stumble: Biological Overview | References
Gene name - stumble
Synonyms - CG30263
Cytological map position - 57F8-57F8
Function - surface transmembrane protein
Symbol - stum
FlyBase ID: FBgn0050263
Genetic map position - chr2R:17563826-17572505
Classification - conserved transmembrane protein of the SPEC3 family
Cellular location - transmembrane
Animal locomotion depends on proprioceptive feedback, which is generated by mechanosensory neurons. A genetic screen for impaired walking was performed in Drosophila, and a gene, stumble (stum), was isolated. The Stum protein has orthologs in animals ranging from nematodes to mammals and is predicted to contain two transmembrane domains. Expression of the mouse orthologs of stum in mutant flies rescued their phenotype, which demonstrates functional conservation. Dendrites of stum-expressing neurons in legs were stretched by both flexion and extension of corresponding joints. Joint angles that induced dendritic stretching also elicited elevation of cellular Ca(2+) levels-not seen in stum mutants. Thus, this study has identified an evolutionarily conserved gene, stum, which is required for transduction of mechanical stimuli in a specific subpopulation of Drosophila proprioceptive neurons that sense joint angles (Desai, 2014).
Animal locomotion is achieved by coordination of motor activity according to proprioceptive mechanosensory inputs. In Drosophila, mechanosensation is mediated either by ciliated or multidendritic receptor neurons. Multidendritic neurons can respond to direct application of mechanical force to their membranes. It is less clear, however, how multidendritic mechanosensory neurons can be tuned to one mechanical modality, such as joint angle, and disregard other mechanical stimuli that may originate from external impacts or changes in the shape of muscles during contraction. In order to identify genes involved in proprioceptive sensation, uncoordination was screened for in a collection of ethyl methanesulfonate-mutagenized Drosophila lines. Lines that exhibited walking impairments were selected, and the phenotype severity was quantified by measuring climbing speed. Three lines, 204, 922, and 4487, were identified that showed lack of coordination in homozygous flies and did not complement one another, which suggested that they represent alleles of the same gene. Two of the lines, 204 and 4487, showed severe uncoordination, and the phenotype in 922 was mild. Deficiency mapping pointed to a gene, CG30263, predicted to encode a large protein with 1870 or 1959 amino acids (depending on the splice variant). Because the mutants had a walking impairment phenotype, this gene was named stumble (stum). The stum ortholog in humans is C1orf95, and it was categorized as a member of the SPEC3 family (UNIPROT, INTERPRO), with unknown function. The stum gene was sequenced in the three mutant lines, and it was found that stum204, stum4487, and stum922 had stop codons at amino acid positions 171, 202, and 1081, respectively (Desai, 2014).
Transgenic flies that express stum cDNA in neurons were generated and it was found that the stum phenotype was partially rescued, which indicated that the mutations in stum were underlying the uncoordination phenotype. It is noted that the mouse ortholog of stum (National Center for Biotechnology Information, the U.S. National Institutes of Health, reference sequence: NP_001074696.1), which is only 141 amino acids long and shares 33% sequence identity with Drosophila stum, was also able to substantially rescue the uncoordination phenotype. Therefore, the function of stum appears to be conserved between distant animal species. Moreover, the rescue of the phenotype with such a short form of stum suggests that the C-terminal region of the fly protein constitutes the functional core (Desai, 2014).
Proprioceptive defects in adult flies have been attributed to malfunction of type I (ciliated) mechanoreceptor neurons. To test whether such defects also underlie the phenotype in the stum mutant, electrophysiological recordings were performed of mechanical responses from the ciliated mechanoreceptor neuron of the anterior notopleural bristle. Type I neurons of stum mutants were indistinguishable from controls. Therefore, unlike known proprioception mutants, the phenotype in stum mutants does not arise from a general defect in type I mechanoreceptor neurons. To identify which cells give rise to the phenotype, the genomic regulatory region of stum was used to drive a CD8 fused to green fluorescent protein (CD8GFP) reporter [stum-Gal4 driving the upstream activation sequence (UAS)–CD8GFP]. It was found that stum expression in the legs was localized to three labeled neurons: one at the femur-tibia joint, the second at the tibia-tarsus joint, and the third spanning the second tarsal segment. The cell bodies of these stum-expressing neurons were located near the distal end of each leg segment, and their dendrites terminated at the corresponding joints (Desai, 2014).
To study whether there are stum-expressing cells within the ventral nerve cord (VNC), it was examined in flies that express CD8GFP using stum-Gal4. The only fluorescent signal in the VNC originated from the axons of the leg neurons. The axons terminated within the neuropil that corresponds to each particular leg, branching into a bowl shape. This pattern is typical of neurons that take part in proprioception, such as the hair plate neurons. Therefore, the stum-expressing cells in the Drosophila body have characteristics of proprioceptive neurons that sense a property of specific joints (Desai, 2014).
