TMF/ARA160 Governs the Dynamic Spatial Orientation of the Golgi Apparatus during Sperm Development.

TMF/ARA160 is known to be a TATA element Modulatory Factor (TMF). It was initially identified as a DNA-binding factor and a coactivator of the Androgen receptor. It was also characterized as a Golgi-associated protein, which is essential for acrosome formation during functional sperm development. However, the molecular roles of TMF in this intricate process have not been revealed. Here, we show that during spermiogenesis, TMF undergoes a dynamic change of localization throughout the Golgi apparatus. Specifically, TMF translocates from the cis-Golgi to the trans-Golgi network and to the emerging vesicles surface, as the round spermatids develop. Notably, lack of TMF led to an abnormal spatial orientation of the Golgi and to the deviation of the trans-Golgi surface away from the nucleus of the developing round spermatids. Concomitantly, pro-acrosomal vesicles derived from the TMF-/- Golgi lacked targeting properties and did not tether to the spermatid nuclear membrane thereby failing to form the acrosome anchoring scaffold, the acroplaxome, around the cell-nucleus. Absence of TMF also perturbed the positioning of microtubules, which normally lie in proximity to the Golgi and are important for maintaining Golgi spatial orientation and dynamics and for chromatoid body formation, which is impaired in TMF-/- spermatids. In-silico evaluation combined with molecular and electron microscopic analyses revealed the presence of a microtubule interacting domain (MIT) in TMF, and confirmed the association of TMF with microtubules in spermatogenic cells. Furthermore, the MIT domain in TMF, along with microtubules integrity, are required for stable association of TMF with the Golgi apparatus. Collectively, we show here for the first time that a Golgi and microtubules associated protein is crucial for maintaining proper Golgi orientation during a cell developmental process

TMF resides in the trans-Golgi compartment of stages 6–8 round spermatids.

<p>Testicular sections prepared as described were subjected to IG-EM analysis using anti-TMF specific antibody followed by an anti-rabbit secondary antibody conjugated to gold particles with a diameter of 10nm (black dots). (A) Stage 6 spermatids. (B) The same spermatid from <b>A</b> under higher magnification showing the positive staining of TMF, which is marked by arrows, in the trans-Golgi and the space between the Golgi and the acrosome. (C) Enlarged boxed area in <b>B</b>. Positive staining of TMF is marked by arrows. (D) Enlarged dashed-boxed area in <b>B</b>. Positive staining of TMF is marked by arrows. (E) A stage 7 spermatid showing the same trans-Golgi localization of TMF, which is marked by arrows. (F) Enlarged boxed area in <b>E</b>. TMF staining is marked by arrows. (G) Stage 8 spermatid showing trans-Golgi and association of TMF along with vesicles association (marked by arrows). (H) Enlarged boxed area in G. Arrows mark TMF associated with the vesicles outer membrane. cG = cis-Golgi, tG = trans-Golgi, Ac = acrosome, Nu = nucleus. Bars represent 1μm (A), 500nm (B-F) and 200nm (G-H). Each image represents one out of twelve different cells selected from three different sections which gave similar results. (I) Comparative quantification of the immuno-gold labeled TMF in the different Golgi compartments. n = 5 (different cells of the same stage from three different sections), histograms represent mean values +/- SD.</p

CC and MIT domains are required for stable association of TMF with the Golgi.

<p>(A) Diagram of the TMF aa sequence with the marked MIT and CC motifs (top). Constructs 1–5 transfected into NIH3T3 cells are depicted below. The TMF aa included in each construct are shown, and the presence of the CC or MIT motifs in each construct are marked in the brackets. (B-F) Confocal microscopy analysis of EGFP-fused TMF segments, ectopically expressed (green) in NIH3T3 cells. (G) Confocal microscopy analysis of TMF segment containing the MIT and c-terminus CC domain fused to EGFP (EGFP-TMF C5) (green) overlapping the immuno-staining of GM130 (Golgi marker, red.). Arrows represent co-localization (yellow color). The different fluorescence channels are presented in the right panels. Bar represents 20μm. Nuclei were visualized with Hoechst solution (blue). (H) As in <b>G</b>, only that these cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (I) Confocal microscope analysis of the full TMF protein fused to EGFP (green) overlapping the immuno-stained Golgi (golgin 97- red.) (J) As in <b>I</b>, only that cells were treated for 20 min with colchicine. Arrow indicates the Golgi apparatus. Nuclei were visualized with Hoechst solution (blue).Images represent typical fluorescence and staining profiles obtained for all transfected cells in four independent transfection experiments. (K) IP of GFP-TMF protein fragments from lysates of NIH3T3 cells expressing either GFP-TMF containing the MIT domain (TMF-MIT) or GFP-TMF fragment containing the CC domains without the MIT motif (TMF-CC).Whole cell lysate (WCL) represents the input of the IP. IgG beads served as a negative control. The IP was followed by a WB analysis to assess the co-IP of the TMF fragment and tubulin. Images represent one out of five independent typical WB analyses which gave similar results.</p

