Native Intranuclear Localization of ACTL7B in Mouse Spermatocytes and Spermatids.

(A-B) Widefield fluorescent microscopy showing intracellular localization of ACTL7B in spermatids (A) and spermatocytes (B) counterstained with DAPI and PNA for nuclear and acrosomal visualization. Subpanels show the fluorescent signal of ACTL7B as a heatmap gradient to better demarcate intracellular zones of ACTL7B enrichment. (C-E) Confocal microscopy images of round spermatids at different developmental stages, in the focal plane with the largest nuclear diameter selected for each cell to ensure intranuclear cross section. Localization of ACTL7B (yellow) is enriched in the nucleus (Blue=DAPI stained DNA) and subacrosomal space (Green= acrosomal PNA). Dashed rectangles in each image indicate the 25-pixel wide line-scan analysis area sampled in each cell to generate the signal plots to the right. Signal plots show the fluorescent intensity of each channel as Relative Fluorescent Units (RFU) across the distance (um) of each line scan. Vertical dashed lines in each signal plot demarcate the nuclear boundary within each cell. All white scalebars per image represent 2.5 um.

Highly Conserved Lys/Arg regions in ACTL7B Identified as Putative Nuclear Localization Signals (NLS).

Two highly conserved Lys/Arg rich sequences were identified through multi-species sequence alignment of ACTL7B and hypothesized to be adequate nuclear localization signal (NLS) candidates. All lysine [K] and arginine [R] residues are highlighted in green for both alignment regions to illustrate sequence conservation and Lys/Arg enrichment regions. The regions of both NLS candidates are mapped onto the predicted structure of human ACTL7B with the color coordinated highlighting of residues showing NLS candidate 1 to be in the N-terminus domain (Red) of ACTL7B while NLS candidate 2 mapped to an external α-helix of subdomain 4 (Blue) of the actin body. Calculated isoelectric points, pH(I), are displaced for both sequences showing general basicity.

The Actin Body of ACTL7B is Responsible for its Nuclear Affinity.

(A-B) Suspension culture HEK293F cells transfected to express YFP conjugated full length human ACTL7B (A), and YFP conjugated to the actin body only of human ACTL7B (B). Both full length and the actin domain of ACTL7B show a strong nuclear affinity as illustrated by the innate fluorescence co-localization of their conjugated YFP (yellow) with DAPI stained nuclei (blue). (C-D) Suspension culture HEK293F cells transfected to express YFP conjugated to the N-terminal domain only of human ACTL7B (C), or YFP alone as a negative control (D). The YFP control and YFP conjugated ACTL7B N-terminus expression patterns show similar cytoplasmic localization indicating no observable NLS activity from the over-expressed N-terminus of ACTL7B. Images are representative of triplicate cultures for all conditions. All white scalebars per image represent 10 ums.

ACTL7B NLS Candidate 2 Induces Intranuclear Localization inHEK293F Cells.

(A-B) Suspension culture HEK293F cells transfected to express YFP conjugated to the NLS of Simian Polyomavirus 40 antigen T [PKKKRKV] (SV40) as a positive control for intranuclear localization. Lateral subpanels deconstruct the merged image to its individual channels showing clear nuclear localization of the YFP fusion protein product. (C-D) Suspension culture HEK293F cells transfected to express YFP conjugated with a 25 residue peptide containing the second NLS candidate of ACTL7B [242-266]. The fluorescent signal of each channel is individually displayed as lateral panels with the YFP-7B NLS channel colored as a heatmap gradient showing both transient cytoplasmic expression and a highly enriched intranuclear signal.

Actl7B Ablation Includes Gross Transcriptomic Consequences Including Altered Transcripts in Protease Inhibition, Inflammation, and Immune Response.

(A) Volcano plot illustrating the transcriptomic differences between WT and Actl7b -/- murine testes. Fold change (FC) cut-offs are indicated as vertical lines at the -1 Log2(FC) position for downregulated genes (light blue) and the +1 Log2(FC) position for upregulated genes (salmon pink). Altered genes belonging to specific cellular mechanisms are color coordinated based on the provided key. (B-C) Gene ontology pathway analysis charts for cellular components (B) and biological processes (C) indicate unique categories for the altered genes between genotypes indicating gene count as circle size and significance of enrichment (p-value) as circle color ranging from a gradient scale of blue to red.

Loss of Actl7B Leads to Transcriptional Changes of General and Germline-Specific Transcription Factors and ARPs.

