EMBO/soda-nel huggingface dataset:
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 1

Figure 1. Developmental expression and localization of SPTAC1L.

A Immunoblotting of SPATC1L in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). An anti-ADAM2 antibody was used as a control.

B Expression of SPATC1L in human testis lysates (TL) and mature sperm (S). An anti-α-tubulin antibody was used as a control.

C Developmental expression pattern of SPATC1L in testis, examined by immunoblotting using total lysates obtained from prepubertal and adult male mice (days 7, 14-21, 28, 35, 42, 49, and 56). An anti-α-tubulin antibody was used as a control.

D Localization of SPATC1L in germ cells during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and γ-tubulin (green). γ-Tubulin was used as a centrosome marker, and cells were counterstained with the nuclear dye Hoechst 33342 (blue). Fluorescence images of individual staining are shown at the top and merged images are shown in the middle. Insets are magnified iamges. Sc, spermatocyte; RS, round spermatid; ES, elongating spermatid; TS, testicular sperm; MS, epididymal mature sperm; BF, overlay with bright field image. Scale bar = 5 μm.
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 2

Figure 2. Identification of SPATC1L-interacting proteins.

A Immunoprecipitation of SPATC1L in the testis using an anti-SPATC1L antibody. IgG was used as a control. In, input; IgG, normal rabbit serum.

B Proteins associated with SPTAC1L, identified by LC-MS/MS analysis. Proteins immunoprecipitated by an anti-SPATC1L antibody were analyzed by LC-MS/MS. Spec., spectrum count; Seq., sequence count (number of non-redundant peptides identified for each protein); Seq. cov., sequence coverage calculated based on the amino acid count; MW, molecular weight calculated using TurboSEQUEST software.

C Interaction between SPATC1L and PKA subunits. Samples precipitated with an anti-SPATC1L antibody were analyzed by immunoblotting with anti-RIα and anti-Cα antibodies. Immunoprecipitation of SPATC1L was confirmed by immunoblotting with anti-SPATC1L antibody. IgG was used as a control. IP, immunoprecipitation; In, input; IgG, immunoglobulin G from normal rabbit serum.

D Expression of PKA subunits during spermatogenesis, examined by immunoblotting for RIα and Cα in testicular cells (TC), testicular sperm (TS), and epididymal sperm (S). Anti-ADAM2 and anti-α-tubulin antibodies were used as controls.

E Localization of SPATC1L and RIα during spermatogenesis. Isolated spermatogenic cells were immunostained for SPATC1L (red) and RIα (green), and counterstained with the nuclear dye Hoechst 33342 (blue). Overlay with bright field image is shown at right in each cell type. TC, testicular cell (spermatocyte); RS, round spermatid; ES, elongating spermatid; TS, testicular sperm. Scale bar = 5 μm.
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 3

Figure 3. Interaction between SPATC1L and RIα, and PKA activity.

A Schematic diagrams of RIα regions subjected to in vitro binding assays. Results from the RIα-SPATC1L interaction assay (Fig. 3B) are indicated as + or - at right. D/D, dimerization/docking domain (binding site for AKAPs); Domain A, catalytic subunit binding domain and cAMP binding domain; Domain B, cAMP binding domain; IS, inhibitor sequence (binding site for the active site of the catalytic subunit).

B Mapping of the SPATC1L binding site on RIα by in vitro binding assay using purified GST-SPATC1L and His-RIα proteins consisting of regions 1-6. Purified GST protein only was used as a control. Binding was confirmed by immunoprecipitation using an anti-SPATC1L antibody followed by immunoblot analyses of RIα, detected using an anti-His antibody. In, input; Sup, supernatant; IP, immunoprecipitated protein.

C PKA activity after overexpression of SPATC1L or SPATC1LΔC. Graphs show the average PKA activity of Spatc1l-transfected cells (white bar) and Spatc1lΔC-transfected cells (black bar) in the presence or absence of cAMP, expressed relative to control cells (mock) as a percentage. Representative signals observed in the assay are shown above. The overexpression of SPATC1L was confirmed in every experiment by immunoblotting (data not shown). Data are presented as means ± s.d. (n >3; * at least P < 0.05 versus mock, **P < 0.01, Student's t-test).

D The amount of Cα associated with RIα in the presence or absence of SPATC1LΔC. After IP of RIα in cells overexpressing SPATC1LΔC or control, immunoblot analysis was performed using anti-Cα antibody. Graph shows densitometric analysis of Cα in relation to immunoprecipitated RIα in immunoblot analysis . In, input; IgG, immunoglobulin G from normal rabbit serum. Data are presented as means ± s.d. (n = 3; *P < 0.05, Student's t-test).
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 4

Figure 4. Generation of Spatc1l-KO mice and assessment of male fertility.

