Cacophony alternative splicing gives rise to different protein isoforms.

(A) Schematic of cacophony with N-terminal sfGFP or mEOS4b tag and two exon pairs that are spliced mutually exclusively. IS4A and IS4B exons encode isoforms of the 4th transmembrane domain (S4) and thus part of the voltage sensor of the first homologous repeat (I) of the calcium channel, while I-IIA and I-IIB give rise to two versions of the intracellular linker between homologous repeats I and II. I-IIA contains a non-well conserved binding site for voltage gated calcium channel β-subunits (Caβ) whereas I-IIB gives rise to a conserved Caβ-binding site as well as a binding site for G-protein βγ-subunits (Gβγ). (B) Genomic removal of one of the mutually exclusively spliced exons of one or two exon pairs by CRISPR/Cas9 mediated double strand breaks in the germ line results in exon out mutants by imprecise excision of the cut exons and cell-intrinsic DNA repair. (C) Cacophony gives rise to 18 annotated transcripts (RA- RU, left). Multiple variants express the same mutually exclusive exons but differ with respect to expression of other alternatively spliced but not mutually exclusive exons. Removal of the mutually exclusive exons IS4A/IS4B and/or I-IIA/I-IIB allows expression of fewer cacophony splice variants. Transcript variants that are possible upon exon excision are marked by +. (D) Western Blots reveal expression of GFP-tagged cacophony protein (cacsfGFP) for all excision variants at the expected band size of ∼240 kDa (cacophony) plus ∼30 kDa (sfGFP, top), while no band can be detected in the Canton S wildtype (CS, top, right) that does not express cacsfGFP. 10 adult brains were used of hemizygous males for all genotypes except for ΔIS4B which is homo-/hemizygous lethal. For ΔIS4B 20 brains of heterozygous females and a heterozygous cacsfGFP control were used (F1 females from cross with Canton S wildtype flies(+)). β-actin was used as loading control (bottom). ΔIS4B shows weak expression (top, left), while all other exon out variants express strongly, although ΔI-IIB shows somewhat weaker expression (top, middle).

The IS4B exon is required for Cav2 localization to active zone and for evoked synaptic transmission.

(A-C) Representative confocal projection views of triple labels for GFP tagged Cav2 channels (green), the active zone marker brp (magenta), and HRP to label axonal membrane (blue) in control animals with all Cav2 exons (cacsfGFP, top row, A), in animals with selective excision of either the alternative exon IS4A ( IS4AsfGFP, middle row, B), or the alternative exon IS4B ( ISABsfGFP, bottom row, C). Excision of IS4B is embryonic lethal, so that localization analysis was conducted in heterozygous animals ( ISABsfGFP/+). The gross morphology of the neuromuscular junctions (muscle fibers, bouton numbers and sizes, active zone numbers) was similar in all three genotypes. GFP tagged Cav2 channels localize to active zones (A) as previously reported (Gratz et al. 2019; Krick et al. 2021). Excision of the IS4A exon does neither impact Cav2 channel active zone localization nor labeling intensity (B). By contrast, upon excision of the IS4B exon, Cav2 channel label (white arrowheads) is faint and not strictly co-localized with active zones (C). (D, E) Representative traces of evoked synaptic transmission as recorded in TEVC from muscle fiber 6 upon extracellular stimulation of the motor nerve from a wildtype control animal (CS, blue), an animal with GFP tagged Cav2 channels (cacsfGFP, green), and animals with GFP tagged Cav2 channels and either IS4A exon excision (ΔISA4, orange) or IS4B excision (ΔISAB, transheterozygous over ΔISA4, black trace). (D) Postsynaptic currents (PSCs) are similarly shaped between CS control (blue) and animals with GFP-tagged Cav2 channels (cacsfGFP, green), and (E) PSC amplitudes are not statistically different (p=0.34, two sided Tukey’s multiple comparison test). In animals with homozygous IS4A exon excision (orange), PSC amplitude is slightly but not significantly increased (p=0.18, two sided Tukey’s multiple comparison test). In transheterozygous animals with IS4A excision on one chromosome and IS4B excision on the other one, PSC amplitude is significantly decreased (p=0.0008), two sided Tukey’s multiple comparison test). (F) Quantal size (mPSC amplitude) and spontaneous release frequency (G) show no significant difference between genotypes. (H, I) Since animals homozygous for IS4B exon excision are lethal, we created mosaic animals that were heterozygous for Cav2 in most neurons but hemizygous for either cacsfGFP or ΔISAB in motoneurons innervating muscle M12 (see methods, cacFlpStop). In control with all Cav2 exons (cacsfGFP, green, top row) cacsfGFP colocalizes with brp (magenta) in presynaptic active zones on M12 (H) and evoked synaptic transmission induces PSCs of about 100 nA amplitude (I). By contrast, upon deletion of IS4B (H, bottom row) in motoneurons to M12 fuzzy Cav2 label is found throughout the motor terminals, but Cav2 (green) does not strictly colocalize with brp (magenta) in active zones (H) and evoked synaptic transmission is reduced by more than 90%, Student’s T-test, p < 0.0001 (I). (J) HVA Cav2 currents as recorded from the somata of adult flight motoneurons in mosaic animals with only one copy of the Cav2 locus in flight motoneurons (see methods). HVA currents are measured by starting from a holding potential of -50 mV (LVA inactivation) followed by step command voltages from -90 mV to +20 mV in 10 mV increments (left). In GFP-tagged controls (cacsfGFP / cacFlpStop) this reveals transient and sustained HVA current components. By contrast, following excision of the IS4B exon ( ISABsfGFP / cacFlpStop), the sustained HVA current is nearly absent. Current-voltage (IV) relation of sustained HVA for controls with all Cav2 exons (cacsfGFP, green circles, n = 8) and following excision of IS4B ( IS4BsfGFP, dark green squares, n = 4).