(A) Heatmap of the top 2500 genes with the highest absolute fold change (FC) between WT and Actl7b -/- murine testes showing general transcriptional homogeneity between individuals of the same genotype. (B) Heatmap of all transcription factors with an absolute FC greater than 2 that were differentially expressed between genotypes. Heatmap is arranged in decreasing order of total FC per transcription factor. (C) Violin plots illustrating a trend in decreasing expression oftranscription factors previously identified as germ-line specific (Green et al., 2018) across developmentally progressive spermatogenic cell types caused by the absence of Actl7b; spermatogonia (SG); preleptotene (PL); spermatocyte (SC); round spermatids (RS); and elongating spermatids (ES). (D) List of testis-specific/enriched ARP genes (Top), and somatic nucleosome remodeling ARP genes (bottom) indicating their expected transcriptional prevalence in transcripts per million (TPM) in testes, and most enriched male germ cell (Robertson et al., 2020), as well as their overall FC between WT and Actl7b -/- testes. (E) Anti-ACTL7A IgG labeled western blot of testicular and sperm lysates from one WT, three Actl7b +/- (7B Het), and three Actl7b -/- (7B KO) mice indicating a severe reduction of ACTL7A expression in Actl7b -/- mice.

In Silico Modeling Indicates Positive Predictive Value for the Binding Capacity of Testis Specific ACTL7A and ACTL7B to HSA Domains of Classical Nucleosome Remodelers.

(A-C) Generated structural model of human PBAF nucleosome remodeling complex (PDB: 7VDV) bound to ACTL6A and ACTB (A), with interface map analysis of protein interactions through ChimeraX (B), and list of protein components (C). (D) Predicted alignment error heatmaps of the top dimeric interactions between ARP6, ACTB, ACTL6A, ACTL6B, ACTL7A, and ACTL7B with the isolated HSA domain of SRCAP, EP400, SMARCA2, and SMARCA4. On each alignment error heatmap red indicates poor to no predicted binding, while blue represents predictive values indicating excellent binding potential between protein residues. The presence of a clear blue vertical strip at the interaction quadrant of each HSA domain with each ARP (bottom left quadrant of each heatmap) indicates strong binding affinity between the two proteins at those residues, absence of a blue line corresponds to poor to no binding affinity. (E) Tabulated results of the interface predicted template modeling (ipTM) score of each ARP’s binding stability with each HSA domain from a scale of 0 to 1.0. An ipTM score of 0.7 or higher for a dimeric prediction is seen as very favorable and biologically relevant. (F) Linear HSA domain primary protein sequence maps with colored brackets indicating all stable predicted ARP interactions (those with an ipTM score higher than 0.7) between each modeled HSA domain and the select ARPs. Amino acid sequence residue numbers corresponding to the parent chromatin remodeling protein are indicated below each represented HSA domain.

ACTL7A and ACTL7B Actin Domains Predicted to Interact with the HSA Domain of SRCAP Cooperatively with Classical Somatic ACTL6A.

(A-B) cooperative binding model of ACTL7A and ACTL7B with ACTL6A and the HSA domain of SRCAP showing an ability of SRCAP to supportively bind at least three ARPs in tandem and in more than one conformation. Accompanying predictive alignment error heatmaps show the direct binding capabilities between these protein components. (C) Multi-ARP competitive binding model between ACTL6A, ACTL7A, ACTL7B, and ACTB with the HSA domain of SRCAP showing the predicted preferential occupancy of ACTB to the HSA binding site adjacent ACTL6A, thus supplanting ACTL7B in this conformation. (D) Additional competitive binding model with ACTL6B instead of ACTB indicating predicted potential to aid in the recruitment or displacement of ACTL7A from its binding site to the HSA domain of SRCAP .

Full length ACTL7A and ACTL7B Orientation in a SRCAP Complex is Predicted to Poise N-terminal Disordered Domains for Possible DNA Interactions, and D-loop Domains for Accessory Protein Interactions.

(A) Predictive interaction between ACTL7A, and ACTL7B with the HSA domain of SRCAP indicating a unilateral enrichment of Lys/Arg residues contained in the HSA domain and in the N-terminal domain of both ARPs indicating a potential special orientation between these components to bind DNA directly. (B) Predictive model of the binding between ACTL7A and ACTL7B with ACTL6A and the HSA domain of SRCAP showing a unilateral arrangement of the D-loop domains of all three ARPs involved. (C) Artistic schematic of the complete chromatin associated human SRCAP nucleosome remodeling complex to illustrate the proposed interaction model contextualizing how testis specific ACTL7A, ACTL7B could structurally integrate with classical somatic ACTL6A and SRCAPto form a putative testis specific regulatory ARP module. This schematic was generated by substituting the recently elucidated SRCAP partial complex (PDB: 6IGM) where the known yeast INO80-RuvB hexamer complex would be relative to its ARP8-Actin-ARP4-HSA-DNA Arp module, and bound nucleosome, as proposed by (Zhang et al. 2022). We then substituted our predictive ACTL7A-ACTL7B-ACTL6A-HSA model in the same position as the INO80 ARP module and added a nucleosome with an extended DNA strand (PDB: 1ZBB) as to exactly emulate the evidence-based structural conformation of the INO80 complex. This diagram assumes that given that SRCAP and INO80 belong to the same protein family then they would form relatively similar structural complexes.

