stmA¹ mutants display a total block of endocytosis in non-neuronal cells and a weaker, partial defect at neuronal synapses.

stmA^rev499/stmA¹ mutants carrying stmA^{S358A.T:Avic\GFP-EGFP} are viable.

stmA¹ mutants show a highly significant decrease in FM1-43 dye loading in the neuromuscular junction compared to wild-type controls.

At permissive temperature (25[o]C) stmA¹ flies expressing stmA^{S358A.T:Avic\GFP-EGFP} in the Garland cells take TR-vidin tracer into discrete puncta/vesicles to the same level as wild-type controls. At the 37[o]C restrictive temperature, while wild-type cells continue to display robust tracer uptake, no tracer-positive vesicles are found. This indicates that there is a complete block in Garland cell endocytosis in stmA¹ flies carrying stmA^{S358A.T:Avic\GFP-EGFP}, as there are in stmA¹ flies that do not carry the transgene.

At 25[o]C, stmA¹/stmA^rev499 exhibit wild-type levels of locomotion and behaviour. At 37[o]C (the restrictive temperature), 100% of these animals paralyse within 5 minutes. In contrast, wild-type flies experience an increase in activity levels. Mutant animals show no detectable movement when paralysed, but fully recover movement after 5 minutes at 25[o]C. Decreasing and increasing the temperature to 35[oC and 39[o]C causes a concomitant change in the time for stmA¹/stmA^rev499 animals to paralyse. Under identical conditions, stmA^{S358A.T:Avic\GFP-EGFP}-expressing stmA¹/stmA^rev499 animals behave as wild-type, with no paralysis.

At the permissive temperature (30[o]C), evoked EJP recordings from the thoracic dorsal longitudinal muscles in stmA¹/stmA^rev499 are indistinguishable from wild-type controls. In contrast, after an acute shift to a restrictive temperature (37[o]C), evoked EJPs in these animals are effectively abolished, whereas control EJP amplitudes remain robust.

stmA¹ homozygous mutants show comparable levels of synaptic vesicle endocytosis at the larval neuromuscular junction at 25[o]C compared to control animals. At 37[o]C, homozygotes show very significant impairment in endocytosis.

stmA¹ homozygous mutants show reduced levels of endocytosis in garland cells at 25[o]C. At 37[o]C a complete block in endocytosis is observed.

The labyrinthine channels of stmA¹ garland cells are elongated and drastically engorged when animals are shifted to 37[o]C compared to 25[o]C, and compared to control animals at 37[o]C. Vacuolar structures develop within the engorged channels. Few endosomal α vacuoles are present, and those that are appear enlarged and irregular in shape.

The total number of vesicles per bouton at the stmA¹ mutant larval neuromuscular junction is slightly elevated compared to control animals. There is a significant increase in the number of synaptic vesicles clustered at the presynaptic active zone, and closely docked at the T-bar. A depolarisation stimulus at 37[o]C eliminates the accumulation of synaptic vesicles, but the elevated clustered and docked vesicle pools persist in these conditions. Stimulation fails to induce the production of more cisternae, compared to the four-fold increase observed in controls.

At the ultrastructural level significant loss of endosome production but significantly more labeled synaptic vesicles are observed in stmA¹ boutons compared to controls at 37[o]C.

In primary neuronal cultures derived from late-stage pupal brains of stmA¹ mutant animals, greatly reduced levels endocytosis are observed at synaptic varicosities along the axon processes compared to controls at 37[o]C.

stmA¹ mutant animals are completely paralysed within minutes of exposure to restrictive temperatures (>37^oC), although control animals appear unaffected. At intermediate temperatures, stmA¹ mutants show intermediate movement impairment. At 29^oC, mutants remain mobile for many hours but become progressively sluggish and die within 1 day. At 33^oC, mutants display reduced movement followed by complete paralysis in approximately 30 minutes.

At permissive temperatures (<25^oC), stmA¹ mutant adult flies appear to act normally and cannot be distinguished from wild-type flies based on locomotion or gross behaviour.