A Schematic diagram of the Spatc1l gene coding region and region deleted using CRISPR/Cas9. The start codon sequence is labeled in red. Blue arrows indicate the forward and reverse primers used for genotyping. The numbers in boxes indicate exons, and filled boxes show Spatc1l coding regions. The 178-bp sequence deleted in the targeted allele of a Spatc1l-/- mouse (red dots) is indicated in partial sequencing results, shown below.

B PCR genotyping analysis of WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice.

C SPATC1L expression levels in testes obtained from WT, Spatc1l+/-, and Spatc1l-/- mice. An anti-GAPDH antibody was used as a control. Graph shows densitometric analysis of SPATC1L in relation to GAPDH in immunoblot analysis. Data are presented a means ± s.d. (n=3, *P < 0.001, Student's t-test).

D Percentage of females that became pregnant after mating with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice. Male mice used: WT, n = 5; Spatc1l+/-, n = 6; Spatc1l-/-, n = 7. WT female mice used for mating with the indicated genotype: WT, n = 30; Spatc1l+/-, n = 36; Spatc1l-/-, n = 42. Vaginal plugs were observed in all mated females. Data are presented a means ± s.d. (*P < 0.001, Student's t-test).

E Average litter size of pups produced by females mated with WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) male mice. Data are presented a means ± s.d. (*P < 0.001, Student's t-test).
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 5

Figure 5. Phenotypic analyses of Spatc1l-KO mice.

A Macroscopic appearance of adult testes from an 8-week-old WT (Spatc1l+/+), heterozygous (Spatc1l+/-) and homozygous (Spatc1l-/-) mice. Scale bar = 2 mm.

B Comparison of testis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The organs were trimmed of fat and weighed. The average testis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.0034±0.0001, 0.0038±0.0003, and 0.0031±0.0002, respectively. Data are presented as means ± s.d. (n = 7).

C Macroscopic appearance of the adult epididymis from WT, Spatc1l+/-, and Spatc1l-/- mice. Scale bar = 2 mm.

D Epididymis weight in WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. The average epididymis weight/body weight ratios for WT, Spatc1l+/-, and Spatc1l-/- males were 0.001±0.0001, 0.001±0.0001, and 0.001±0.00008, respectively. Data are presented as means ± s.d. (n = 7).

E Histological analysis of hematoxylin and eosin-stained sections of seminiferous tubules prepared from testes of WT and KO mice. Scale bar = 100 μm (upper) and 50 μm (lower).

F Number of mature epididymal sperm from WT (white bar), Spatc1l+/- (gray bar), and Spatc1l-/- (black bar) mice. Sperm were collected from the vas deferens. The average number of mature sperm in WT, Spatc1l+/-, and Spatc1l-/- males was (4.06 ± 0.40) × 106, (3.7 ± 0.7) × 106, and (3.93 ± 0.90) × 106, respectively. Data are presented as means ± s.d. (n = 5).

G Morphological analyses of epididymal sperm in WT, He and KO mice. Epididymal sperm were observed under an inverted microscope. The arrows indicate separated tails, black arrowheads indicate separated heads and white arrowheads indicate folded heads. Scale bar = 50 μm.

H Percentage of headless sperm from cauda epididymides of WT (black), Spatc1l+/- (gray), and Spatc1l-/- (white) mice. The average percentage of headless sperm in WT, Spatc1l+/-, and Spatc1l-/- males was 6.3, 52.9 and 100, respectively. Each dot represents percentage of headless sperm in an individual mouse, and medians are represented as horizontal lines. (n=13, *P < 0.001, Student's t-test).

I Immunostaining analysis of sperm heads and tails in WT and KO mice. Sperm tails were observed by immunostaining for α-tubulin antibody (red), and counterstaining with the nuclear dye Hoechst 33342 (blue). Sperm were also observed by differential interference contrast (DIC) microscopy. Scale bars = 20 μm (upper) and 5 μm (lower).
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 6

Figure 6. Ultrastructures of sperm in Spatc1l-KO mice.

A Morphological analyses of Spatc1l-KO epididymal sperm by SEM. Sperm were obtained from the vas deferens of 8-week-old WT (Spatc1l+/+) and KO (Spatc1l-/-) mice. Sperm neck regions are magnified in lower panels. AC, acrosomal cap; MP, mid piece; BP, basal plates. Scale bar = 2 μm (upper) and 0.5 μm (lower).

