Mutated hVAPB is degraded faster than wildtype hVAPB through a proteasome-dependent mechanism.

<p>(A) Western blot analysis of COS-7 cells transfected with the indicated expression vectors and treated or not with the protein biosynthesis inhibitor cycloheximide (CHX, 100 µg/ml) for 3, 6, 8 and 10 h and/or with the proteasome blocking agent MG-132 (10 µM) for 10 h. Actin served as a loading control. (B) hVAPB immunoreactive bands were quantified by densitometry and values were normalized to actin and expressed relative to values obtained in untreated cells. (C) Densitometric quantification of hVAPB levels in transfected cells following 10 h of treatment with CHX and MG-132. (D-E) Immunolabeling of hVAPB in transfected COS-7 cells treated for 12 h with MG-132 (5 µM)(D). The number of cells showing a perinuclear accumulation of hVAPB was determined 36 h after transfection with the indicated vectors (E). Scale bar, 20 µm. Results shown in (B), (C) and (E) are the mean values ± S.D of three independent experiments.</p

Wildtype and mutated hVAPB associate with the proteasome.

<p>(A) Both hVAPB^WT and hVAPB^P56S colocalize with the alpha 5 subunit of the proteasome, as indicated by the double immunostaining of cells transfected with empty, hVAPB^WT and hVAPB^P56S vectors. Images were acquired with the same exposure time and camera settings. White arrows indicates non-transfected cells. Scale bar, 20 µm. (B–C) hVAPB^WT and hVAPB^P56S were immunoprecipitated from COS-7 cells transfected with hVAPB^WT, hVAPB^P56S, myc-tagged Sar1, myc-tagged Arf1, myc-tagged hSOD1 and hVAPA. Endogenous alpha 5 (B) or alpha 1–7 (C) subunits of the proteasome that co-immunoprecipitated with hVAPBs was detected by Western blotting using specific antibodies. hVAPB, myc (Sar1, Arf1 and hSOD1) and hVAPA input levels are shown. Immunoprecipitation of alpha 1–7 by Rpt2 served as a positive control.</p

Accumulation of hVAPB^WT leads to the formation of cytoplasmic inclusions.

<p>(A) Sequential detergent extraction of cellular proteins at different times following transfection of COS-7 cells with indicated an empty vector, hVAPB^WT and hVAPB^P56S expression vectors. (B) Accumulation of wildtype hVAPB leads to the formation of insoluble inclusions that disrupt ER structure as documented by the co-immunostaining of hVAPB with KDEL 72 h after transfection. Scale bar, 20 µm.</p

Overexpression of wildtype and mutated hVAPB impairs proteasome activity.

<p>Levels of proteasome YFP reporters in cells co-transfected (or not) for 36 h with hVAPB^WT, hVAPB^P56S, hVAPA, hSOD1 and Ub-R-YFP (A), Ub-G76V-YFP (B) or CD3δ-YFP (C) were examined by Western blotting using anti-GFP antibodies. (D) Cells were co-transfected with vectors encoding hVAPB^WT, hVAPB^P56S or hVAPA and HA-tagged Fat10, an ubiquitin-independent signal for proteasomal degradation. Protein extracts were prepared 36 h post-transfection, resolved by SDS-PAGE, Western blotted and probed with HA, hVAPB, hVAPA and actin antibodies.</p

hVAPB^P56S partially colocalizes with ubiquitinated conjugates but increases the general ubiquitin levels in the cells.

<p>(A) When co-transfected with hVAPB^WT or hVAPB^P56S, GFP-Ubi forms cytoplasmic aggregates that occasionally (white arrow) colocalize with hVAPB^WT or hVAPB^P56S. (B) The GFP-tagged mutated ubiquitin Ubi_AA-GFP does not form detectable aggregates. Scale bar, 20 µm. (C) Ubiquitin immunoblot profile of COS-7 cells transfected with empty, hVAPB^WT and hVAPB^P56S vectors following differential detergent extraction. (D) Western blot analysis of total HA-tagged ubiquitin levels in cells expressing either form of hVAPB or hVAPA. In (C) and (D), protein extracts were prepared 36 h after transfection and actin was used as a loading control.</p

hVAPB-mediated ER stress contributes to the accumulation of proteasome substrates.

