Unraveling the CLCC1 interactome: Impact of the Asp25Glu variant and its interaction with SigmaR1 at the Mitochondrial-Associated ER Membrane (MAM).

The endoplasmic reticulum (ER) plays an indispensable role in cellular processes, including maintenance of calcium homeostasis, and protein folding, synthesized and processing. Disruptions in these processes leading to ER stress and the accumulation of misfolded proteins can instigate the unfolded protein response (UPR), culminating in either restoration of balanced proteostasis or apoptosis. A key player in this intricate balance is CLCC1, an ER-resident chloride channel, whose essential role extends to retinal development, regulation of ER stress, and UPR. The importance of CLCC1 is further underscored by its interaction with proteins localized to mitochondria-associated endoplasmic reticulum membranes (MAMs), where it participates in UPR induction by MAM proteins. In previous research, we identified a p.(Asp25Glu) pathogenic CLCC1 variant associated with retinitis pigmentosa (RP) (CLCC1 hg38 NC_000001.11; NM_001048210.3, c.75C > A; UniprotKB Q96S66). In attempt to decipher the impact of this variant function, we leveraged liquid chromatography-mass spectrometry (LC-MS) to identify likely CLCC1-interacting proteins. We discovered that the CLCC1 interactome is substantially composed of proteins that localize to ER compartments and that the Asp25Glu variant results in noticeable loss and gain of specific protein interactors. Intriguingly, the analysis suggests that the CLCC1Asp25Glu mutant protein exhibits a propensity for increased interactions with cytoplasmic proteins compared to its wild-type counterpart. To corroborate our LC-MS data, we further scrutinized two novel CLCC1 interactors, Calnexin and SigmaR1, chaperone proteins that localize to the ER and MAMs. Through microscopy, we demonstrate that CLCC1 co-localizes with both proteins, thereby validating our initial findings. Moreover, our results reveal that CLCC1 co-localizes with SigmaR1 not merely at the ER, but also at MAMs. These findings reinforce the notion of CLCC1 interacting with MAM proteins at the ER-mitochondria interface, setting the stage for further exploration into how these interactions impact ER or mitochondria function and lead to retinal degenerative disease when impaired.

A blended future? A cross-sectional study demonstrating the impacts of the COVID-19 pandemic on student experiences of well-being, teaching and learning.

INTRODUCTION: The COVID-19 pandemic necessitated emergency changes to teaching, learning and assessment across higher education. Healthcare courses were particularly affected because of their interdependence with overstretched health services. We used this unprecedented situation to provide insight into how students react to unexpected crises and how institutions can most effectively support them. MATERIALS AND METHODS: This cohort study explored students' experiences of the pandemic across programmes and stages from five schools (medicine, dentistry, biomedical sciences, psychology and health professions) in a health faculty in a UK university. We carried out an inductive thematic analysis on the data collected. RESULTS: Many students reported fluctuating emotions and struggled to adapt to home working. Students' changes in motivation and coping strategies varied, many found structure, recreation and social interaction important. Opinions on how well online learning worked relative to face-to-face diverged across programmes. CONCLUSION: A one-size-fits-all blended learning response is unlikely to be appropriate. Our study shows students across one faculty, within one institution, responded diversely to an emergency affecting them all. Educators need to be flexible and dynamic in delivering curricula and supporting students responding to an unexpected crisis during their higher education.

The metabolic response to inflammation in astrocytes is regulated by nuclear factor-kappa B signaling.

Inflammation and metabolism are intrinsically linked with inflammatory stimuli inducing metabolic changes in cells and, in turn, metabolic capacity determining cellular inflammatory responses. Although well characterized in peripheral immune cells there is comparatively less known about these "immunometabolic" responses in astrocytes. In this study, we tested the hypothesis that the astrocytic inflammatory response driven by nuclear factor-kappa B (NF-κB) signaling is dependent on glycolytic metabolism. Using mouse primary cortical astrocyte cultures, we assessed changes in cellular metabolism after exposure to lipopolysaccharide (LPS), with cytokine ELISAs and immunoblotting being used to measure inflammatory responses. Results indicate temporally distinct metabolic adaptations to pro-inflammatory stimulation in astrocytes: 3 hr LPS treatment increased glycolysis but did not alter mitochondrial metabolism, while following 24 hr of LPS treatment we observed increased oxidative phosphorylation, and decreased glycolytic capacity and glucose uptake, partly due to reduced glucose transporter 1 expression. Inhibition of NF-κB signaling with the IKK-beta inhibitor TPCA-1 prevented the LPS induced changes to glycolysis and oxidative phosphorylation. Furthermore, TPCA-1 treatment altered both glycolysis and oxidative phosphorylation independently from inflammatory stimulation, indicating a role for NF-κB signaling in regulation of basal metabolism in astrocytes. Inhibition of glycolysis with 2-deoxyglucose significantly attenuated LPS-induced cytokine release and NF-κB phosphorylation, indicating that intact glycolysis is required for the full inflammatory response to LPS. Together our data indicate that astrocytes display immunometabolic responses to acute LPS stimulation which may represent a potential therapeutic target for neuroinflammatory disorders.