B TEM analyses of flagella in WT and KO testicular sperm. TEM images represent ultrastructures of cross-sections of the midpiece (upper) and end piece (lower) of WT and KO testicular sperm. Ax, axonemal microtubules; Mt, mitochondrial sheath; ODF, outer dense fibers. Scale bars = 200 nm (upper) and 100 nm (lower).

C TEM analyses of the development of the connecting piece in WT (left) and KO (right) sperm. TEM images represent ultrastructures of longitudinal sections of elongating spermatids (ES) and testicular sperm (TS) in WT and KO mice. An, annulus; BP, basal plates; Cp, capitulum; Mc, manchette; Mt, mitochondria; Nu, nucleus; PCV, proximal centriolar vault; PM, plasma membrane; SC, segmented column. Scale bar = 500 nm.

D Ultrastructures of epididymal sperm in KO mice. TEM images represent ultrastructures of separated sperm head (upper) and tail (lower) in the epididymis of KO mice. Images in the boxes are enlarged at right. BP, basal plates; Cp, capitulum; SC, segmented column. Scale bar = 500 nm.

E TEM analysis of morphologically abnormal KO sperm. Detached heads abnormally located to the midpiece of sperm flagella were observed by TEM (left) and confocal differential interference contrast (DIC) microscopy (right). PM, plasma membrane. Scale bars = 500 nm (left) and 5 μm (right).
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 7

Figure 7. Identification of CAPZB as a candidate target of phosphorylation by PKA in the neck region of testicular sperm.

A PKA activities in testicular sperm from WT (+/+) and KO (-/-) mice. Graph shows average PKA activity in testicular sperm from KO mice (black bar) relative to that from WT mice (white bar), expressed as a percentage. Data are presented as means ± s.d. (n = 3; *P < 0.05, Student's t-test).

B Representative ProQ Diamond-stained 2D gel images of isolated proteins from testes in WT (left) and KO (right) mice. Testes lysates were separated by 2DE, and phosphoproteins were stained with ProQ diamond. Images in the boxes are enlarged in insets shown below. Circles with numbers indicate spots with significantly changed volume relative to those on WT gels.

C Comparative phosphorylation intensities of selected spots in 2D gels. The intensities of selected spots in KO gels were compared with intensities in WT gels. Average intensities of the indicated spot numbers (#) in WT (white bar) and KO (black bar): #1205, 1092.2 (WT) and 244.98 (KO); #1207, 729.9 (WT) and 140.43 (KO); #3303, 1165.5 (WT) and 279.5 (KO); #4302, 2725.8 (WT) and 1 (KO); #4405, 984.5 (WT) and 1 (KO). An intensity value of 1 indicates no spot, but is provided for calculation of fold change. Data are presented as means ± s.d. (n = 3; *P < 0.05, **P < 0.001, Student's t-test).

D Identification of proteins with altered phosphorylation patterns in Spatc1l-KO testis by PMF. SS, standard spot; Seq. cov., sequence coverage calculated based on the amino acid count; MW, molecular weight; pI, isoelectric point.

E, F Immunoblot analyses of five candidate proteins in testis (E) and sperm (F) of WT and KO mice. Anti-GAPDH, anti-ADAM2, and anti-α-tubulin antibodies were used as controls. Loss of SPATC1L in KO testis was confirmed by immunoblotting with anti-SPATC1L antibody.

G Localization of SPATC1L (red) and CAPZB (green) in testicular sperm. Overlay with bright field image is shown at right. The white arrow indicates the spatial overlap between SPATC1L and CAPZB. Scale bar = 5 μm.
SPATC1L maintains the integrity of the sperm head-tail junction (2018)
Figure 8

Figure 8. Characterization of phosphorylation in CAPZB by PKA.

A The amount of CAPZB in mature sperm from WT (+/+) and KO (-/-) mice. Graph shows densitometric analysis of CAPZB shown in immunoblot analyses (Figure 7F), expressed relative to WT as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of α-Tubulin. Data are presented a means ± s.d. (n=3, *P < 0.001, Student's t-test).

B The amount of CAPZB in mock and Spatc1lΔC-transfected cells. Anti-GAPDH antibody was used as control. Overexpression of SPATC1L in TM4 cells was confirmed by immunoblotting with anti-SPATC1L antibody. Graph shows densitometric analysis of CAPZB in pcDNA3.1 vector-transfected control cells (mock; white bar) and SPATC1LΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. The density of CAPZB in immunoblot analysis was calculated in relation to density of GAPDH. Data are presented a means ± s.d. (n=3, *P < 0.001, Student's t-test).

