FGF negatively regulates muscle membrane extension in Caenorhabditis elegans

Striated muscles from Drosophila and several vertebrates extend plasma membrane to facilitate the formation of the neuromuscular junction (NMJ) during development. However, the regulation of these membrane extensions is poorly understood. In C. elegans, the body wall muscles (BWMs) also have plasma membrane extensions called muscle arms that are guided to the motor axons where they form the postsynaptic element of the NMJ. To investigate the regulation of muscle membrane extension, we screened 871 genes by RNAi for ectopic muscle membrane extensions (EMEs) in C. elegans. We discovered that an FGF pathway, including let-756(FGF), egl-15(FGF receptor), sem-5(GRB2) and other genes negatively regulates plasma membrane extension from muscles. Although compromised FGF pathway activity results in EMEs, hyperactivity of the pathway disrupts larval muscle arm extension, a phenotype we call muscle arm extension defective or MAD. We show that expression of egl-15 and sem-5 in the BWMs are each necessary and sufficient to prevent EMEs. Furthermore, we demonstrate that let-756 expression from any one of several tissues can rescue the EMEs of let-756 mutants, suggesting that LET-756 does not guide muscle membrane extensions. Our screen also revealed that loss-of-function in laminin and integrin components results in both MADs and EMEs, the latter of which are suppressed by hyperactive FGF signaling. Our data are consistent with a model in which integrins and laminins are needed for directed muscle arm extension to the nerve cords, while FGF signaling provides a general mechanism to regulate muscle membrane extension.     

MADD-4 is a secreted cue required for midline-oriented guidance in Caenorhabditis elegans

The netrins and slits are two families of widely conserved cues that guide axons and cells along the dorsal-ventral (D-V) axis of animals. These cues typically emanate from the dorsal or ventral midlines and provide spatial information to migrating cells by?forming gradients along the D-V axis. Some cell types, however, extend processes to both the dorsal and ventral midlines, suggesting the existence of additional guidance cues that are secreted from both midlines. Here, we report that a previously uncharacterized protein called MADD-4 is secreted by the dorsal and ventral nerve cords of the nematode C.?elegans to attract sensory axons and muscle?membrane extensions called muscle arms. MADD-4's activity is dependent on UNC-40/DCC, a netrin receptor, which functions cell-autonomously to direct membrane extension. The biological role of MADD-4 orthologs, including ADAMTSL1 and 3 in mammals, is unknown. MADD-4 may therefore represent the founding member of a family of guidance proteins.     

The MADD-3 LAMMER Kinase Interacts with a p38 MAP Kinase Pathway to Regulate the Display of the EVA-1 Guidance Receptor in Caenorhabditis elegans

The proper display of transmembrane receptors on the leading edge of migrating cells and cell extensions is essential for their response to guidance cues. We previously discovered that MADD-4, which is an ADAMTSL secreted by motor neurons in Caenorhabditis elegans, interacts with an UNC-40/EVA-1 co-receptor complex on muscles to attract plasma membrane extensions called muscle arms. In nematodes, the muscle arm termini harbor the post-synaptic elements of the neuromuscular junction. Through a forward genetic screen for mutants with disrupted muscle arm extension, we discovered that a LAMMER kinase, which we call MADD-3, is required for the proper display of the EVA-1 receptor on the muscle’s plasma membrane. Without MADD-3, EVA-1 levels decrease concomitantly with a reduction of the late-endosomal marker RAB-7. Through a genetic suppressor screen, we found that the levels of EVA-1 and RAB-7 can be restored in madd-3 mutants by eliminating the function of a p38 MAP kinase pathway. We also found that EVA-1 and RAB-7 will accumulate in madd-3 mutants upon disrupting CUP-5, which is a mucolipin ortholog required for proper lysosome function. Together, our data suggests that the MADD-3 LAMMER kinase antagonizes the p38-mediated endosomal trafficking of EVA-1 to the lysosome. In this way, MADD-3 ensures that sufficient levels of EVA-1 are present to guide muscle arm extension towards the source of the MADD-4 guidance cue.     