Homozygous stmA¹, heterozygous stmA¹/stmA^rev499, stmA¹/stmA^18-2, and stmA¹/Df(2R)H3D3 mutants display 100% paralysis within 3-4 minutes when shifted from room temperature (22^oC) to 37^oC, and recover with a similar time course after a return to room temperature. No difference in the kinetics of paralysis or recovery is observed between stmA¹ homozygotes and stmA¹/stmA^rev499, stmA¹/stmA^18-2, and stmA¹/Df(2R)H3D3 mutants.

stmA¹ mutants do not show any visible change in death-rate after exposure to veratridine for 2, 4, 8, and 12 hours at 18, 22, and 28^oC, respectively.

At permissive temperature (25^oC), stmA¹/stmA^rev499 mutants exhibit evoked EJP amplitudes from the thoracic dorsal longitudinal muscle that are indistinguishable from wild-type. In contrast, after a shift to 37^oC, evoked EJPs in stmA¹/stmA^rev499 mutants are undetectable and remain completely suppressed at 37^oC, but then recover after a return to 25^oC (in wild-type, EJP amplitudes are reduced by ~25% at 37^oC, but then persist throughout the entire time course of the temperature shift recording). There isn't a similar acute requirement for stmA at the larval neuromuscular junction. EJC amplitudes at room temperature (20-22^oC) are indistinguishable between stmA¹/stmA^rev499 and control animals. Incubation of stmA¹/stmA^rev499 wandering third instar larvae at 37^oC does not cause significant change in EJC amplitudes. At a stimulation frequency of 0.5Hz, the mean amplitude is approximately 243nA for stmA¹/stmA^rev499 animals and 224nA for control animals, respectively. Assays of SV cycling with FM1-43 dye loading/unloading at the larval neuromuscular junction fail to reveal deficits in stmA¹/stmA^rev499 mutants.

Thoracic dorsal longitudinal muscle EJP recordings in stmA¹/stmA^rev499 mutants after a shift to restrictive temperature (25 to 37^oC) initially decline, consistent with a progressive weakening of neuromuscular junction transmission, but then display a complete loss. In contrast, TTM (tergotrochanteral muscle) recordings of stmA¹/stmA^rev499 mutants show a gradual loss of EJP amplitude over several minutes after a shift to the restrictive temperature, and a similar gradual recovery after a return to permissive temperature.

When stimulated by an extracellular electrode placed in the thoracic ganglion to directly excite the DLM (dorsal longitudinal muscle) motor neuron axon, the DLM in stmA¹/stmA^rev499 mutants shows graded loss/recovery of EJP amplitude with a time course comparable with that seen in TTM (tergotrochanteral muscle).

During temperature elevation, both wild-type and stmA¹/stmA^rev499 mutant DLM EJP latencies first transiently shorten, but then begin to increase at ~33^oC. Above 33-35^oC, stmA¹/stmA^rev499 latencies are significantly prolonged compared with controls.

Using two-electrode voltage-clamps, the EJC can be recorded from DLMe/d cells. At 25^oC, EJC amplitude in wild-type and stmA¹/stmA^rev499 mutants are indistinguishable. At 36^oC, stmA¹/stmA^rev499 EJCs are completely abolished, but wild-type EJCs remain robust throughout the entire course of the recording. After a return to 25^oC, mutant transmission recovers to 87% of the initial amplitude.

At permissive temperature (22^oC), the ultrastructure of NMJ terminals in stmA¹/stmA^rev499 mutants and wild-type controls appear essentially indistinguishable.

After a 10 minute shift to 37^oC, the synaptic vesicle number in stmA¹/stmA^rev499 mutants is almost tripled, while in wild-type the synaptic vesicle number is not changed. The conditional increase in synaptic vesicle number is consistent with a block of exocytosis in stmA¹/stmA^rev499 mutants. The temperature shift from 22 to 37^oC results in a slight reduction of bouton area in stmA¹/stmA^rev499 mutants, although this change is not statistically significant.

