Heterotrimeric Kinesin II is required for flagellar assembly and elongation of nuclear morphology during spermiogenesis in Schmidtea mediterranea

Abstract

Development of sperm requires microtubule-based movements that drive assembly of a compact head and flagellated tails. Much is known about how flagella are built given their shared molecular core with motile cilia, but less is known about the mechanisms that shape the sperm head. The Kinesin Superfamily Protein 3A (KIF3A) pairs off with a second motor protein (KIF3B) and the Kinesin Associated Protein 3 (KAP3) to form Heterotrimeric Kinesin II. This complex drives intraflagellar transport (IFT) along microtubules during ciliary assembly. We show that KIF3A and KAP3 orthologs in Schmidtea mediterranea are required for axonemal assembly and nuclear elongation during spermiogenesis. Expression of Smed-KAP3 is enriched during planarian spermatogenesis with transcript abundance peaking in spermatocyte and spermatid cells. Disruption of Smed-kif3A or Smed-KAP3 expression by RNA-interference results in loss of spermatozoa and accumulation of unelongated spermatids. Confocal microscopy of planarian testis lobes stained with alpha-tubulin antibodies revealed that spermatids with disrupted Kinesin II function fail to assemble flagella, and visualization with 4',6-diamidino-2-phenylindole (DAPI) revealed reduced nuclear elongation. Disruption of Smed-kif3A or Smed-KAP3 expression also resulted in edema, reduced locomotion, and loss of epidermal cilia, which corroborates with somatic phenotypes previously reported for Smed-kif3B. These findings demonstrate that heterotrimeric Kinesin II drives assembly of cilia and flagella, as well as rearrangements of nuclear morphology in developing sperm. Prolonged activity of heterotrimeric Kinesin II in manchette-like structures with extended presence during spermiogenesis is hypothesized to result in the exaggerated nuclear elongation observed in sperm of turbellarians and other lophotrochozoans.

Keywords: Cilia; Flagella; Heterotrimeric kinesin II; Manchette; Planarian; Spermatogenesis; Spermiogenesis.

Figure 1.. Smed-KAP3 is required for sperm development in S. mediterranea.

(A) Whole-mount DAPI-stained control sexual planarian viewed by fluorescence stereomicroscopy reveals areas of high cellular density, such as the pharynx and dorso-lateral testis lobes. Sperm accumulated in the copulatory apparatus is also visible in this sample. (B) Single confocal section reveals normal anatomy of developing cells in testis lobes of DAPI-stained planarians after four weeks of luciferase dsRNA feedings. Inset shows 2-fold magnified view of round spermatids (r.spd), elongating spermatids (e.spd), and spermatozoa (spz) in the inner lumen of a testis lobe. (C) Reverse transcription quantitative PCR results showing effective reduction of targeted mRNA levels in planarians subjected to Smed-kifC3-like, Smed-KAP3, or Smed-ARMC4-like dsRNA, but not in planarians fed Smed-kif17-like dsRNA. (D-F) Nuclear morphology comparable to control animals (B) is observed in testis lobes of Smed-kifC3-like (D) and Smed-ARMC4-like (F) knockdowns, including filamentous nuclei in spermatozoa that are largely absent in testis lobes of Smed-KAP3 RNAi samples (E). The fraction of biological replicates that displayed the phenotype represented by each image is shown in parenthesis. Scale bars = 1 mm in A, 50 μm in B, D-F.

Figure 2.. Smed-KAP3 expression is enriched during sperm development.

(A) Whole-mount in situ hybridization analysis in sexual planarian shows enriched Smed-KAP3 expression in testis lobes (5x magnified view inset). (B) Parallel analysis using luciferase sequence riboprobe shows background signal. (C-E) Confocal sections of samples analyzed by double fluorescence in situ hybridization using Smed-KAP3 (green; C’-C”; D’-D”; and E’-E”) riboprobes with markers (magenta) of spermatogonia (gH4; C and C”), spermatocytes (tkn-1; D and D”), and spermatids (pka, E and E”). DAPI staining is shown in blue. Scale bar = 1 mm in A-B, and 50 μm in C-E.