UNC-119 suppresses axon branching in C. elegans

The architecture of the differentiated nervous system is stable but the molecular mechanisms that are required for stabilization are unknown. We characterized the gene unc-119 in the nematode Caenorhabditis elegans and demonstrate that it is required to stabilize the differentiated structure of the nervous system. In unc-119 mutants, motor neuron commissures are excessively branched in adults. However, live imaging demonstrated that growth cone behavior during extension was fairly normal with the exception that the overall rate of migration was reduced. Later, after development was complete, secondary growth cones sprouted from existing motor neuron axons and cell bodies. These new growth cones extended supernumerary branches to the dorsal nerve cord at the same time the previously formed axons retracted. These defects could be suppressed by expressing the UNC-119 protein after embryonic development; thus demonstrating that UNC-119 is required for the maintenance of the nervous system architecture. Finally, UNC-119 is located in neuron cell bodies and axons and acts cell-autonomously to inhibit axon branching.     

The giant neurone system in ophiuroids

Summary The radial nerve cords of members of the class Ophiuroidea consist of two parts, the ectoneural and the hyponeural tissues, which are separated by an acellular basal lamina. The hyponeural tissue is composed entirely of motor fibres. The cell bodies of the hyponeural neurones are arranged in ganglia, one to each segment of the arm, and each containing approximately one hundred cell bodies. Synaptic contact between the two tissues occurs across the basal lamina. Ultrastructural evidence shows that the majority of these synapses operate in the ectoneural to hyponeural direction. Three pairs of nerve bundles, each containing approximately thirty five large motor fibres arise from each ganglion and innervate the intervertebral muscles. The large motor fibres divide into a number of pre-terminal axons in the region in which the motor fibre enters the muscle block. The terminal axons run at right-angles across the muscle fibres and neuromuscular junctions are found at the points of contact between the two; each terminal axon makes contact with a large number of muscle fibres. The hyponeural axons also pass through the juxtaligamental tissue before they reach the muscle blocks and there is some evidence of synaptic contact with the juxtaligamental cells. The juxtaligamental tissue is thought to be associated with changes in the structural properties of the collagenous ligaments of the arm during arm autotomy (Wilkie 1979). Degeneration studies confirmed the layout of the hyponeural motor axons.     

Dual roles of tropomyosin as an F-actin stabilizer and a regulator of muscle contraction in Caenorhabditis elegans body wall muscle

Tropomyosin is a well-characterized regulator of muscle contraction. It also stabilizes actin filaments in a variety of muscle and non-muscle cells. Although these two functions of tropomyosin could have different impacts on actin cytoskeletal organization, their functional relationship has not been studied in the same experimental system. Here, we investigated how tropomyosin stabilizes actin filaments and how this function is influenced by muscle contraction in Caenorhabditis elegans body wall muscle. We confirmed the antagonistic role of tropomyosin against UNC-60B, a muscle-specific ADF/cofilin isoform, in actin filament organization using multiple UNC-60B mutant alleles. Tropomyosin was also antagonistic to UNC-78 (AIP1) in vivo and protected actin filaments from disassembly by UNC-60B and UNC-78 in vitro, suggesting that tropomyosin protects actin filaments from the ADF/cofilin-AIP1 actin disassembly system in muscle cells. A mutation in the myosin heavy chain caused greater reduction in contractility than tropomyosin depletion. However, the myosin mutation showed much weaker suppression of the phenotypes of ADF/cofilin or AIP1 mutants than tropomyosin depletion. These results suggest that muscle contraction has only minor influence on the tropomyosin's protective role against ADF/cofilin and AIP1, and that the two functions of tropomyosin in actin stability and muscle contraction are independent of each other.     