After a 10 minute shift to 37^oC, the cross-sectional bouton area in stmA¹/stmA^rev499 mutants is reduced approximately 38%. The bouton area of wild-type controls show no significant change during the same temperature shift.

Active zones in stmA¹/stmA^rev499 mutants at the permissive temperature (22^oC) contain one or two synaptic vesicles in contact (docked) with the active zone membrane, as in wild-type. In contrast, after a 10 minute shift to 37^oC, there is a significant accumulation of docked synaptic vesicles in stmA¹/stmA^rev499 mutants when compared to both wild-type at 37^oC and stmA¹/stmA^rev499 mutants at 22^oC. Typically, all available active zone membrane in the mutant appears occupied with docked synaptic vesicles, suggesting that synaptic vesicle docking sites may be nearly or completely saturated.

At 33^oC, stmA¹ homozygotes and stmA¹/stmA^rev499 trans-heterozygotes are only slowly paralysed over a time course of approximately 30 minutes. For the first 10 minutes at 33^oC, stmA¹ homozygotes remain fully upright and mobile.

At the permissive temperature of 25^oC, stmA¹/stmA^rev499 flies show normal light-induced electroretinogram (ERG) responses. At the non-permissive temperature of 37^oC, the ERG is completely eliminated in these flies. This block in phototransduction is activity dependent; at 37^oC, continuous light blocks phototransduction within 40 seconds, but repetitive 1 second light flashes at low frequency (3 minute intervals) induce robust ERGs even after 30 minutes at 37^oC. Repetitive 1 second light flashes at an interval of 20 seconds block phototransduction in less than 5 minutes. At the non-permissive temperature, recovery (in darkness) of the initial ERG peak and the sustained ERG peak after exposure to continuous light is dark interval dependent, with the initial ERG depolarisation recovering faster and more completely, whereas the amplitude of sustained depolarisation at 10 seconds does not recover appreciably even after 3 minutes in darkness.

Brief anoxia after phototransduction blockade at 37^oC induces a rapid depolarisation in the photoreceptors in stmA¹/stmA^rev499 mutants similar to that seen in wild-type flies under these conditions.

stmA¹ is hypoactive at 24^oC, and weakly negatively geotactic. Adults are almost always flightless. 18 hour stmA¹ embryos reared at 23^oC show a mild hypertrophy of the embryonic brain lobes and ventral nerve cord. Embryos made inviable by raised temperature show cuticle loss over the embryonic brain and ventral nerve cord.

Double mutants with mle^nap-ts1 or para^ts1 show no exaggeration of the paralytic phenotypes, nor any efect on their viability and fertility. These results suggest stmA does not interact with para or mle.

Adult males are resistant to the lethal effects of the sodium channel neuropoison veratridine.

Shows a temperature-dependent, maternally determined embryonic lethality. Adult flies are less sensitive to veratrine than wild-type. Sensitivity to tetrodotoxin is normal.

Two-hour heat shocks applied throughout development show 0-8 h and 56-140 h to be highly sensitive, with no survival; 8-56 h and after 140 h show survival with normal phenotype. Early temperature shock leads to blastoderm arrest with no gastrulation. Embryos produced by homozygous stm-A mothers are exceedingly sensitive to a two-hour exposure to 36^oC; leads to complete lethality from 0-3 h irrespective of paternal contribution. paralyzed at 35^oC; rapid paralysis at 39^oC; larval paralysis; complements mle^nap-ts1 One of three EMS-induced temperature-sensitive paralytic mutations at different loci on chromosome 2. Paralysis dependent on time and temperature of exposure; the recovery time is proportional to the severity of the paralyzing exposure.

stmA¹ is an enhancer of paralytic | heat sensitive phenotype of Syx1A^3-69

stmA[+]/stmA¹ is an enhancer of paralytic | heat sensitive phenotype of Syx1A^3-69

Syx1A^3-69, stmA¹ has NMJ bouton | larval stage | temperature conditional phenotype

Syx1A^3-69, stmA¹ has garland cell | wandering third instar larval stage | temperature conditional phenotype