STIM1 Is Required for Remodeling of the Endoplasmic Reticulum and Microtubule Cytoskeleton in Steering Growth Cones.

The spatial and temporal regulation of calcium signaling in neuronal growth cones is essential for axon guidance. In growth cones, the endoplasmic reticulum (ER) is a significant source of calcium signals. However, it is not clear whether the ER is remodeled during motile events to localize calcium signals in steering growth cones. The expression of the ER-calcium sensor, stromal interacting molecule 1 (STIM1) is necessary for growth cone steering toward the calcium-dependent guidance cue BDNF, with STIM1 functioning to sustain calcium signals through store-operated calcium entry. However, STIM1 is also required for growth cone steering away from semaphorin-3a, a guidance cue that does not activate ER-calcium release, suggesting multiple functions of STIM1 within growth cones (Mitchell et al., 2012). STIM1 also interacts with microtubule plus-end binding proteins EB1/EB3 (Grigoriev et al., 2008). Here, we show that STIM1 associates with EB1/EB3 in growth cones and that STIM1 expression is critical for microtubule recruitment and subsequent ER remodeling to the motile side of steering growth cones. Furthermore, we extend our data in vivo, demonstrating that zSTIM1 is required for axon guidance in actively navigating zebrafish motor neurons, regulating calcium signaling and filopodial formation. These data demonstrate that, in response to multiple guidance cues, STIM1 couples microtubule organization and ER-derived calcium signals, thereby providing a mechanism where STIM1-mediated ER remodeling, particularly in filopodia, regulates spatiotemporal calcium signals during axon guidance.SIGNIFICANCE STATEMENT Defects in both axon guidance and endoplasmic reticulum (ER) function are implicated in a range of developmental disorders. During neuronal circuit development, the spatial localization of calcium signals controls the growth cone cytoskeleton to direct motility. We demonstrate a novel role for stromal interacting molecule 1 (STIM1) in regulating microtubule and subsequent ER remodeling in navigating growth cones. We show that STIM1, an activator of store-operated calcium entry, regulates the dynamics of microtubule-binding proteins EB1/EB3, coupling ER to microtubules, within filopodia, thereby steering growth cones. The STIM1-microtubule-ER interaction provides a new model for spatial localization of calcium signals in navigating growth cones in the nascent nervous system.

Copy number variation of LINGO1 in familial dystonic tremor.

OBJECTIVE: To elucidate the genetic cause of a large 5 generation South Indian family with multiple individuals with predominantly an upper limb postural tremor and posturing in keeping with another form of tremor, namely, dystonic tremor. METHODS: Whole-genome single nucleotide polymorphism (SNP) microarray analysis was undertaken to look for copy number variants in the affected individuals. RESULTS: Whole-genome SNP microarray studies identified a tandem duplicated genomic segment of chromosome 15q24 present in all affected family members. Whole-genome sequencing demonstrated that it comprised a ∼550-kb tandem duplication encompassing the entire LINGO1 gene. CONCLUSIONS: The identification of a genomic duplication as the likely molecular cause of this condition, resulting in an additional LINGO1 gene copy in affected cases, adds further support for a causal role of this gene in tremor disorders and implicates increased expression levels of LINGO1 as a potential pathogenic mechanism.

Basal fatty acid oxidation increases after recurrent low glucose in human primary astrocytes.