UNC-87 isoforms,Caenorhabditis elegans calponin-related proteins,interact with both actin and myosin and regulate actomyosin contractility

Calponin-related proteins are widely distributed among eukaryotes and involved in signaling and cytoskeletal regulation. Calponin-like (CLIK) repeat is an actin-binding motif found in the C-termini of vertebrate calponins. Although CLIK repeats stabilize actin filaments, other functions of these actin-binding motifs are unknown. The Caenorhabditis elegans unc-87 gene encodes actin-binding proteins with seven CLIK repeats. UNC-87 stabilizes actin filaments and is essential for maintenance of sarcomeric actin filaments in striated muscle. Here we show that two UNC-87 isoforms, UNC-87A and UNC-87B, are expressed in muscle and nonmuscle cells in a tissue-specific manner by two independent promoters and exhibit quantitatively different effects on both actin and myosin. Both UNC-87A and UNC-87B have seven CLIK repeats, but UNC-87A has an extra N-terminal extension of ∼190 amino acids. Both UNC-87 isoforms bind to actin filaments and myosin to induce ATP-resistant actomyosin bundles and inhibit actomyosin motility. UNC-87A with an N-terminal extension binds to actin and myosin more strongly than UNC-87B. UNC-87B is associated with actin filaments in nonstriated muscle in the somatic gonad, and an unc-87 mutation causes its excessive contraction, which is dependent on myosin. These results strongly suggest that proteins with CLIK repeats function as a negative regulator of actomyosin contractility.     

Patterns of motor activity in the isolated nerve cord of the octopus arm

The extremely flexible octopus arm provides a unique opportunity for studying movement control in a highly redundant motor system. We describe a novel preparation that allows analysis of the peripheral nervous system of the octopus arm and its interaction with the muscular and mechanosensory elements of the arm's intrinsic muscular system. First we examined the synaptic responses in muscle fibers to identify the motor pathways from the axial nerve cord of the arm to the surrounding musculature. We show that the motor axons project to the muscles via nerve roots originating laterally from the arm nerve cord. The motor field of each nerve is limited to the region where the nerve enters the arm musculature. The same roots also carry afferent mechanosensory information from the intrinsic muscle to the axial nerve cord. Next, we characterized the pattern of activity generated in the dorsal roots by electrically stimulating the axial nerve cord. The evoked activity, although far reaching and long lasting, cannot alone account for the arm extension movements generated by similar electrical stimulation. The mismatch between patterns of activity in the isolated cord and in an intact arm may stem from the involvement of mechanosensory feedback in natural arm extension.     

Caenorhabditis elegans UNC-45 is a component of muscle thick filaments and colocalizes with myosin heavy chain B, but not myosin heavy chain A

In the nematode Caenorhabditis elegans, animals mutant in the gene encoding the protein product of the unc-45 gene (UNC-45) have disorganized muscle thick filaments in body wall muscles. Although UNC-45 contains tetratricopeptide repeats (TPR) as well as limited similarity to fungal proteins, no biochemical role has yet been found. UNC-45 reporters are expressed exclusively in muscle cells, and a functional reporter fusion is localized in the body wall muscles in a pattern identical to thick filament A-bands. UNC-45 colocalizes with myosin heavy chain (MHC) B in wild-type worms as well as in temperature-sensitive (ts) unc-45 mutants, but not in a mutant in which MHC B is absent. Surprisingly, UNC-45 localization is also not seen in MHC B mutants, in which the level of MHC A is increased, resulting in near-normal muscle thick filament structure. Thus, filament assembly can be independent of UNC-45. UNC-45 shows a localization pattern identical to and dependent on MHC B and a function that appears to be MHC B-dependent. We propose that UNC-45 is a peripheral component of muscle thick filaments due to its localization with MHC B. The role of UNC-45 in thick filament assembly seems restricted to a cofactor for assembly or stabilization of MHC B.     

