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. 2015 Aug 19;5(8):150067. doi: 10.1098/rsob.150067

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© 2015 The Authors.

Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original author and source are credited.

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Figure 2. — The ER associates with astral MTs in meiotic spermatocytes. (a) A fixed GFP-Sec61α (green) spermatocyte at metaphase of the first meiotic division was immunostained for α-tubulin (red), asterless (Asl, blue) to localize centrosomes (blue arrows) and phosphorylated histone 3 (pH3, blue). ER structures are closely aligned with astral MTs (yellow arrowheads). (b) A spermatocyte expressing GFP-Sec61α (green) and H2A-mRFP (red) was imaged live throughout the course of the first meiotic division. NEB was determined based on the sudden loss of background H2A-mRFP fluorescence throughout the nucleus. The approximate outline of the cell (yellow dotted lines) and the astral ER domains at metaphase (arrow heads) are indicated. (c) asl^mecD/TM6 control (left panel) and asl^mecD/asl^mecD (right panel) GFP-Sec61α expressing spermatocytes were imaged throughout meiosis I. Spindle poles (arrows) recruit ER in control cells, but fail to do so in asl^mecD/asl^mecD spermatocyte; abnormal ER structures (arrowheads) are prominent in the asl^MecD/asl^MecD spermatocyte. (d) A fixed GFP-Sec61α (green) metaphase I asl^MecD/asl^MecD spermatocyte was immunostained for α-tubulin (red), asl (blue) and pH3 (blue). Indicated are spindle poles (arrows) and abnormal ER structures associated with MTs (arrowheads). (e) Fixed GFP-Sec61α (green) metaphase I asp^MB/TM6 control (left panel) and asp^MB/asp^MB (right panel) spermatocytes were fixed and immunostained for α-tubulin (red). DAPI staining of DNA is shown in blue in the merged image. Abnormal ER structures are prominent in asp^MB/asp^MBspermatocyte (arrows). Times, min:s; scale bar, 5 µm.