AIMS/HYPOTHESIS: Hypoglycaemia is a major barrier to good glucose control in type 1 diabetes. Frequent hypoglycaemic episodes impair awareness of subsequent hypoglycaemic bouts. Neural changes underpinning awareness of hypoglycaemia are poorly defined and molecular mechanisms by which glial cells contribute to hypoglycaemia sensing and glucose counterregulation require further investigation. The aim of the current study was to examine whether, and by what mechanism, human primary astrocyte (HPA) function was altered by acute and recurrent low glucose (RLG). METHODS: To test whether glia, specifically astrocytes, could detect changes in glucose, we utilised HPA and U373 astrocytoma cells and exposed them to RLG in vitro. This allowed measurement, with high specificity and sensitivity, of RLG-associated changes in cellular metabolism. We examined changes in protein phosphorylation/expression using western blotting. Metabolic function was assessed using a Seahorse extracellular flux analyser. Immunofluorescent imaging was used to examine cell morphology and enzymatic assays were used to measure lactate release, glycogen content, intracellular ATP and nucleotide ratios. RESULTS: AMP-activated protein kinase (AMPK) was activated over a pathophysiologically relevant glucose concentration range. RLG produced an increased dependency on fatty acid oxidation for basal mitochondrial metabolism and exhibited hallmarks of mitochondrial stress, including increased proton leak and reduced coupling efficiency. Relative to glucose availability, lactate release increased during low glucose but this was not modified by RLG. Basal glucose uptake was not modified by RLG and glycogen levels were similar in control and RLG-treated cells. Mitochondrial adaptations to RLG were partially recovered by maintaining euglycaemic levels of glucose following RLG exposure. CONCLUSIONS/INTERPRETATION: Taken together, these data indicate that HPA mitochondria are altered following RLG, with a metabolic switch towards increased fatty acid oxidation, suggesting glial adaptations to RLG involve altered mitochondrial metabolism that could contribute to defective glucose counterregulation to hypoglycaemia in diabetes.

Truncating SLC5A7 mutations underlie a spectrum of dominant hereditary motor neuropathies.

OBJECTIVE: To identify the genetic cause of disease in 2 previously unreported families with forms of distal hereditary motor neuropathies (dHMNs). METHODS: The first family comprises individuals affected by dHMN type V, which lacks the cardinal clinical feature of vocal cord paralysis characteristic of dHMN-VII observed in the second family. Next-generation sequencing was performed on the proband of each family. Variants were annotated and filtered, initially focusing on genes associated with neuropathy. Candidate variants were further investigated and confirmed by dideoxy sequence analysis and cosegregation studies. Thorough patient phenotyping was completed, comprising clinical history, examination, and neurologic investigation. RESULTS: dHMNs are a heterogeneous group of peripheral motor neuron disorders characterized by length-dependent neuropathy and progressive distal limb muscle weakness and wasting. We previously reported a dominant-negative frameshift mutation located in the concluding exon of the SLC5A7 gene encoding the choline transporter (CHT), leading to protein truncation, as the likely cause of dominantly-inherited dHMN-VII in an extended UK family. In this study, our genetic studies identified distinct heterozygous frameshift mutations located in the last coding exon of SLC5A7, predicted to result in the truncation of the CHT C-terminus, as the likely cause of the condition in each family. CONCLUSIONS: This study corroborates C-terminal CHT truncation as a cause of autosomal dominant dHMN, confirming upper limb predominating over lower limb involvement, and broadening the clinical spectrum arising from CHT malfunction.

How does calcium interact with the cytoskeleton to regulate growth cone motility during axon pathfinding?

The precision with which neurons form connections is crucial for the normal development and function of the nervous system. The development of neuronal circuitry in the nervous system is accomplished by axon pathfinding: a process where growth cones guide axons through the embryonic environment to connect with their appropriate synaptic partners to form functional circuits. Despite intense efforts over many years to understand how this process is regulated, the complete repertoire of molecular mechanisms that govern the growth cone cytoskeleton and hence motility, remain unresolved. A central tenet in the axon guidance field is that calcium signals regulate growth cone behaviours such as extension, turning and pausing by regulating rearrangements of the growth cone cytoskeleton. Here, we provide evidence that not only the amplitude of a calcium signal is critical for growth cone motility but also the source of calcium mobilisation. We provide an example of this idea by demonstrating that manipulation of calcium signalling via L-type voltage gated calcium channels can perturb sensory neuron motility towards a source of netrin-1. Understanding how calcium signals can be transduced to initiate cytoskeletal changes represents a significant gap in our current knowledge of the mechanisms that govern axon guidance, and consequently the formation of functional neural circuits in the developing nervous system.