Confocal imaging was performed of the dendritic region of stum-expressing neurons, and it was found that, close to its tip, the dendrite branches toward the lateral aspect of the joint, and this side branch terminates at a short distance from the cuticle. The tips of dendrite were not associated with a cuticular structure or a scolopale. These structural features are typical features of type II (multidendritic) neurons but are incompatible with type I mechanoreceptor neurons that terminate with a ciliary structure. Thus, stum uncoordination mutations affect type II mechanoreceptor neurons. Furthermore, the entire dendritic terminal of these neurons was located in a region that is devoid of musculature, which suggests that they do not sense the mechanical properties of muscles. Taken together, these data suggest that stum-expressing neurons sense a mechanical property of the joint (Desai, 2014).
To test whether stum-positive neurons encode joint angles, high-resolution imaging of the tibia-tarsus joint area was performed at different angles. At each angle of the joint, the total length of the sensory dendrite and its side branch was measured. It was found that the total dendrite length had a minimum typically at 130o to 170o, and it increased when the joint was shifted to either more obtuse or more acute angles. These morphological changes indicate that these neurons are mechanically affected by the position of the joint. Because the tip of the side branch is stationary, it is likely that the change in total length results from the coupling of the tip of the main dendrite to the motion of the distal joint segment. The position of the dendrite and the susceptibility of its morphology to joint angle suggest that the role of stum-positive neurons is to sense and encode the angle of the joint (Desai, 2014).
Ca2+ fluorescence was measured while forcing the tibia-tarsus joint to different angles, and the Ca2+ fluorescence in stum-expressing neurons was found to correlate with the angle of the joint. As in the morphological changes, the responses correlated with joint angle in a U-shaped manner, where both acute and obtuse angles induced increasing Ca2+ elevations. These results indicate that the stum-positive neurons encode proprioceptive information about the angle of joints. It is noteworthy that a similar U-shaped encoding of joint angles has also been described in receptor neurons of mammalian joints, which suggests that sensing the deviations from a neutral joint range is universally critical for motor function (Desai, 2014).
The walking impairment in the stum mutant fly suggests that the gene is necessary for generating the proper proprioceptive responses in stum-expressing neurons. To test whether the stum mutations affect coupling between joint angles and dendritic stretching, the stretching was quantitated in mutant flies that have stum-expressing neurons labeled with CD8GFP. In the stum mutant, the dendritic stretching in response to joint angles was comparable to that of control flies. Thus, stum is not required for the mechanical coupling between joint angles and stretching of the sensory dendrites (Desai, 2014).
Although the dendritic stretching was not significantly affected in the stum mutant, the Ca2+ responses to both acute and obtuse joint angles were abolished in the mutant. Therefore, it is concluded that stum is essential for transducing dendrite stretching into cellular responses (Desai, 2014).
The morphology of stum-expressing neurons were examined, and it was found that, although axons and cell bodies of stum mutants were indistinguishable from controls, the sensory dendrite in mutants exhibited abnormalities. Most notably, in some of the stum-expressing neurons the tip of the dendrite was overgrown and extended into the distal segment of the joint. Thus, the absence of stum leads to a morphological defect, possibly because of the lack of mechanical responsiveness. The occasional morphological differences may account for the slight difference in the stretching profile between control and mutant dendrites. The fraction of neurons demonstrating the abnormal morphology in the mutant increased from the day of eclosion to the following day. As the addition of morphological changes takes place after eclosion, it is possible that stum-dependent activity is essential for late shape determination that can take place in adult multidendritic neurons (Desai, 2014).
Transgenic flies were generated that express a GFP fused with the N terminus of Stum under UAS regulation (UAS-GFP-Stum). The Stum fusion protein was found to be specifically localized to the distal part of the sensory dendrite, although it did not accumulate substantially in any other part of the cell. The fluorescent signal started at the region of bifurcation and extended to both distal tips of the dendrite. This specific localization suggests that Stum functions in the part of the dendrite that senses stretching (Desai, 2014).
Taken together, stum expression in mechanosensory neurons, Stum localization to the sensory dendrite, and the abolition of responses to stretching in the stum mutant suggest that stum has an essential role in mediating mechanical sensing in receptor neurons. Because the Stum protein in most species is very small and because Drosophila stum is expressed in limited populations of receptor neurons, it is proposed that stum is not the mechanically activated channel. Rather, stum may serve as an accessory module that is essential for the proper localization or function of the transduction channels (Desai, 2014).
The stretch-receptor neurons that express stum present an elegant engineering solution for generating specificity to the modality of mechanical stimulus. The distal part of their dendrite bifurcates into two branches whose tips are anchored to parts of the joint that shift their relative positions. Sensing the stretching only between the two dendritic tips may tune the nerve responses to joint motions and filter out the effect of irrelevant mechanical impacts. This specificity enables the sensory neuron to relay reliable proprioceptive information to the central nervous system (Desai, 2014).
Search PubMed for articles about Drosophila Stumble
Desai, B. S., Chadha, A. and Cook, B. (2014). The stum gene is essential for mechanical sensing in proprioceptive neurons. Science 343(6176): 1256-9. PubMed ID: 24626929
date revised: 18 March 2014
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