Abnormal Golgi orientation in TMF^-/- spermatids.

<p>TEM analysis of testicular sections from wt (A-D) and TMF^-/- mice (E-H). (A) Stage 1 wt spermatid. (B) Boxed area in <b>A</b> under higher magnification. The Golgi apparatus is marked by an arrow head. Pro-acrosomal vesicles are marked by an arrow. (C) Stage 3 wt spermatid. (D) Boxed area in <b>C</b> under higher magnification. The Golgi apparatus is marked by an arrow head. Pro-acrosomal vesicles are marked by arrow. (E) Stage 3 TMF^-/- spermatid. (F) Boxed area in <b>E</b> under higher magnification. The Golgi apparatus is marked by an arrow head. Pro-acrosomal vesicles are marked by an arrow. (G) Different TMF^-/- round spermatids of the same stage as in <b>E</b>. The Golgi apparatus is marked by an arrow head. Pro-acrosomal vesicles are marked by an arrow. (H) Stage 1 TMF^-/- spermatid. The Golgi apparatus is marked by an arrow head. Pro-acrosomal vesicles are marked by an arrow. Ac = acrosome, Nu = Nucleus, cG = cis-Golgi. Bars represent 2μm (A,C and E), 1μm (B, D, F and H) and 500 nm (G). Each image represents one out of twenty different cells selected from five different sections which gave similar results. Immunofluorescence staining of the trans-Golgi marker TGN38 in wt (I, K), and TMF^-/- round spermatids (J, L) (Green). Immunofluorescence staining of the trans-Golgi marker Golgin 97 in wt (M and O), and TMF^-/- round spermatids (N, P) (Green). Nuclei were visualized with Hoechst (Blue). Bars represent 5 μm.</p

TMF^-/- round spermatids lack the CB compartment.

<p>Testicular sections from wt (A and C) or TMF^-/- (B and D) mice were subjected to TEM analysis. (A) Stage 4 round spermatid of wt mice. The Golgi apparatus is marked by an arrow. The CB is marked by an arrow head. (B) Stage 4 TMF^-/- round spermatid. The Golgi is marked by an arrow. No CB is observed in this spermatid. (C) Early stage 1 spermatid of wt mice. The Golgi is marked by an arrow. The CB is marked by an arrow head. (D) TMF^-/- spermatid of an early stage 1. The Golgi is marked by an arrow. No CB is observed in this spermatid. Bars represent 2μm. (E and G) Testicular round spermatids of wt mice were subjected to KIF17B immunofluorescence staining using specific antibodies (green, aggregations of KIF17B are marked by arrows). (F and H) TMF^-/- testicular round spermatids were subjected to KIF17B immunofluorescence staining using specific antibodies (green). Nuclei were visualized with Hoechst. Bars represent 10μm.</p

TMF resides in the cis-Golgi of stages 3–4 round spermatids.