Axons get ahead: Insights into axon guidance and congenital cranial dysinnervation disorders.

Cranial nerves innervate head muscles in a well-characterized and highly conserved pattern. Identification of genes responsible for human congenital disorders of these nerves, combined with the analysis of their role in axonal development in animal models, has advanced understanding of how neuromuscular connectivity is established. Here, we focus on the ocular motor system, as an instructive example of the success of this approach in unravelling the aetiology of human strabismus. The discovery that ocular motility disorders can arise from mutations in transcription factors, including HoxA1, HoxB1, MafB, Phox2A, and Sall4, has revealed gene regulatory networks that pattern the brainstem and/or govern the differentiation of cranial motor neurons. Mutations in genes involved in axon growth and guidance disrupt specific stages of the extension and pathfinding of ocular motor nerves, and have been implicated in human strabismus. These genes encompass varied classes of molecule, from receptor complexes to dynamic effectors to cytoskeletal components, including Robo3/Rig1, Alpha2-chimaerin, Kif21A, TUBB2, and TUBB3. A current challenge is to understand the protein regulatory networks that link the cell surface to the cytoskeleton and to dissect the co-ordinated signalling cascades and motile responses that underpin axonal navigation. Here we review recent insights derived from basic and clinical science approaches, to show how, by capitalising on the strengths of each, a more complete picture of the aetiology of human congenital cranial dysinnervation disorders can be achieved. This elucidation of these principles illustrates the success of clinical genetic studies working in tandem with molecular and cellular models to enhance our understanding of human disease. © 2016 Wiley Periodicals, Inc. Develop Neurobiol 77: 861-875, 2017.

Axon guidance in the developing ocular motor system and Duane retraction syndrome depends on Semaphorin signaling via alpha2-chimaerin.

Eye movements depend on correct patterns of connectivity between cranial motor axons and the extraocular muscles. Despite the clinical importance of the ocular motor system, little is known of the molecular mechanisms underlying its development. We have recently shown that mutations in the Chimaerin-1 gene encoding the signaling protein α2-chimaerin (α2-chn) perturb axon guidance in the ocular motor system and lead to the human eye movement disorder, Duane retraction syndrome (DRS). The axon guidance cues that lie upstream of α2-chn are unknown; here we identify candidates to be the Semaphorins (Sema) 3A and 3C, acting via the PlexinA receptors. Sema3A/C are expressed in and around the developing extraocular muscles and cause growth cone collapse of oculomotor neurons in vitro. Furthermore, RNAi knockdown of α2-chn or PlexinAs in oculomotor neurons abrogates Sema3A/C-dependent growth cone collapse. In vivo knockdown of endogenous PlexinAs or α2-chn function results in stereotypical oculomotor axon guidance defects, which are reminiscent of DRS, whereas expression of α2-chn gain-of-function constructs can rescue PlexinA loss of function. These data suggest that α2-chn mediates Sema3-PlexinA repellent signaling. We further show that α2-chn is required for oculomotor neurons to respond to CXCL12 and hepatocyte growth factor (HGF), which are growth promoting and chemoattractant during oculomotor axon guidance. α2-chn is therefore a potential integrator of different types of guidance information to orchestrate ocular motor pathfinding. DRS phenotypes can result from incorrect regulation of this signaling pathway.

Drebrin controls neuronal migration through the formation and alignment of the leading process.

Formation of a functional nervous system requires neurons to migrate to the correct place within the developing brain. Tangentially migrating neurons are guided by a leading process which extends towards the target and is followed by the cell body. How environmental cues are coupled to specific cytoskeletal changes to produce and guide leading process growth is unknown. One such cytoskeletal modulator is drebrin, an actin-binding protein known to induce protrusions in many cell types and be important for regulating neuronal morphology. Using the migration of oculomotor neurons as a model, we have shown that drebrin is necessary for the generation and guidance of the leading process. In the absence of drebrin, leading processes are not formed and cells fail to migrate although axon growth and pathfinding appear grossly unaffected. Conversely, when levels of drebrin are elevated the leading processes turn away from their target and as a result the motor neuron cell bodies move along abnormal paths within the brain. The aberrant trajectories were highly reproducible suggesting that drebrin is required to interpret specific guidance cues. The axons and growth cones of these neurons display morphological changes, particularly increased branching and filopodial number but despite this they extend along normal developmental pathways. Collectively these results show that drebrin is initially necessary for the formation of a leading process and subsequently for this to respond to navigational signals and grow in the correct direction. Furthermore, we have shown that the actions of drebrin can be segregated within individual motor neurons to direct their migration independently of axon guidance.