The I-II exon does not affect active zone localization but release probability.

(A-C) Representative confocal projection views of triple labels for GFP tagged Cav2 channels (green), the active zone marker brp (magenta), and HRP to label axonal membrane (blue) in control animals with all Cav2 exons (cacsfGFP, top row, A), with selective excision of either the alternative exon I-IIA ( I-IIAsfGFP, middle row, B), or the alternative exon I-IIB ( I-IIBsfGFP, bottom row, C). The gross morphology of the neuromuscular junctions (muscle fibers, bouton numbers and sizes, active zone numbers) was similar in all three genotypes (not shown). Excision of the I-IIA exon does neither impact Cav2 channel active zone localization nor labeling intensity (B). Excision of I-IIB does not impact Cav2 channel active zone localization but labeling intensity seems lower (C). (D-F) Quantification of Cav2 channel co-localization with the active zone marker brp yields a similar Pearson’s colocalization coefficient (D) as well as similar Manders 1 (E) and Manders 2 (F) coefficients for controls and both exon-out variants of the I-II locus. (G) I-IIBsfGFP shows fainter immunofluorescence signals in the active zone as compared to control (cacsfGFP) and I-IIAsfGFP. (H) Quantification confirms a significant reduction in I-IIBsfGFP labeling intensity (Kruskal Wallis ANOVA with Dunn’s post hoc test, p < 0.0001) and no differences between I-IIAsfGFP and control (p > 0.99). (I) Evoked synaptic transmission as recorded in TEVC from muscle fiber 6 upon extracellular stimulation of the motor nerve. Postsynaptic currents (PSCs) are of similar shape and amplitude for CS control (blue) and animals with GFP-tagged Cav2 channels (cacsfGFP, green, p=0.34, two sided Tukey’s multiple comparison test). Excision of I-IIA ( I-IIAsfGFP, magenta) has no effect on evoked release amplitude (p=0.52, two sided Tukey’s multiple comparison test), but excision of I-IIB ( I-IIBsfGFP, orange) reduces evoked release significantly (p < 0.0001). (J) Quantification of PSC amplitude reveals a highly significant reduction in I-IIBsfGFP(orange) as compared cacsfGFP controls (green), but neither animals with excision of the I-IIA exon (magenta), nor transheterozygous animals with excision of I-IIA on one and I-IIB on the chromosome (brown) show differences to control (p=0.97, two sided Tukey’s multiple comparison test). (K) Quantal size (mPSC amplitude) and spontaneous release frequency (L) show no significant difference among genotypes.

