absence of dystrophin, there is a disorganization of the costameric lattice, as well as disruption of the sarcolemma integrity. Disruption of the costameric lattice correlates with functional studies that show reduction of contractile force in muscles lacking dystrophin. Ervasti's minireview provides insights into the growing costameric protein network and illustrates how these proteins can interact with many components of both the sarcolemma and cytoskeleton. In addition, the newly identified proteins suggest a role for costameric proteins in converting mechanical stimuli to alterations in cell signaling and gene expression. Finally, Ervasti discusses non-sarcolemmal mechanical defects associated with the loss of costameric proteins. It is well recognized that the function and maintenance of skeletal muscle cell integrity is dependent upon interactions of the muscle cell with the surrounding basement membrane and underlying cytoskeleton. Trans-sarcolemma receptors are known to be involved in providing critical mechanical links between the basement membrane and the cytoskeleton. In addition, recent data suggest that these receptors transmit signals from the basement membrane into the muscle cell. Over the past 10 years there has been great progress in the identification and characterization of the two sarcolemma protein complexes that connect cytoskeleton to the basement membrane in skeletal muscle: integrins and the dystrophin-glycoprotein complex (DGC).1 Integrins form a large family of cell surface receptors that mediate cell-extracellular matrix interactions. In the third minireview, entitled "Integrins, Redundant or Important Players in Skeletal Muscle?," Ulrike Mayer focuses on the role of integrins in skeletal muscle (11Mayer U. J. Biol. Chem. 2003; 278: 14587-14590Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). Of the current members of this family, a subset is expressed in skeletal muscle, in particular, at the sarcolemma, neuromuscular junction, myotendinous junction, and costameres. Expression of the integrin family is regulated during skeletal muscle development. Integrins play a major role in muscle differentiation, and α7β1 integrin is a critical receptor for myoblast migration. In adult skeletal muscle, integrins are concentrated at the neuromuscular junction and the myotendinous junction and provide an important link to the basement membrane in these specialized regions of muscle. α7β1 integrin is the major form found in adult skeletal muscle. Integrins have also been implicated in skeletal muscle diseases, and the absence of α7 integrin leads to a mild muscular dystrophy in both mice and humans. Myotendinous junctions were severely disturbed in mouse models with α7integrin deficiency. Both mouse and human studies suggest that muscle weakness arises from destruction of the myotendinous junction, rather than from compromised sarcolemma integrity. Finally, α7β1 integrin is reduced in several muscular dystrophies, and α7β1 integrin overexpression may provide a possible therapeutic approach for Duchenne muscular dystrophy. In the final minireview, entitled "Dystrophin-Glycoprotein Complex: Post-translational Processing and Dystroglycan Function," Daniel E. Michele and Kevin P. Campbell focus on the major sarcolemma membrane complex in adult skeletal muscle that links the cytoskeleton to the basement membrane (12Michele D.E. Campbell K.P. J. Biol. Chem. 2003; (January 29, 10.1074/jbc.R200031200)Google Scholar). DGC is a large oligomeric complex of proteins in the sarcolemma of skeletal muscle. The DGC is composed of both integral and peripheral membrane proteins and provides a structural connection between the basement membrane and the actin cytoskeleton and has been hypothesized to protect the sarcolemma from mechanical damage during muscle contraction. Several forms of muscular dystrophy arise from primary mutations in genes encoding components of the DGC. The DGC is grouped into three subcomplexes, dystroglycan (α- and β-dystroglycan), the sarcoglycan-sarcospan subcomplex, and the cytoskeletal components dystrophin, syntrophyin, and dystrobrevin. Michele and Campbell review the current status of our understanding of the DGC, and in particular focus on the structure and post-translational processing of dystroglycan. Interestingly, recent genetic data have demonstrated that proteins with homology similar to glycosyltransferases are linked to muscular dystrophy and appear to preferentially or exclusively modify α-dystroglycan. The role of glycosylation in the function of dystroglycan is discussed, and the mechanisms whereby the loss of functional dystroglycan leads to clinical symptoms, including muscular dystrophy, and abnormal central nervous system development and function are presented. Finally, new insights into dystroglycan function revealed from the studies of mouse models and patients with incomplete glycosylation (dystroglycanopathies) are reviewed. Much progress has been made in our understanding of the role of basement membrane-sarcolemma-cytoskeleton interactions in the development, structure, and function of striated muscle. The field continues to be a dynamic arena for future research, in which new functional molecules, their interactions, and their functional significance, continue to be identified from molecular genetics of human diseases, biochemistry, cell biology, and gene targeting in the mouse. Given the essential role of these molecules in human disease, understanding the interactions of the skeletal muscle basement membrane-sarcolemma-cytoskeleton will hopefully lead to a better understanding of disease pathogenesis and therapeutic opportunity.

