Fig. 6: P56S-VAPB leads to abnormal stress granules formation and alters their dynamics. | Cell Death & Disease

Fig. 6: P56S-VAPB leads to abnormal stress granules formation and alters their dynamics.

From: Pathomechanisms of ALS8: altered autophagy and defective RNA binding protein (RBP) homeostasis due to the VAPB P56S mutation

Fig. 6

a, b Co-labelling of HeLa cells expressing wt-VAPB and P56S-VAPB with antibodies against SG markers TIAR1 (a) and G3BP (b), showing cytoplasmic SG formation (arrowheads) in P56S-VAPB expressing cells. Scale bar: 10 µm. c Immunofluorescence analysis of HeLa cells stably expressing SG protein G3BP, showing SG formation in P56S-VAPB transfected cells compared to wt-VAPB transfected cells after 24 h. Scale bar: 10 µm. d Quantification of TIAR1 in HeLa cells and G3BP stable cell lines for SGs formation due to overexpression of either the wt-VAPB or P56S-VAPB after 36 h of transfection. e, f FRAP analysis by live cell imaging on the above cell lines showing reduced recovery of G3BP-SGs in P56S-VAPB transfected cells compared to wt-VAPB transfected cells, Quantifications (f). Scale bar: 10 µm. g Immunoblot analysis subcellular fractions of HeLa cells expressing wt-VAPB and P56S-VAPB, showing increased levels of TIAR1 in cytoplasmic fraction (red arrowhead, CE) in P56S-VAPB expressing cells. h, i DAB immunohistochemistry using antibodies against SG marker TIAR1 in lumbar spinal cord α-MNs of wt and P56S-VAPB Tg mice (h) and in ALS8 muscle biopsy (i), showing altered nuclear immunoreactivity (arrows) and diffuse albeit increased cytoplasmic immunoreactivity (arrows) in both α-MNs of P56S- VAPB tg mice as well as in ALS8 muscle biopsy. (Scale bars: 15 µm in h, 60 µm in i. j Accumulated globular immunoreactive TIAR1 aggregates co-localises with VAPB aggregates (arrowheads) in ALS8 muscle biopsy. Scale bars: 25 µm.

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