<p>Testicular 80 nm thick sections from 10 weeks old wt mice were subjected to IG-EM analysis using anti-TMF antibodies followed by an anti-rabbit secondary antibody conjugated to gold particles with a diameter of 10 nm (black dots). (A) Stage 4 spermatids. (B) The same spermatid from <b>A</b> under higher magnification, showing the staining for TMF in the peripheral cis-Golgi (indicated by arrows). (C) Enlarged boxed area in <b>B</b>. TMF staining is marked by arrows. (D) A late stage 3 spermatid showing the same peripheral cis-Golgi localization of TMF. (E) Enlarged Boxed area in <b>D</b>. TMF positive staining is marked by arrows. cG = cis-Golgi, tG = trans-Golgi, Nu = Nucleus, Ac = acrosome. Bars represent 1μm (A), and 500nm (B-E). Each image represents one out of twelve different cells selected from three different sections which gave similar results. (F) Comparative quantification of the immuno-gold labeled TMF in the different Golgi compartments. n = 5 (different cells of the same stage from three different sections), histograms represent mean values +/- SD.</p

TMF contains an MIT domain and associates with tubulin and microtubules.

<p>(A) The protein sequence of TMF was analyzed using the SMART algorithm. CC = coiled-coil. MIT = microtubule interacting domain. Underneath the motifs location scheme is the MIT sequence of TMF and its exact location inside TMF protein sequence. In bold are the key preserved amino acids of the sequence which are identical to the consensus MIT sequence. (B) Protein extracts from wt and TMF^-/- mice testes were subjected to immuno-precipitation (IP) analysis using anti-TMF antibodies followed by western-blot analysis to detect tubulin co-precipitation. WCL = whole cell lysate from wt testes, which is the IP input. (C) Co-precipitation of microtubules and TMF from mice spermatogenic cells. S denotes the supernatant which contains all the soluble proteins. P represents the precipitate which contains the sedimented microtubules. (D) Immuno-fluorescence co-staining of TMF (Red) and microtubules (Green) in round spermatid. (E) Enlarged boxed area in <b>D</b>. Co-localization of TMF and microtubules (Yellow) is depicted by white arrow heads. Nuclei were visualized with Hoechst solution (blue). Bar in <b>D</b> represents 10μm. (F) IG-EM analysis of TMF (black dots, presented by arrows) carried-out on testicular sections from wt mouse. (G) Enlarged boxed area in <b>F</b>. TMF positive staining is indicated by arrow heads. The FS is indicated by arrows. Bar represents 500nm. Immunocytochemical analysis of the Golgi marker GM130 (red) and microtubules (green) in wt (H-K) and TMF^-/- spermatids (L-O). The Golgi apparatus is marked by arrows in H-I and L-M. Nuclei were visualized with Hoechst solution (blue). Bars represent 10μm (H-J and L-N) and 20μm (K and O). Images represent one out of four independently prepared cell suspensions that gave similar staining profiles.</p

TMF^-/- spermatids lack an acroplaxome.

<p>Testicular sections from wt (A-D) and TMF^-/- mice (E-F) were subjected to EM analyses. (A) Stage 2/early 3 wt spermatid. (B) Boxed area in <b>A</b> under higher magnification. The acroplaxome is marked by arrows. (C) Stage 4 wt spermatid. (D) The boxed area in <b>C</b> under higher magnification. The acroplaxome is marked by arrows. (E) Stage 4 TMF^-/- spermatid. (F) The boxed area in <b>E</b> under higher magnification. The supposed localization of the acroplaxome is marked by arrows. Ac = acrosome, Nu = Nucleus. Bars represent 2μm (A, C and E) and 1μm (B, D and F). Each image represents one out of twenty different cells selected from three different sections which gave similar results. Immunocytochemical staining of F-actin (red) and acrosome (green) in round spermatids from wt (G-I) and TMF^-/- (J-L) mice, which were exposed to hypotonic shock.DIC images of the stained spermatids are shown in <b>I</b> and <b>L</b>. Acroplaxome margins are marked by arrows in <b>G</b>. (M) Immunocytochemical staining of F-actin (red) in wt round spermatid. (N) Immunocytochemical staining of F-actin (red) in TMF^-/- round spermatid. (O) Immunocytochemical staining of F-actin (red) and TMF (Green) in wt round spermatid. (P) Immunocytochemical staining of F-actin (red) in wt elongated spermatid. (Q) Immunocytochemical staining of F-actin (red) in TMF^-/- elongated spermatid. DIC images of the spermatids are also shown in <b>P</b> and <b>Q</b>. Nuclei were visualized with Hoechst solution (blue). Bars represent 10μm. Images represent one out of five independently prepared cell suspensions that gave similar staining profiles.</p