The dynamic interaction between positive and negative signals is necessary for remodeling of postsynaptic structures at the neuromuscular junction. Here we report that Wnt3a negatively regulates acetylcholine receptor (AChR) clustering by repressing the expression of Rapsyn, an AChR-associated protein essential for AChR clustering. In cultured myotubes, treatment with Wnt3a or overexpression of beta-catenin, the condition mimicking the activation of the Wnt canonical pathway, inhibited Agrin-induced formation of AChR clusters. Moreover, Wnt3a treatment promoted dispersion of AChR clusters, and this effect was prevented by DKK1, an antagonist of the Wnt canonical pathway. Next, we investigated possible mechanisms underlying Wnt3a regulation of AChR clustering in cultured muscle cells. Interestingly, we found that Wnt3a treatment caused a decrease in the protein level of Rapsyn. In addition, Rapsyn promoter activity in cultured muscle cells was inhibited by the treatment with Wnt3a or beta-catenin overexpression. Forced expression of Rapsyn driven by a promoter that is not responsive to Wnt3a prevented the dispersing effect of Wnt3a on AChR clusters, suggesting that Wnt3a indeed acts to disperse AChR clusters by down-regulating the expression of Rapsyn. The role of Wnt/beta-catenin signaling in dispersing AChR clusters was also investigated in vivo by electroporation of Wnt3a or beta-catenin into mouse limb muscles, where ectopic Wnt3a or beta-catenin caused disassembly of postsynaptic apparatus. Together, these results suggest that Wnt/beta-catenin signaling plays a negative role for postsynaptic differentiation at the neuromuscular junction, probably by regulating the expression of synaptic proteins, such as Rapsyn.

Glia are an indispensable structural and functional component of the synapse. They modulate synaptic transmission and also play important roles in synapse formation and maintenance. The vertebrate neuromuscular junction (NMJ) is a classic model synapse. Due to its large size, simplicity and accessibility, the NMJ has contributed greatly to our understanding of synapse development and organization. In the past decade, the NMJ has also emerged as an effective model for studying glia-synapse interactions, in part due to the development of various labeling techniques that permit NMJs and associated Schwann cells (the glia at NMJs) to be visualized in vitro and in vivo. These approaches have demonstrated that Schwann cells are actively involved in synapse remodeling both during early development and in post-injury reinnervation. In vivo imaging has also recently been combined with serial section transmission electron microscopic (ssTEM) reconstruction to directly examine the ultrastructural organization of remodeling NMJs. In this review, we focus on the anatomical studies of Schwann cell dynamics and their roles in formation, maturation and remodeling of vertebrate NMJs using the highest temporal and spatial resolution methods currently available.

BackgroundMyosin Va is a motor protein involved in vesicular transport and its absence leads to movement disorders in humans (Griscelli and Elejalde syndromes) and rodents (e.g. dilute lethal phenotype in mice). We examined the role of myosin Va in the postsynaptic plasticity of the vertebrate neuromuscular junction (NMJ).Methodology/Principal FindingsDilute lethal mice showed a good correlation between the propensity for seizures, and fragmentation and size reduction of NMJs. In an aneural C2C12 myoblast cell culture, expression of a dominant-negative fragment of myosin Va led to the accumulation of punctate structures containing the NMJ marker protein, rapsyn-GFP, in perinuclear clusters. In mouse hindlimb muscle, endogenous myosin Va co-precipitated with surface-exposed or internalised acetylcholine receptors and was markedly enriched in close proximity to the NMJ upon immunofluorescence. In vivo microscopy of exogenous full length myosin Va as well as a cargo-binding fragment of myosin Va showed localisation to the NMJ in wildtype mouse muscles. Furthermore, local interference with myosin Va function in live wildtype mouse muscles led to fragmentation and size reduction of NMJs, exclusion of rapsyn-GFP from NMJs, reduced persistence of acetylcholine receptors in NMJs and an increased amount of punctate structures bearing internalised NMJ proteins.Conclusions/SignificanceIn summary, our data show a crucial role of myosin Va for the plasticity of live vertebrate neuromuscular junctions and suggest its involvement in the recycling of internalised acetylcholine receptors back to the postsynaptic membrane.

Many studies worldwide have shown that students and pre-service teachers have misconceptions about cell biology concepts. In most cases, teachers are the source of their students’ misconceptions. In this regard, we conducted this study to investigate if Moroccan pre-service teachers of life and earth sciences have a good understanding of cell biology concepts, and to find which concepts they misconceive. We developed and validated a three-tier diagnostic test with 20 items concerning cell biology concepts. The test was based on knowledge related to cell biology included in the Moroccan secondary school life and earth sciences’ curriculum, which the pre-service teachers will teach. The use of a three-tier test allowed us to differentiate incorrect answers due to misconceptions from those related to a lack of knowledge and correct answers related to strong knowledge, from lucky guess. This study shows that pre-service teachers share several cell biology misconceptions, and that they misconceive the following concepts: cell, gene, hormone, nerve impulse and immune response. Misconceptions detected may impede pre-service teachers’ learning and may be transmitted to their students, which requires development of a training strategy to overcome these obstacles.