Interlimb interactions during cyclic in-phase and antiphase movements of arms and legs and their dependence on afferent influences

Coordinated arm and leg movements imply neural interactions between the rhythmic generators of the upper and lower extremities. In ten healthy subjects in the lying position, activity of the muscles of the upper and lower extremities was recorded during separate and joint cyclic movements of the arms and legs with different phase relationships between the movements of the limbs and under various conditions of the motor task. Antiphase active arm movements were characterized by higher muscle activity than during the inphase mode. The muscle activity during passive arm movements imposed by the experimentalist was significantly lower than muscle activity during passive arm movements imposed by the other arm. When loading one arm, the muscle activity in the other, passively moving, arm increased independently from the synergy of arm movements. During a motor task implementing joint antiphase movements of both upper and lower extremities, compared to a motor task implementing their joint in-phase movements, we observed a significant increase in activity in the biceps brahii muscle, the tibialis anterior muscle, and the biceps femoris muscle. Loading of arms in these motor tasks has been accompanied by increased activity in some leg muscles. An increase in the frequency of rhythmic movements resulted in a significant growth of the muscle activity of the arms and legs during their cooperative movements with a greater rate of rise in the flexor muscle activity of the arms and legs during joint antiphase movements. Thus, both the spatial organization of movements and the type of afferent influences are significant factors of interlimb interactions, which, in turn, determine the type of neural interconnections that are involved in movement regulation.     

The role of co-activation in strength and force modulation in the elbow of children with unilateral cerebral palsy

To study the role of coactivation in strength and force modulation in the elbow joint of children and adolescents with cerebral palsy (CP), we investigated the affected and contralateral arm of 21 persons (age 8-18) with spastic unilateral CP in three tasks: maximal voluntary isokinetic concentric contraction and passive isokinetic movement during elbow flexion and extension, and sub-maximal isometric force tracing during elbow flexion. Elbow flexion-extension torque and surface electromyography (EMG) of the biceps brachii (BB) and triceps brachii (TB) muscles were recorded. During the maximal contractions, the affected arm was weaker, had decreased agonist and similar antagonist EMG amplitudes, and thus increased antagonist co-activation (% of maximal activity as agonist) during both elbow flexion and extension, with higher coactivation levels of the TB than the BB. During passive elbow extension, the BB of the affected arm showed increased resistance torque and indication of reflex, and thus spastic, activity. No difference between the two arms was found in the ability to modulate force, despite increased TB coactivation in the affected arm. The results indicate that coactivation plays a minor role in muscle weakness in CP, and does not limit force modulation. Moreover, spasticity seems particularly to increase coactivation in the muscle antagonistic to the spastic one, possibly in order to increase stability.     

Role of netrin UNC-6 in patterning the longitudinal nerves of Caenorhabditis elegans

The nervous system of Caenorhabditis elegans comprises circumferential and longitudinal axon tracts. Netrin UNC-6 is required for the guidance of circumferential axon migrations and is expressed by ventral neuroglia and neurons in temporally and spatially regulated patterns. Migrating axons mediate the UNC-6 signal through the UNC-5 and UNC-40 receptors. It is thought that UNC-6 is secreted and becomes associated with basement membranes and cell surfaces to form gradients that direct circumferentially migrating axons toward or away from the ventral UNC-6 sources. Little is known about the effects of UNC-6 on longitudinally migrating axons. In unc-6, unc-5, and unc-40 null mutants, some longitudinal nerves are dorsally or ventrally misdirected. Furthermore, the organization of axons are disrupted within nerves. We show that cells ectopically expressing UNC-6 can redirect the migrations of some neighboring longitudinal axons, suggesting that the gradients postulated to direct circumferential migration also help specify the dorsoventral positions of these longitudinal nerves. We also manipulated the temporal and spatial expression pattern of UNC-6 by two different means. First, we removed the PVT midline neuron which expresses UNC-6 for a short time during axon outgrowths. Second, we expressed UNC-6 uniformly in the nervous system throughout development. The results suggest that changing UNC-6 expression patterns modify the distribution of the cue by providing new localized sources. This new guidance information is critical for organizing the axons of longitudinal nerves.     