Dual color STED imaging reveals equal nanoscale channel localization in AZs of Cav2 for all exon-out variants, and live sptPALM imaging reduced channel numbers in AZs for ΔI-IIB.

(A) Representative intensity projection image of the active zone marker bruchpilot (labeled with anti-brp, green) and Cav2 clusters (cacsfGFP labeled with anti-GFP, magenta) as imaged with dual color STED at motoneuron axon terminal boutons on larval muscle M6. The dotted white box demarks one bouton that is enlarged in (B). Each Cav2 cluster (magenta) is in close spatial proximity to the active zone marker brp (green). In 3D STED the active zone Cav2 -brp is viewed from different angles. Top views (see C1 in B and in selective enlargement) show 4 brp puncta that symmetrically surround the central Cav2 cluster. Viewing active zones at the edge of the bouton shows the Cav2 cluster facing to the outside and the brp puncta in close proximity (see 2-6). (C) Selective enlargements of each active zone that is numbered in B. (D) Top views (left column) and side views (right column) of the Cav2-brp arrangement in active zones in controls with GFP-tagged Cav2 channels, (cacsfGFP, top row), with excision of exon IS4A ( ISA4sfGFP, second row), with excision of exon I/IIA ( I/IIAsfGFP, third row), and with excision of exon I/IIB ( I/IIBsfGFP, bottom row). (E) Quantification of the distances between the center of each Cav2 punctum to the nearest brp punctum in the same focal plane. (F-G) Live sptPALM imaging of mEOS4b tagged Cav2 channels from AZs of MN terminals on muscle 6 in controls with full isoform diversity (cacmEOS4b, green) and following the removal of either I-IIA (ΔI-IIAmEOS4b, purple) or I-IIB (ΔI-IIBmEOS4b, orange). (F) Quantification of channel numbers from bleaching curves (G) reveals ∼ 9-11 Cav2 channels per AZ for tagged controls, which matches previous reports (Ghelani et al. 2023). Counts for ΔI-IIA reveal no significant differences (Kruskal-Wallis test with Dunn’s posthoc comparison, p=0.94), but Cav2 channel number in AZs is reduced by ∼50 % in ΔI-IIB (p<0.0001). (G) Bleaching curves of single active zones were illuminated after ∼10 s and then imaged under constant illumination for another 240 s. Discrete bleaching steps (dotted lines) indicate the bleaching of single mEOS4b tagged Cav2 channels. Comparing the amplitudes of single events and their integer multiples (dotted lines) to the maximum fluorescence at illumination start allows estimates of the total channel number per AZ.

Alternative splicing in the I-II linker affects short term plasticity and motor behavior.