<p>(A) The immunoreactivity of CHOP in cells expressing hVAPB^WT and hVAPB^P56S was monitored by Western blotting (36 h after transfection). Thapsigargin treatment (for 16 h) was used as a positive control for ER stress-dependent CHOP upregulation. (B) Levels of BiP and phosphorylation status of IRE1 were examined by Western blotting 36 h following the transfection of cells with empty, hVAPB^WT and hVAPB^P56S plasmids. (C) Protein extracts of cells transfected with the proteasome reporters Ub-R-YFP and Ub^G76V-YFP and treated (or not) for 16 h with the ER stress inducer thapsigargin (10 µM) were subjected to Western blotting using anti-GFP (referred to as YFP), and anti-CHOP antibodies. (D) Quantification of the YFP immunoreactive bands (C) normalized to actin signals (arbitrary densitometry units). (E) Salubinal treatment (20 µm) diminished the accumulation of the proteasome reporter (YFP) as indicated by Western blotting of COS-7 cells co-transfectd with hVAPB^WT or hVAPB^P56S. (F) Differential detergent extraction and Western blot analysis of ubiquitin in cells expressing hVAPB^WT and hVAPB^P56S and treated or not with salubrinal (20 µM).</p

Wildtype and mutated hVAPB associate with components of the secretory pathway in non-human primate cells.

<p>(A–D) Thirty-six hours following transfection of COS-7 cells, hVAPB^WT and hVAPB^P56S mainly colocalize with components of the secretory pathway as demonstrated by the immunostaining of hVAPB with the ER marker KDEL (A), the COPI vesicle marker β-COP-CFP (B) and ERGIC marker ERGIC-53 (C). hVAPB^P56S forms cytoplasmic aggregates that colocalize with ER, COPI and ERGIC markers. Both hVAPB^WT and hVAPB^P56S seldom colocalize (white arrow) with the COPII marker Sec23-YFP (D). Scale bar, 20 µm.</p

Effects of pattern on spinal motoneurons polarity and electrical properties.

<p>(A) Image of triple staining of a patterned <i>Hb9::GFP</i> embryonic motoneuron with anti-laminin (pink) to reveal the pattern, anti-GFP (green) to enhance motoneuron morphology and the axonal marker anti-SMI312 (red). Motoneuron shows polarity with one long axon (red) and short dendrites (d, green). Hb9⁺-GFP motoneurons were fixed at 2 DIV (d, dendrite; s, soma and a, axon). (B) Patterned embryonic motoneurons begin to express electrical activity at 2DIV following the injection of 2 ms current. (C) Voltage clamp recordings on the same neuron evidenced the expression of sodium current (INa). Voltage protocol is shown below current trace.</p

Immunostaining of patterned sensory neuron polarity.

<p>Image of double immunostaining with the neuronal cytoskeletal marker anti-βIII tubulin (green) and axonal marker anti-SMI312 (red) shows that both neurites expressed the axonal marker (merge image).</p

Effects of pattern on excitability of large sensory neurons.

<p>(A) Typical recordings of an action potential elicited by threshold current (all or nothing response). Black lines: 2 ms current amplitude does not trigger an AP. Red lines: increasing current amplitude triggers a full AP. (B) Unlike unpatterned substrate, the amplitude of current that triggers an action potential is not significantly different between control and axotomized sensory neurons grown on patterned substrate (***<i>p</i><0.001, <i>t</i> test). (C) Typical firing action potentials of patterned neurons induced by long depolarizing current (500 ms duration). (D) Maintained firing was recorded in roughly 20–30% of patterned control and conditioned neurons. No firing activity was observed on unpatterned neurons recorded at 2 DIV. (*<i>p</i><0.05, Chi-square, Fisher test).</p