UNC-97/PINCH is involved in the assembly of integrin cell adhesion complexes in Caenorhabditis elegans body wall muscle

UNC-97/PINCH is an evolutionarily conserved protein that contains five LIM domains and is located at cell-extracellular matrix attachment sites known as cell adhesion complexes. To understand the role of UNC-97/PINCH in cell adhesion, we undertook a combined genetic and cell biological approach to identify the steps required to assemble cell adhesion complexes in Caenorhabditis elegans. First, we have generated a complete loss of function mutation in the unc-97 coding region. unc-97 null mutants arrest development during embryogenesis and reveal that the myofilament lattice and its attachment structures, which include PAT-4/ILK (integrin-linked kinase) and integrin fail to assemble into properly organized arrays. Although in the absence of UNC-97/PINCH, PAT-4/ILK and integrin fail to organize normally, they are capable of colocalizing together at the muscle cell membrane. Alternatively, in integrin and pat-4 mutants, UNC-97/PINCH fails to localize to the muscle cell membrane and instead is found diffusely throughout the muscle cell cytoplasm. In agreement with mammalian studies, we show that LIM domain 1 of UNC-97/PINCH is required for its interaction with PAT-4/ILK in yeast two-hybrid assays. Additionally, we find, by LIM domain deletion analysis, that LIM1 is required for the localization of UNC-97/PINCH to cell adhesion complexes. Our results provide evidence that UNC-97/PINCH is required for the development of C. elegans and is required for the formation of integrin based adhesion structures.     

UNC-73/trio RhoGEF-2 activity modulates Caenorhabditis elegans motility through changes in neurotransmitter signaling upstream of the GSA-1/Galphas pathway

Rho-family GTPases play regulatory roles in many fundamental cellular processes. Caenorhabditis elegans UNC-73 RhoGEF isoforms function in axon guidance, cell migration, muscle arm extension, phagocytosis, and neurotransmission by activating either Rac or Rho GTPase subfamilies. Multiple differentially expressed UNC-73 isoforms contain a Rac-specific RhoGEF-1 domain, a Rho-specific RhoGEF-2 domain, or both domains. The UNC-73E RhoGEF-2 isoform is activated by the G-protein subunit Gαq and is required for normal rates of locomotion; however, mechanisms of UNC-73 and Rho pathway regulation of locomotion are not clear. To better define UNC-73 function in the regulation of motility we used cell-specific and inducible promoters to examine the temporal and spatial requirements of UNC-73 RhoGEF-2 isoform function in mutant rescue experiments. We found that UNC-73E acts within peptidergic neurons of mature animals to regulate locomotion rate. Although unc-73 RhoGEF-2 mutants have grossly normal synaptic morphology and weak resistance to the acetylcholinesterase inhibitor aldicarb, they are significantly hypersensitive to the acetylcholine receptor agonist levamisole, indicating alterations in acetylcholine neurotransmitter signaling. Consistent with peptidergic neuron function, unc-73 RhoGEF-2 mutants exhibit a decreased level of neuropeptide release from motor neuron dense core vesicles (DCVs). The unc-73 locomotory phenotype is similar to those of rab-2 and unc-31, genes with distinct roles in the DCV-mediated secretory pathway. We observed that constitutively active Gαs pathway mutations, which compensate for DCV-mediated signaling defects, rescue unc-73 RhoGEF-2 and rab-2 lethargic movement phenotypes. Together, these data suggest UNC-73 RhoGEF-2 isoforms are required for proper neurotransmitter signaling and may function in the DCV-mediated neuromodulatory regulation of locomotion rate.     

MAX-1, a novel PH/MyTH4/FERM domain cytoplasmic protein implicated in netrin-mediated axon repulsion

The netrin UNC-6 repels motor axons by activating the UNC-5 receptor alone or in combination with the UNC-40/DCC receptor. In a genetic screen for C. elegans mutants exhibiting partial defects in motor axon projections, we isolated the max-1 gene (required for motor neuron axon guidance). max-1 loss-of-function mutations cause fully penetrant but variable axon guidance defects. Mutations in unc-5 and unc-6, but not in unc-40, dominantly enhance the mutant phenotypes of max-1, whereas overexpression of unc-5 or unc-6, but not of unc-40, bypasses the requirement for max-1. MAX-1 proteins contain PH, MyTH4, and FERM domains and appear to be localized to neuronal processes. Human MAX-1 and UNC5H2 colocalize in discrete subcellular regions of transfected cells. Our results suggest a possible role for MAX-1 in netrin-induced axon repulsion by modulating the UNC-5 receptor signaling pathway.     