(A- B) Paired pulse ratio (PPR, ratio of second PSC divided by first PSC amplitude) as measured in 0.5 mM external calcium at different interpulse intervals (IPSs ranging from 10 ms to 100 ms) in control animals with GFP-tagged Cav2 (cacsfGFP, A), in animals with removal of I-IIA (ΔI-IIAsfGFP, B), and in animals with removal of I-IIB (ΔI-IIBsfGFP, C). (E-G) Synaptic depression as measured in 0.5 mM external calcium in response to stimulus trains of 1 minute duration at 1 Hz frequency for animals with GFP-tagged Cav2 (cacsfGFP, E), following removal of I-IIA (ΔI-IIAsfGFP, F), and with removal of I-IIB (ΔI-IIBsfGFP, G). The upper traces show representative TEVC recordings from the postsynaptic muscle cell, and the diagrams mean values (n=5 for E and F, N=6 for G, error bars are SD). For all 3 genotypes, depression reaches steady state at ∼ 80 % of the original PSC amplitude, but upon excision of I-IIB it is more variable (G) and slower (H) as compared to control and I-IIA excision (E, F, H). (I-L) Synaptic depression in response to stimulus trains at 10 Hz frequency for animals with GFP-tagged Cav2 (cacsfGFP, I), following removal of I-IIA (ΔI-IIAsfGFP, J), and with removal of I-IIB (ΔI- IIBsfGFP, K). Again, depression is most variable between animals upon excision of I-IIB (K, L) and it is slower as compared to control and ΔI-IIA (L). Motoneuron stimulation at 60 (N) or 100 Hz (M) frequency, both for durations of 200 ms in animals with GFP-tagged Cav2 (cacsfGFP, upper traces), following removal of I-IIA (ΔI-IIAsfGFP, middle traces), and with removal of I-IIB (ΔI-IIBsfGFP, lower traces). To compare charge transfer during across the NMJ during high frequency bursts the total PSC area below baseline (prior to stimulation) was measured during each 200 ms burst and plotted for each genotype for 60 Hz stimulation in (O) and for 100 Hz stimulation in (P). (Q) shows single evoked PSC half amplitude width. (R-U) show different measurements during larval crawling for control animals with GFP-tagged Cav2 (cacsfGFP), removal of I-IIA (ΔI-IIAsfGFP), removal of I-IIB (ΔI- IIBsfGFP), and in transheterozygous animals with removal of I-IIA on one and removal of I-IIB on the other chromosome (ΔI-IIAsfGFP/ΔI-IIBsfGFP). The measured parameters are mean speed during 10 minutes of crawling (R), mean speed without any stops (S), the relative time spent stopping (T) and the maximum speed reached (U). In all diagrams each dot demarks a measurement from a different animal and horizontal bars the medians. For statistics, non-parametric Kruskal Wallis ANOVA with planned Dunn’s posthoc comparison to control was conducted.

Removal of I-IIB impairs presynaptic homeostatic potentiation.

(A-D) Presynaptic homeostatic potentiation (PHP) can be induced in control animals with GFP-tagged Cav2 (cacsfGFP) by bath application of the glutamate IIA receptor blocker philanthotoxin (PhTx). As compared to control (A, black traces), bath application of PhTx (A, red traces) reduces the amplitude of miniature postsynaptic currents (B, mEPSCs) significantly, but EPSC amplitude upon evoked synaptic transmission remains unaltered (C) because the number of SVs that are released upon one presynaptic action potential (D, mean quantal content, mQC) is significantly increased to compensate for the smaller postsynaptic response to a given amount of neurotransmitter release. (E-H) PHP is also measured upon PhTx application to animals with removal of I-IIA. (I-L) By contrast, in animals with excision of I-IIB the reduction of mEPSCs by bath application of PhTx (I and J) causes a significant reduction in the amplitude of evoked synaptic transmission (EPSC amplitude, K), because no compensatory upregulation of mQC is observed (L). Therefore, PHP induction requires the I-IIB exon. (M) PHP maintenance is typically assessed in GluRIIA mutants. PHP maintenance is observed in control animals with GFP-tagged Cav2 (cacsfGFP) because mQC is increased in a GluRIIA mutant background (GluRSP16). By contrast, in ΔI-IIB animals the GluRIIA mutant background does not cause an increase in mQC. (N) In animals that are transheterozygous for the removal of I-IIA and the removal of I-IIB, and carry GFP-tagged I-IIA and mEOS4b-tagged I-IIB Cav2, triple immunolabel for the AZ marker brp, GFP, and mEOS4b show that most active zones (cyan) contain both, GFP-tagged I-IIA (green) and mEOS4b-tagged I-IIB (magenta) channels. (O) Quantification shows that >95 % of all brp positive AZs contain I-IIA and I-IIB channels (gray dots), few AZs (∼5 %) contain only I- IIB (magenta), and no AZ contains only I-IIA (orange).

gRNA sequences used for cas9 target site. Vertical lines depict intended break points. Bold and underlined nucleotides indicated PAM (protospacer adjacent motif) sequences.

Cycler settings for exon out verification.

Exon out verification primers