Figure 3.. Smed-KAP3 is required for spermatid elongation and flagellar assembly.

(A) Illustration of processing steps for high-magnification analysis of spermatogenesis by DAPI and acetylated alpha-Tubulin (AcTub) staining of isolated testis lobes. (B-C) Representative image of filamentous structures of spermatozoa nuclei (B and B”; arrows) and flagella (B’ and B”; asterisks) observed in testis lobes of luciferase(RNAi) planarians stained with DAPI (blue in B” and C”) and anti-acetylated alpha-tubulin (green in B” and C”), whereas only partially elongated nuclear structures (C and C”; arrowheads) and short flagella (C’ and C”) were observed in lobes of Smed-KAP3(RNAi) planarians after 4-weeks of RNAi. Insets show 3x-magnified views. Scale bar = 10 μm. (D) Depiction of normal steps in progression of planarian spermiogenesis and failure to progress through spermatid elongation upon Smed-KAP3 RNAi.

Figure 4.. Spermatid elongation defects of Smed-KAP3(RNAi) are not upon knockdown of Smed-DAW1, a regulator of ciliary function in the planarian soma.

(A-C) Assessment of planarian motility in luciferase(RNAi) (A), Smed-DAW1(RNAi) (B), and Smed-KAP3(RNAi) (C), reveals progression from normal (green) to slower (yellow) and inchworm (red) phenotype develop first in Smed-DAW1(RNAi) (B) and later in Smed-KAP3(RNAi) (C). The y-axis denotes the percent (%) of animals (n = 13 to 14) per group scored under the different phenotypes. The x-axis represents days into the RNAi treatment starting with the day of the first feeding (1). (D-F) Single confocal section reveals normal nuclear anatomy of developing cells in testis lobes of DAPI-stained luciferase(RNAi) (D) and Smed-DAW1(RNAi) (E) planarians, but loss of elongated structures in Smed-KAP3(RNAi) (F). Parentheses in (D-F) show the fraction of sexually mature animals (defined as per presence of copulatory structures) from the groups analyzed in (A-C) upon fixation on day 26 from the initial dsRNA feeding that show the phenotype displayed. Scale bar = 50 μm.

Figure 5.. Spermatid elongation defects in Smed-KAP3(RNAi) precede collapse of mitotic divisions in testis lobes.

(A-C) Maximal projection of partial z-stack confocal images of testis lobes from luciferase(RNAi) (A) and Smed-KAP3(RNAi) (B-C) stained with DAPI (blue) and anti-PH3 after 4 (A and B) and 6-week (C) RNAi treatments. (D-E) Quantification of PH3(+) cysts classified as composed of 1–2, 4, 8, or 16 cells testis lobes of luciferase(RNAi) and Smed-KAP3(RNAi) after four (D) and six (E) weeks of RNAi. Each timepoint includes analysis of 18 to 27 testis lobes visualized from each of at least four biological samples by confocal z-stack imaging throughout as much of the samples as technically feasible. Bars show the mean and error bars the standard deviation. Asterisks indicate p < 0.01, Student’s t-test).

Figure 6.. Smed-kif3A(RNAi) phenocopies Smed-KAP3(RNAi).

(A) Confocal section of samples analyzed by double fluorescence in situ hybridization using Smed-kif3A (green; A’ and A”’) and Smed-KAP3 (magenta; A” and A”’) riboprobes. DAPI staining shows nuclear structures of developing sperm (white; A-A”’). Insets show 3-fold magnified views. (B-C) Single confocal sections of whole-mount samples stained with DAPI reveal morphology of developing sperm in testis lobes of luciferase(RNAi) (B) and Smed-kif3A(RNAi) (C) planarians fixed after 4-weeks of RNAi. Filamentous nuclear structures of spermatozoa (B, inset) are largely absent in testis lobes of Smed-kif3A(RNAi) (C, inset). Insets show 2-fold magnified views. The fraction of biological replicates showing the phenotype displayed is shown in parenthesis (B and C). (D) RT-qPCR measurement of Smed-kif3A mRNA abundance relative to levels in luciferase(RNAi) shows efficacy of Smed-kif3A RNAi. Scale bars = 50 μm.