C Phosphorylation of CPAZB3 by cAMP-dependent protein kinase (PKA). CAPZB3 was incubated with PKA and 32P, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower).

D Phosphorylation of CPAZB3 in the presence or absence of SPATC1L. Phosphorylation of purified CAPZB3 was labeled with 32P via PKA in the presence or absence of SPATC1L, and estimated by autoradiography. The amount of CAPZB3 protein was quantified by Coomassie Brilliant Blue (CBB) staining (lower). Graph shows densitometric analysis of phopho-CAPZB3 (p-CAPZB3) in reactions without (control; white bar) or with SPATC1L (black bar), expressed relative to control as a percentage. Data are presented a means ± s.d. (n=4, *P < 0.001, Student's t-test).

E Observation of filamentous actin (F-actin) in the presence (lower) or absence (upper) of SPATC1L. F-actin in pEGFP vector-transfected control cells (mock; upper, green) and cells overexpressing SPATC1LΔC (lower, green) was visualized by staining using Phalloidin (red). White bar shows the length of transfected cell. Scale bar = 50 μm.

F The length of cell overexpressing SPATC1LΔC compared with control. Graph shows the average of measured cell length in pEGFP vector-transfected control cells (mock; white bar) and Spatc1lΔC-transfected cells (black bar), expressed relative to control cells (mock) as a percentage. Data are presented as means ± s.d. (n = 3; *P < 0.001, Student's t-test).

G Proposed model for the function of SPATC1L in maintaining the stability of the sperm head-tail junction. SPATC1L is localized to the centrosome in a round spermatid, but separated from the centrosome in an elongating seprmatid. Finally, SPATC1L is localized to the neck region close to the centriole in testicular sperm. SPATC1L acts through interactions with RIα to increase PKA activity, and thereby increases the phosphorylation of CAPZB and stability of F-actin responsible for the integrity of the head-tail junction.

Expanded View Figure Legends
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 1

Figure 1 - amiRSUL moves intercellularly, and over long-distances via sieve elements

(A) Schematized pSUC2::amiRSUL reporter. T35S: CaMV 35S terminator.

(B) amiRSUL-silencing phenotype in pSUC2::amiRSUL transgenic- versus WT- Arabidopsis.

(C) GUS-staining pattern in pSUC2::GUS transcriptional reporter plants.

(D) RT-qPCR analysis of SUL mRNA levels in WT or pSUC2::amiRSUL (amiR) rootstocks grafted onto WT or amiR scions. Error-bars: SD. Welch's t-test p-value are indicated. n=5.

(E) amiRSUL northern analysis in WT or amiR scions (S) or rootstocks (R) in the indicated grafting combinations. U6 RNA hybridization provides an internal RNA loading control.

(F) Top: aphids' feeding-stylets selectively reach the phloem sieve-elements. Bottom: amiRSUL northern analysis, in biological duplicates, from total RNA extracted 14 days-post-infestation from aphids fed on WT or amiR plants. U6: as in (E).

(G) Same as (E) with amiRhen1-14 scions and non-transgenic WT or hen1-14 rootstocks. Average relative U6-normalized band-intensity quantifications from two biological replicates are indicated.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 2

Figure 2 - hst suppresses amiRSUL-silencing

(A) HST genomic sequence spanning exons 11-to-13 and position of the hst-1/-25/-26 mutations. EMS-induced nucleotide transitions (arrows) in hst-25 and hst-26 are in red; upper-/lower-case fonts: exonic/intronic sequences.

(B) HST western analysis, in biological duplicates, in the indicated genotypes. Coomassie blue staining (Coom.) provides a control for total protein loading.

(C) Phenotype of pSUC2::amiRSUL plants in hst-1/-25/-26 backgrounds.

(D) RT-qPCR analysis of SUL mRNA levels in leaves with the indicated genotypes. Error-bars: SD. Welch's t-test p-values are indicated. n=5.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 3

Figure 3 - amiRSUL levels are reduced in hst, unlike its loading into AGO1 or its silencing activity

(A) Northern analysis of indicated miRNAs, in biological duplicates, in leaves with the indicated genotypes. U6 was probed as endogenous control. Average relative U6-normalized band-intensity quantifications are indicated.

(B) Left: silencing phenotype of homozygous (+/+) versus hemizygous (+/-) pSUC2::amiRSUL plants. Right: RT-qPCR analysis of SUL mRNA levels in leaves of (+/+) or (+/-) pSUC2::amiRSUL versus WT Arabidopsis. Error-bars: SD. t-test p-values are indicated. n=3.

