Fig 1.
Kismet affects the expression of gene products involved in both CME and ADBE.
PI3K92E and shaggy/GSK3β (sgg) transcripts are differentially expressed in kis mutants. Relative expression of CNS transcripts was assessed via RT-qPCR. 2-ΔΔC(t) values are indicated. Data includes at least four biological replicates each including three technical replicates. Technical replicates are represented by circles for the representative genotypes. Bars indicate means with the error bars showing the SEM.
Fig 2.
Kismet and Liquid Facets promote ADBE.
eEJCs were measured during 60 sec of 20 Hz HFS to induce ADBE and during a 50 sec recovery period with 0.2 Hz stimulation. (A) Mean eEJC amplitudes are shown relative to the amplitude measured after the first stimuli for w1118 controls (n = 10) and kis (n = 9) and lqf (n = 12) mutants. Error bars represent the SEM. (B) Representative eEJC recordings from the genotypes listed.
Fig 3.
Inhibition of CME using Dynasore does not produce a change in evoked currents in kis or lqf mutants.
eEJCs were measured during five min of 10 Hz stimulation to induce CME. (A) Mean eEJC amplitudes are shown relative to the amplitude measured after the first stimuli for w1118 controls (n = 9) and kis (n = 10) and lqf (n = 10) mutants. Error bars represent the SEM. (B) Representative eEJC recordings from the genotypes listed. (C) eEJC amplitudes were obtained after animals were pretreated with 100 μM Dynasore. Mean eEJC amplitudes are shown relative to the amplitude measured after the first stimuli for w1118 controls (n = 9) and kis (n = 10) and lqf (n = 10) mutants. Error bars represent the SEM. (D) Representative eEJC recordings from the genotypes listed after pretreatment with 100 μM Dynasore.
Fig 4.
Kismet mutants exhibit minimal changes in endocytosis when ADBE is inhibited.
Endocytosis was assessed by measuring internalization of the lipophilic dye FM 1-43FX after one min stimulation with 90 mM KCl. Animals were pretreated with either 100 μM BAPTA-AM, 25 μM EGTA-AM, or 200 μM Roscovitine to inhibit ADBE. (A) Panels show high resolution confocal micrographs of terminal presynaptic motor neuron boutons (HRP, magenta) after internalization of the lipophilic dye FM 1-43FX (green). (B) Data for each condition were normalized to w1118 controls. Bars indicate means, points represent individual larvae, and error bars represent SEM. Endocytic and kis mutants show reduced endocytosis. Endocytosis was unchanged in kis mutants pretreated with BAPTA or EGTA but reduced when pretreated with Roscovitine. Scale bar = 5 μm.
Fig 5.
Kismet mutants do not show changes in endocytosis when CME is inhibited.
Endocytosis was assessed by measuring internalization of the lipophilic dye FM 1-43FX after one min stimulation with 90 mM KCl. Larvae were pretreated with either 200 μM Chlorpromazine or 100 μM Dynasore to inhibit CME. (A) Panels show high resolution confocal micrographs of terminal presynaptic motor neuron boutons (HRP, magenta) after internalization of the lipophilic dye FM 1-43FX (green). (B) Data for each condition were normalized to w1118 controls. Bars indicate means, points represent individual larvae, and error bars represent SEM. Endocytosis was unchanged in kis mutants pretreated with Chlorpromazine or Dynasore. Scale bar = 5 μm.
Fig 6.
Kismet helps maintain vesicle pools.
Representative responses (top) in controls (n = 9) and kis mutants (n = 10) to 3 (A) or 10 (B) Hz stimulation in the presence of 2 μM Bafilomycin, which inhibits vesicle refilling. Muscles were clamped at -60 mV and recordings were obtained in HL-3 + 1.0 mM Ca2+. 3 Hz stimulation depletes the readily releasable and recycling pool of vesicles while 10 Hz stimulation deplete the reserve pool of vesicles. Vesicle pools are smaller in kis mutants as indicated by reduced eEJCs induced by stimulation. Bottom graphs show mean eEJC amplitudes normalized to the first stimulus for each condition. Error bars represent the SEM.
Fig 7.
Kismet is required in postsynaptic muscles for proper endocytosis of presynaptic vesicles.
Tissue-specific drivers were used to restore kis expression in specific tissues of kis mutants including presynaptic motor neurons (using the elav-Gal4 driver), postsynaptic muscle (using the 24B-Gal4 driver), or glial cells (using the repo-Gal4 driver). A) High resolution confocal micrographs of terminal presynaptic motor neuron boutons (HRP, magenta) after internalization of the lipophilic dye FM 1-43FX (green) after one min stimulation with 90 mM KCl in genotypes as listed. Scale bar = 5 μm. B) Quantification of FM 1-43FX fluorescence intensity indicates that when kis is restored in postsynaptic muscles of kis mutants, presynaptic endocytosis returns to control levels. C) Quantification of FM 1-43FX fluorescence intensity after tissue-specific knockdown of Kis. Knockdown of Kis in all tissues using the Actin5c-Gal4 driver or in postsynaptic muscles using the 24B-Gal4 driver resulted in impaired endocytosis. D) Histograms show quantification as determined by wrMTrck of larval crawling behavior on an agar arena for 30 s. Distance traveled, maximum velocity, average velocity, and crawling velocity normalized to body size (body lengths per second). Restoring kis expression in all tissues led to enhanced larval crawling.
Fig 8.
Expression of human CHD7 in kis mutants restores behavior but not endocytosis.
Tissue-specific drivers were used to express human CHD7 in all tissues (using the Actin5c-Gal4 driver), presynaptic motor neurons (using the elav-Gal4 driver), or postsynaptic muscle (using the 24B-Gal4 driver) of kis mutants. A) Histograms show quantification as determined by wrMTrck of larval crawling behavior on an agar arena for 30 s. Distance traveled, maximum velocity, average velocity, and crawling velocity normalized to body size (body lengths per second). B) Representative traces of larval crawling behavior for the genotypes listed. C) Quantification of FM 1-43FX fluorescence intensity indicates that expression of human CHD7 does not restore endocytosis in kis mutants. D) High resolution confocal micrographs of terminal presynaptic motor neuron boutons (HRP, magenta) after internalization of the lipophilic dye FM 1-43FX (green) after 1 min stimulation with 90 mM KCl in genotypes as listed. Scale bar = 5 μm.
Fig 9.
Expression of an ATPase deficient Kis, KisK2060R, in kis mutants restores behavior and endocytosis.
Tissue-specific drivers were used to express KisK2060R in all tissues (using the Actin5c-Gal4 driver), presynaptic motor neurons (using the elav-Gal4 driver), or postsynaptic muscle (using the 24B-Gal4 driver) of kis mutants. A) Histograms show quantification as determined by wrMTrck of larval crawling behavior on an agar arena for 30 s. Distance traveled, maximum velocity, average velocity, and crawling velocity normalized to body size (body lengths per second). B) Representative traces of larval crawling behavior for the genotypes listed. C) Quantification of FM 1-43FX fluorescence intensity indicates that expression of KisK2060R restores endocytosis in kis mutants. D) High resolution confocal micrographs of terminal presynaptic motor neuron boutons (HRP, magenta) after internalization of the lipophilic dye FM 1-43FX (green) after one min stimulation with 90 mM KCl in genotypes as listed. Scale bar = 5 μm.