stmArev499/stmA1 mutants carrying stmAS358A.T:Avic\GFP-EGFP are viable.
At permissive temperature (25[o]C) stmA1 flies expressing stmAS358A.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 stmAS358A.T:Avic\GFP-EGFP, as there are in stmA1 flies that do not carry the transgene.
At 25[o]C, stmA1/stmArev499 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/stmArev499 animals to paralyse. Under identical conditions, stmAS358A.T:Avic\GFP-EGFP-expressing stmA1/stmArev499 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/stmArev499 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.
At restrictive temperatures (>37oC), all stmArev499 mutant animals are completely paralysed within minutes, although control animals appear unaffected. At intermediate temperatures, stmArev499 mutants show intermediate movement impairment. At 29oC, mutants remain mobile for many hours but become progressively sluggish and die within 1 day. At 33oC, mutants display reduced movement followed by complete paralysis in approximately 30 minutes.
At permissive temperatures (<25oC), stmArev499 mutant adult flies appear to act normally and cannot be distinguished from wild-type flies based on locomotion or gross behaviour.
Heterozygous stmA1/stmArev499 mutants display 100% paralysis within 3-4 minutes when shifted from room temperature (22oC) to 37oC) and recover with a similar time course after a return to room temperature.
At permissive temperature (25oC), stmA1/stmArev499 mutants exhibit evoked EJP amplitudes from the thoracic dorsal longitudinal muscle that are indistinguishable from wild-type. In contrast, after a shift to 37oC, evoked EJPs in stmA1/stmArev499 mutants are undetectable and remain completely suppressed at 37oC, but then recover after a return to 25oC (in wild-type, EJP amplitudes are reduced by ~25% at 37oC, 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 (20-22oC) are indistinguishable between stmA1/stmArev499 and control animals. Incubation of stmA1/stmArev499 wandering third instar larvae at 37oC does not cause significant change in EJC amplitudes. At a stimulation frequency of 0.5Hz, the mean amplitude is 243 for stmA1/stmArev499 animals and 224 for control animals, respectively. Assays of SV cycling with FM1-43 dye loading/unloading at the larval neuromuscular junction fails to reveal deficits in stmA1/stmArev499 mutants.
Thoracic dorsal longitudinal muscle EJP recordings in stmA1/stmArev499 mutants after a shift to restrictive temperature (25 to 37oC) 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/stmArev499 mutants show a gradual loss of EJP amplitude over several minutes after shift to the restrictive temperature, and a similar gradual recovery after return to permissive temperature.
At the permissive temperature of 25oC, stmA1/stmArev499 flies show normal light-induced electroretinogram (ERG) responses. At the non-permissive temperature of 37oC, the ERG is completely eliminated in these flies. This block in phototransduction is activity dependent; at 37oC, 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 37oC. 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 37oC induces a rapid depolarisation in the photoreceptors in stmA1/stmArev499 mutants similar to that seen in wild-type flies under these conditions.
cmp44Erev499/Df(2R)cmp44E-rev390 animals die as embryos. Identifiable clones are not detected in females capable of forming homozygous cmp44Erev499 germline clones. A high frequency of leg defects and occasional eye defects are seen in females capable of forming homozygous cmp44Erev499 germline clones.
stmA1/stmArev499 is rescued by stmAS358A.EGFP
stmA1/stmArev499 is rescued by stmAEGFP
stmA1/stmArev499 is rescued by stmAEGFP
stmA1/stmArev499 is rescued by stmAEGFP
stmArev499 is rescued by stmA+t17
stmArev499 is rescued by stmA+t8.5
stmArev499 is not rescued by stmAS358A.EGFP
stmArev499 is not rescued by stmAEGFP
stmAS358A.T:Avic\GFP-EGFP-expressing stmA1/stmArev499 animals behave as wild-type, with no paralysis, compared to stmA1/stmArev499 mutants, that experience paralysis within 5 minutes of exposure to the restrictive temperature.
Endocytosis, which is defective in stmA1 animals, is restored to wildtype levels in stmA1/stmArev499; stmAT:Avic\GFP-EGFP/stmAT:Avic\GFP-EGFP animals.
A single copy of stmAT:Avic\GFP-EGFP fully recues the behavioural paralysis found in heterozygous stmA1/stmArev499 mutants.