Control of cell shape and plasticity during development and disease by the actin-binding protein Drebrin.

Drebrin is an actin-binding protein, originally identified in neuronal cells, involved in the regulation of actin filament organisation, especially during the formation of neurites and cell protrusions of motile cells. Drebrin is found in diverse non-neuronal cells, primarily in association with cell processes and intercellular junctions where it again plays a key role in actin remodelling. The downregulation of Drebrin in Alzheimer's Disease and Down Syndrome and conversely its upregulation in various carcinomas indicate that Drebrin is an important component of the pathogenesis of multiple diseases.

The specific targeting of guidance receptors within neurons: who directs the directors?

Guidance molecules present in both axonal and dendritic growth cones mediate neuronal responses to extracellular cues thereby ensuring correct neurite pathfinding and development of the nervous system. Little is known though about the mechanisms employed by neurons to deliver these receptors, specifically and efficiently, to the extending growth cone. A deeper understanding of this process is crucial if guidance receptors are to be manipulated to promote nervous system repair. Studies in other polarised cells, notably epithelial, have elucidated fundamental routes to the intracellular segregation of molecules mediated by endosomal pathways. Due to their extreme complexity and specialisation, neurons appear to have built upon these generic systems to evolve sophisticated trafficking networks. A striking feature is the axon initial segment which acts like a valve to tightly regulate the flux of molecules both entering and leaving the axon. Once in the growth cone, further controls operate to enhance the retention or rejection, as appropriate, of membrane receptors. We discuss the current state of knowledge regarding the intracellular trafficking of axon guidance receptors and how this relates to their developmental roles. We highlight the various facets still to be properly elucidated and by building on existing data regarding neuronal polarity and intracellular sorting mechanisms suggest ways to fill these gaps.

Targeting of the F-actin-binding protein drebrin by the microtubule plus-tip protein EB3 is required for neuritogenesis.

Interactions between dynamic microtubules and actin filaments (F-actin) underlie a range of cellular processes including cell polarity and motility. In growth cones, dynamic microtubules are continually extending into selected filopodia, aligning alongside the proximal ends of the F-actin bundles. This interaction is essential for neuritogenesis and growth-cone pathfinding. However, the molecular components mediating the interaction between microtubules and filopodial F-actin have yet to be determined. Here we show that drebrin, an F-actin-associated protein, binds directly to the microtubule-binding protein EB3. In growth cones, this interaction occurs specifically when drebrin is located on F-actin in the proximal region of filopodia and when EB3 is located at the tips of microtubules invading filopodia. When this interaction is disrupted, the formation of growth cones and the extension of neurites are impaired. We conclude that drebrin targets EB3 to coordinate F-actin-microtubule interactions that underlie neuritogenesis.

Tubulin tyrosination is a major factor affecting the recruitment of CAP-Gly proteins at microtubule plus ends.

Tubulin-tyrosine ligase (TTL), the enzyme that catalyzes the addition of a C-terminal tyrosine residue to alpha-tubulin in the tubulin tyrosination cycle, is involved in tumor progression and has a vital role in neuronal organization. We show that in mammalian fibroblasts, cytoplasmic linker protein (CLIP) 170 and other microtubule plus-end tracking proteins comprising a cytoskeleton-associated protein glycine-rich (CAP-Gly) microtubule binding domain such as CLIP-115 and p150 Glued, localize to the ends of tyrosinated microtubules but not to the ends of detyrosinated microtubules. In vitro, the head domains of CLIP-170 and of p150 Glued bind more efficiently to tyrosinated microtubules than to detyrosinated polymers. In TTL-null fibroblasts, tubulin detyrosination and CAP-Gly protein mislocalization correlate with defects in both spindle positioning during mitosis and cell morphology during interphase. These results indicate that tubulin tyrosination regulates microtubule interactions with CAP-Gly microtubule plus-end tracking proteins and provide explanations for the involvement of TTL in tumor progression and in neuronal organization.

Molecular mechanisms of axon guidance.