Loss of ACTL7A and ACTL7B Affect Intra-cellular Patterning Changes in Acetylated-Lysine and Histone H3 Across Spermiogenesis.

(A-E) Widefield immunofluorescent images of round spermatids showing differential expression patterns of acetylated lysine in their intranuclear spaces with strong nuclear associated expression in WT (A), which is diminished in both Actl7a -/- (7A KO) (B-C), and Actl7b - /- (7B KO) (D-E) mice. (F-J) Accompanying images of elongating spermatids for each of respective genotype highlighting the reduction of acetylated lysine during these steps of development in both WT and 7AKO, but persistent signal in 7BKO. (K-O) Immunofluorescent images of round spermatids from across the three depicted genotypes illustrating a somewhat conserved expression patterns of histone H3 in their intranuclear spaces. (P-T) Accompanying images of elongating spermatids for each of respective genotypes showing varying degrees of H3 incorporation onto the forming postacrosomal sheath with reduced signal apparent in the nuclear associated postacrosomal sheath of 7AKO. All white scalebars per image represent 2.5 um.

HDAC1 and HDAC3 Localization is Altered within ACTL7A and ACTL7B Null Spermatids.

(A-E) Representative widefield immunofluorescent images of round spermatids showing contrasting patterns of HDAC1 expression with WT (A) exhibiting nuclear localization and enhanced expression in a nucleolar associated puncta. Actl7a -/- spermatids (7A KO) (B-C) lose these expression patterns and instead exhibit cytoplasm enriched localization, and Actl7b -/- (7B KO) (D-E) mice exhibit variable intermediate HDAC1 localization to both cytoplasm and nuclear compartments with reduced observation of nucleolar associated HDAC1 puncta. (F-J) Accompanying images of elongating spermatids for each of respective genotype showing no clear HDAC1 expression differences between them. (K-O) Immunofluorescent images of round spermatids from each genotype illustrating altered intranuclear HDAC3 incorporation. Nuclear localization is diminished in both KOs (L-O) compared to the WT control (K), while cytoplasmic localization is comparable between genotypes. Round spermatids in both KOs also lose acrosomal granule associated HDAC3 localization seen in WT (K). (P-T) Accompanying images of elongating spermatids exhibiting similar cytoplasmic HDAC3 expression patterns between genotypes. All white scalebars per image represent 2.5 um.

Plasmids.

A) YFP only negative control. B) YFP conjugated full length human ACTL7B. C) YFP conjugated N-terminus of human ACTL7B. D) YFP conjugated actin body of human ACTL7B. E) YFP conjugated SV40 NLS positive control for nuclear localization. F) YFP conjugated NLS candidate 2 of human ACTL7B [242-266].

Transfection optimization.

A) fluorescent microscopy showing YFP localization patters when conjugated to ACTL7B 24 to 40 hours post transfection. The 40 hours incubation time after YFP-ACTL7B transfection readily led to cell death. B) Tabulated results of cell viability and transfection efficiency indicating a clear decrease in cell viability in YFP-ACTL7B transfected cells compared to the YFP control after 40 hours, but not 17 or 24 hours.

SRCAP HSA-ARP modeling controls.

A) Predicted alignment error heatmap of interactions between SRCAP(1-706) and ACTL6A serving as a positive control for a known interaction. Red indicates poor to no binding, while blue on the heatmap indicates excellent binding between the two proteins. The presence of a clear blue vertical strip between the interaction quadrant of ACTL6A with SRCAP indicates the placement of their interaction allowing identification of the HSA domain. B) Generated PDB file of the predicted interaction between ACTL6A and the identified HSA domain of SRCAP. Subpanel illustrates a transparent cross section of the protein-protein interaction showing a vast number of buried (interacting) amino acid residues that stabilize the binding of ACTL6A with SRCAP and associated H-bonds shown as blue dash lines (buried/interacting residues were analytically identified by the interface mapping function in ChimeraX). C-D) Predictive interaction between SRCAP(1-706) and ARP6 serving as a negative control given that ARP6 is known to not interact with SRCAP in this region of the protein. Cross sectional protein mapping of the amino acid residues that interact between the HSA domain of SRCAP and ARP6 show minimal cooperative binding.

Antibodies.

Comprehensive list of all used antibodies and other labels; key experimental parameters are indicated.