(C) amiRSUL northern analysis, in biological triplicates, in leaves of (B). miR159 and U6 were probed as endogenous controls. Band-intensity quantifications: as in (A).

(D) amiRSUL northern analysis in input and AGO1-immunoprecipitated (AGO1-IP) fractions isolated from leaves of pSUC2::amiRSUL plants in the specified genotypes. No Ab: No antibody added to the HST input extract (IP negative control). miR165/166 and U6 were probed as endogenous controls. AGO1 western analysis and Coomassie blue (Coom.) staining of the western blot membrane are provided as controls. amiRSUL band-intensity quantifications, U6-normalized for input samples, are indicated.

(E) amiRSUL northern analysis in pSUC2::amiRSUL (amiR) scions (S) and non-transgenic WT and hst-1 rootstocks (R) in the indicated grafting combinations. U6: as in (A).

(F) RT-qPCR analysis of SUL mRNA levels in WT and hst-1 rootstocks grafted onto WT or amiR scion. Error-bars: SD. Tukey's multiple comparisons test p-values are indicated. n=4.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 4

Figure 4 - HST is required for amiRSUL cell-to-cell and long-distance movement

(A) amiRSUL northern analysis, in two independent replicates (rep.), in Meselect-separated leaf-vasculature versus -epidermis of pSUC2::amiRSUL plants with the specified genotypes (see also Appendix Fig.S12A). An enhanced contrast is shown for the epidermis. miR165/166 and U6 were probed as endogenous controls. U6-normalized band-intensity quantifications relative to HST are indicated for each tissue.

(B) Average epidermis/vasculature ratio of amiRSUL levels presented in (A) and Appendix Fig.S12A. Error-bars: SD. t-test p-values are indicated. n=4.

(C) amiRSUL northern analysis, in biological duplicates, in scions (S) and rootstocks (R) in the specified genotypes' grafting combinations (amiR: pSUC2::amiRSUL) (see also Appendix Fig.S12B). U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated for each tissue.

(D) Average rootstock/scion ratio of amiRSUL levels as measured in (C), Fig.1G, Appendix Fig.S3A and Fig.S12B. Error-bars: SD. t-test p-values are indicated. n≥4.

(E) amiRSUL northern analysis, in biological duplicates, from total RNA extracted from aphids fed on WT or amiR plants, in the specified genetic backgrounds. U6: as in (A). Average relative U6-normalized band-intensity quantifications are indicated.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 5

Figure 5 - A cell-autonomous activity of HST enables movement of amiRSUL, but not of free GFP

(A) Representative vein-chlorosis phenotype caused by the genetic mosaic rescue of pSUC2::HST:GFP co-expressed in pSUC2::amiRSULhst-1 plants (see also Appendix Fig.S11A).

(B) Bright field and GFP fluorescence images of pSUC2::GFP and pSUC2::tmGFP9 seedlings.

(C-D) Localization of HST:GFP and, as a reference, of tmGFP9 expressed under the pSUC2 promoter in Arabidopsis primary leaves (C) or roots (D). CW: calcofluor white staining. Scale bars: 20 µm.

(E) Confocal fluorescence images of the differentiation and division zones of Arabidopsis root tips expressing pSUC2::HST:GFP, pSUC2::GFP or pSUC2::tmGFP9. Scale bars: 100 µm.

(F) Bright field and GFP fluorescence images of pSUC2::GFPhst-1 and pSUC2::tmGFP9hst-1 seedlings.

(G) Confocal fluorescence images of the differentiation and division zones of pSUC2::GFPhst-1 or pSUC2::tmGFP9hst-1 root tips. Scale bars: 100 µm.
HASTY, the Arabidopsis EXPORTIN5 ortholog, regulates cell-to-cell and vascular microRNA movement (2021)
Figure 6

Figure 6 - HST's nucleo-cytosolic shuttling is dispensable for its control of amiRSUL movement

(A) Subcellular localizations of HST:GFP expressed from the UBQ10 promoter (pUBQ) in Arabidopsis roots (i). Distinct planes of the same root cells imaged by confocal microscopy are shown (ii-iii). PI: propidium iodide staining. Scale bars: 50µm (i); 10µm (ii-iii).

(B) Phenotype of pSUC2::amiRSUL in the HST or hst-3 background.

(C) Subcellular localizations of hst-3:GFP expressed from pUBQ in root cells. Distinct planes of the same root cells imaged by confocal microscopy are shown (i-ii). PI: as in (A). Scale bars: 10µm.

(D) Phenotype of SUC-SUL plants expressing, from the pSUC2 promoter, an inverted-repeat transgene producing siRNA populations targeted against SUL, in the HST or hst-1 background.