In order to form a functional nervous system, neurones extend axons, often over long distances, to reach their targets. This process is controlled by extracellular receptors and their ligands, several families of which have been identified. These proteins may act to either repel or attract growth cones and a given receptor may transduce either type of signal, depending on the cellular context. In addition to these archetypal axon guidance molecules, it is becoming apparent that molecules previously known for their role in patterning can also direct axonal outgrowth. The growth cone receptors do not act in isolation and combine with members of the same or other families to produce a graded response or even a complete reversal in its polarity. These signals can be further combined and/or modulated by processing of the molecule both directly at the cell surface and by the network of intracellular signalling pathways which are activated. The result is a sophisticated and dynamic set of cues that enable a growth cone to successfully navigate to its destination, modulating its response to changing environmental cues along its pathway.

Semaphorin/neuropilin signaling influences the positioning of migratory neural crest cells within the hindbrain region of the chick.

Within the hindbrain region, neural crest cell migration is organized into three streams that follow the segmentation of the neuroepithelium into distinct rhombomeric compartments. Although the streaming of neural crest cells is known to involve signals derived from the neuroepithelium, the molecular properties underlying this process are poorly understood. Here, we have mapped the expression of the signaling component of two secreted class III Semaphorins, Semaphorin (Sema) 3A and Sema 3F, at time points that correspond to neural crest cell migration within the hindbrain region of the chick. Both Semaphorins are expressed within rhombomeres at levels adjacent to crest-free mesenchyme and expression of the receptor components essential for Semaphorin activity by neural crest cells suggests a function in restricting neural crest cell migration. By using bead implantation and electroporation in ovo, we define a role for both Semaphorins in the maintenance of neural crest cell streams in proximity to the neural tube. Attenuation of Semaphorin signaling by expression of soluble Neuropilin-Fc resulted in neural crest cells invading adjacent mesenchymal territories that are normally crest-free. The loss or misguidance of specific neural crest cell populations after changes in Semaphorin signaling also affects the integration of the cranial sensory ganglia. Thus, Sema 3A and 3F, expressed and secreted by the hindbrain neuroepithelium contributes to the appropriate positioning of neural crest cells in proximity to the neural tube, a process crucial for the subsequent establishment of neuronal connectivity within the hindbrain region.

Cranial expression of class 3 secreted semaphorins and their neuropilin receptors.

The semaphorin family of chemorepellents and their receptors the neuropilins are implicated in a variety of cellular processes, including axon guidance and cell migration. Semaphorins may bind more than one neuropilin or a heterodimer of both, thus a detailed knowledge of their expression patterns may reveal possible cases of redundancy or mutual antagonism. To assess their involvement in cranial development, we cloned fragments of the chick orthologues of Sema3B and Sema3F. We then carried out mRNA in situ hybridisation of all six class 3 semaphorins and both neuropilins in the embryonic chick head. We present evidence for spatiotemporal regulation of these molecules in the brainstem and developing head, including the eye, ear, and branchial arches. These expression patterns provide a basis for functional analysis of semaphorins and neuropilins in the development of axon projections and the morphogenesis of cranial structures.

Isoform-specific binding of the tyrosine phosphatase PTPsigma to a ligand in developing muscle.

PTPsigma is a receptor tyrosine phosphatase that is expressed widely in the developing nervous system and that controls the growth and retinotopic mapping of retinal axons. PTPsigma is also expressed in motor neurons where its function is unclear. Given that invertebrate relatives of PTPsigma can control motor axon guidance, target contact, and synaptogenesis, we have asked if extracellular ligands exist for cPTPsigma, the avian PTPsigma orthologue, in the neuromuscular system. Of the two major isoforms cPTPsigma1 and cPTPsigma2, only the shorter cPTPsigma1 isoform is expressed in developing spinal motor neurons and their axons. We show that ectodomains of cPTPsigma1, but not of cPTPsigma2, bind specifically to developing skeletal myotubes. The putative myotube ligand is not related to the previously described binding of cPTPsigma to heparan sulfates within the proteoglycans agrin and collagen XVIII, since heparinase treatment of myotubes does not alter cPTPsigma1 binding and since most mutations that abolish binding of cPTPsigma1 to heparin do not affect myotube binding. The expression of cPTPsigma1 in motor axons and its direct binding to target myotubes suggest an isoform-specific role for axonally expressed cPTPsigma1 during establishment or maintenance of neuromuscular contacts.