UNC-6 expression by the vulval precursor cells of Caenorhabditis elegans is required for the complex axon guidance of the HSN neurons

Netrin is an evolutionarily conserved axon guidance molecule that has both axonal attraction and repulsion activities. In Caenorhabditis elegans, Netrin/UNC-6 is secreted by ventral cells, attracting some axons ventrally and repelling some axons, which extend dorsally. One axon guided by UNC-6 is that of the HSN neuron. The axon guidance process for HSN neurons is complex, consisting of ventral growth, dorsal growth, branching, second ventral growth, fasciculation with ventral nerve cords, and then anterior growth. The vulval precursor cells (VPC) and the PVP and PVQ neurons are required for the HSN axon guidance; however, the molecular mechanisms involved are completely unknown. In this study, we found that the VPC strongly expressed UNC-6 during HSN axon growth. Silencing of UNC-6 expression in only the VPC, using a novel tissue-specific RNAi technique, resulted in abnormal HSN axon guidance. The expression of Netrin/UNC-6 by only the VPC in unc-6 null mutants partially rescued the HSN ventral axon guidance. Furthermore, the expression of Netrin/UNC-6 by the VPC and the ventral nerve cord (VNC) in unc-6 null mutants restored the complex HSN axon guidance. These results suggest that UNC-6 expressed by the VPC and the VNC cooperatively regulates the complex HSN axon guidance.     

Increased neuromuscular activity causes axonal defects and muscular degeneration

Before establishing terminal synapses with their final muscle targets, migrating motor axons form en passant synaptic contacts with myotomal muscle. Whereas signaling through terminal synapses has been shown to play important roles in pre- and postsynaptic development, little is known about the function of these early en passant synaptic contacts. Here, we show that increased neuromuscular activity through en passant synaptic contacts affects pre- and postsynaptic development. We demonstrate that in zebrafish twister mutants, prolonged neuromuscular transmission causes motor axonal extension and muscular degeneration in a dose-dependent manner. Cloning of twister reveals a novel, dominant gain-of-function mutation in the muscle-specific nicotinic acetylcholine receptor alpha-subunit, CHRNA1. Moreover, electrophysiological analysis demonstrates that the mutant subunit increases synaptic decay times, thereby prolonging postsynaptic activity. We show that as the first en passant synaptic contacts form, excessive postsynaptic activity in homozygous embryos severely impedes pre- and postsynaptic development, leading to degenerative defects characteristic of the human slow-channel congenital myasthenic syndrome. By contrast, in heterozygous embryos, transient and mild increase in postsynaptic activity does not overtly affect postsynaptic morphology but causes transient axonal defects, suggesting bi-directional communication between motor axons and myotomal muscle. Together, our results provide compelling evidence that during pathfinding, myotomal muscle cells communicate extensively with extending motor axons through en passant synaptic contacts.     

Effect of Rabbit Anti-Asialo-GM1 (GA1) Polyclonal Antibodies on Neuromuscular Transmission and Acetylcholine-Induced Action Potentials: Neurophysiological and Immunohistochemical Studies

We produced anti-asialo-GM1 (GA1) polyclonal antibodies by sensitizing New Zealand rabbits with GA1 and investigated the epitopes and pathogenic role of anti-GA1 antibodies that appeared in serum. The serum blocked neuromuscular transmission, but not acetylcholine (ACh)-induced potentials, in muscle-spinal cord cocultured cells. The effect was complement independent. The antibodies inhibited voltage-gated Ca2+ channel (VGCC). The epitopes recognized by the antibodies were located in the outer membrane of Schwann cells and motor axons of Wistar rat ventral roots and on motor axons extended from spinal cord to muscle cells in muscle-spinal cocultured cells. The ACh-induced potential was not reduced by the addition of sera, suggesting the blockade is presynaptic. Thus, anti-GA1 antibodies may block neuromuscular transmission by suppressing VGCC on axonal terminals of motor nerves.