Filamin and phospholipase C-ε are required for calcium signaling in the Caenorhabditis elegans spermatheca

Abstract

The Caenorhabditis elegans spermatheca is a myoepithelial tube that stores sperm and undergoes cycles of stretching and constriction as oocytes enter, are fertilized, and exit into the uterus. FLN-1/filamin, a stretch-sensitive structural and signaling scaffold, and PLC-1/phospholipase C-ε, an enzyme that generates the second messenger IP3, are required for embryos to exit normally after fertilization. Using GCaMP, a genetically encoded calcium indicator, we show that entry of an oocyte into the spermatheca initiates a distinctive series of IP3-dependent calcium oscillations that propagate across the tissue via gap junctions and lead to constriction of the spermatheca. PLC-1 is required for the calcium release mechanism triggered by oocyte entry, and FLN-1 is required for timely initiation of the calcium oscillations. INX-12, a gap junction subunit, coordinates propagation of the calcium transients across the spermatheca. Gain-of-function mutations in ITR-1/IP3R, an IP3-dependent calcium channel, and loss-of-function mutations in LFE-2, a negative regulator of IP3 signaling, increase calcium release and suppress the exit defect in filamin-deficient animals. We further demonstrate that a regulatory cassette consisting of MEL-11/myosin phosphatase and NMY-1/non-muscle myosin is required for coordinated contraction of the spermatheca. In summary, this study answers long-standing questions concerning calcium signaling dynamics in the C. elegans spermatheca and suggests FLN-1 is needed in response to oocyte entry to trigger calcium release and coordinated contraction of the spermathecal tissue.

Figure 1. fln-1(tm545) brood size defect is suppressed by increased IP₃ signaling.

Brood sizes of fln-1(tm545) animals carrying itr-1(gf) and lfe-2(lf) mutations. The genotypes are indicated along the y-axis. Points represent individual animals, the bar indicates the mean, and the error bar is the standard deviation. Student's t-test was used to compare the genotypes, and p values are indicated with *** for p = 0.0001.

Figure 2. Oocyte entry into the spermatheca triggers calcium oscillations.

(A–B′) A representative first ovulation in xbIs1101[fln-1p::GCaMP] (n = 26) transgenic animals. (A) A plot of GCaMP mean pixel intensity normalized to the pre-ovulation mean pixel intensity is shown, along with a (A′) corresponding kymogram at the same time scale. The top and bottom of the kymogram represent the distal and proximal spermatheca, respectively. Darker regions indicate higher calcium levels. Oocyte entry begins at 30 seconds and is complete by 60 seconds, and the zygote exit is marked by a magenta arrowhead in (A) and is visible in (A′) as a reduction in the size of the spermatheca. (A′) The leftward tilt of dark stripes is indicative of calcium transient propagation from the distal spermatheca. (B–B′) A detailed view of several calcium transients is shown as a (B) trace and (B′) kymogram from the region indicated by the blue line in (A). (B) GCaMP fluorescence intensity was quantified in 1 µm strips (distal is black, and proximal is gray), 50 µm apart as indicated in (B′). The mean peak-to-peak transit time across the spermatheca is 8±1 seconds and is indicated on the trace in (B). (C) A representative first ovulation in xbEx0811[fln-1p::GFP] (n = 6) transgenic animals. The fln-1p::GFP signal does not change during oocyte entry or spermathecal transit. (C′) A corresponding kymogram of fln-1p::GFP spermathecal transit with oocyte entry and sp-ut valve dilation visible as dark horizontal stripes.

Figure 3. Gap junction subunit INX-12 is required for spermathecal coordination.

(A) A representative calcium signal trace observed during the first ovulation in inx-12(RNAi) (n = 4) animals imaged with xbIs1101[fln-1p::GCaMP]. The oocyte fails to exit into the uterus and changes direction thrice before returning into the ovary. The arrows indicate direction of movement with blue arrows indicating movement towards the uterus. (A′) Arrowheads in the corresponding kymogram indicate bursts of calcium and correspond to the contractions that stop the oocyte from being refluxed into the ovary. In the kymogram, darker values indicate higher calcium levels. Top is distal, bottom is proximal, and the kymogram is at the same time scale as the calcium trace shown above it.

Figure 4. FLN-1 and PLC-1 are required for different aspects of calcium signaling.

Representative calcium signal traces observed during the first ovulation in (A) fln-1(tm545) (n = 19), (B) plc-1(rx1) (n = 5), and (C) fln-1(tm545); plc-1(rx1) (n = 3) animals imaged using xbIs1101[fln-1p::GCaMP]. Corresponding kymograms (A′–C′) are also shown. In the kymograms, darker values indicate higher calcium levels. Top is distal, bottom is proximal, and each kymogram is at the same time scale as the calcium trace shown above it.

Figure 5. itr-1(sa73ts) disrupts calcium signaling at the semi-permissive temperature.

Representative calcium signaling traces in itr-1(sa73ts) animals at the semi-permissive temperature of 20°C imaged with xbIs1101[fln-1p::GCaMP]. (A) At the non-permissive temperature, 66% (n = 6) of the itr-1(sa73ts) successfully propelled oocytes from the spermatheca into the uterus. (B) The remaining ovulations resulted in embryos trapped within the spermatheca. Corresponding kymograms (A′ and B′) are also shown. In the kymograms, darker values indicate higher calcium levels. Top is distal, bottom is proximal, and each kymogram is at the same time scale as the calcium trace shown above it. In both groups the calcium pulse intensity was markedly reduced compared to wildtype animals. In all cases the itr-1(sa73ts) ovulations resulted in a portion of the oocyte being pinched off during ovulation.

Figure 6. Increased IP₃ signaling suppresses the fln-1(tm545) calcium signaling defect.

Representative calcium signal traces observed during the first ovulation in (A) fln-1(tm545) itr-1(sy290gf) (n = 3) and (B) fln-1(tm545); lfe-2(sy326) (n = 3) animals imaged with xbIs1101[fln-1p::GCaMP]. The corresponding kymograms (A′ and B′) are shown below the plots. Arrowheads indicate the beginning of partial exit of the embryo from the spermatheca. In the kymograms, darker values indicate higher calcium levels. Top is distal, bottom is proximal, and each kymogram is at the same time scale as the calcium trace shown above it.

Figure 7. NMY-1 is required for spermathecal contractility.

The spermatheca appears compact before the first ovulation (A, C, E), however, the sp-ut valve is constricted only in (A′) wildtype animals but not in (C′) fln-1(tm545) and (E′) nmy-1(RNAi) animals. (B) The wildtype spermatheca and (B′) sp-ut valve fully expel the embryo and re-constrict following every ovulation. In contrast, (D) fln-1(tm545) and (F) nmy-1(RNAi) spermathecae retain embryos, and the sp-ut valves (D′ and F′, respectively) remain open. The spermatheca is labeled with ezIs2[fkh-6p::GFP] (images merged with DIC) and the sp-ut valve is labeled with xbEx1019[tag-312p::GFP]. Arrowheads indicate the sp-ut valve. Vertical lines next to the sp-ut valves indicate the width of the opening. The scale bar indicates 25 µm.

Figure 8. MEL-11 modulates spermathecal contractility.

(A) mel-11(RNAi) results in hyper-constriction and rupture of the spermatheca. The asterisk indicates an escaped embryo and arrowheads indicate cell fragments in the pseudocoelom. mel-11(RNAi) in the (B) fln-1(tm545) or (C) plc-1(rx1) genetic background does not result in spermathecal rupture. The spermatheca is labeled with ezIs2[fkh-6p::GFP] and the fluorescent image is merged with DIC. The scale bar indicates 25 µm.

Figure 9. Proposed genetic control of calcium signaling in the spermatheca.

The diagram represents a working model of the spermathecal calcium signaling pathway. Green lines indicate interactions that promote cell contraction, and magenta lines indicate those which inhibit contraction. Dashed lines indicate possible regulatory interactions. We propose FLN-1 acts upstream or in parallel to PLC-1 to initiate calcium release via ITR-1. FLN-1 also plays a downstream role in contraction and maintenance of the F-actin cytoskeleton.