Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: ( a , b ) Immunoblot for endogenous HDAC6 ( a ) in newborn (P0) and adult cerebral cortex and ( b ) in primary cerebrocortical neurons at div2 and 3. Each lane respectively contained 50/10 μg total protein. ( c ) Mutual co-immunoprecipitation of endogenous SEPT7 and HDAC6. Lysates from div2 cerebrocortical neurons were incubated with protein A beads coated with nonimmune IgG, anti-SEPT7 antibody or anti-HDAC6 antibody, and these proteins were detected by immunoblot in a reciprocal manner. ( d ) Co-expression and mutual co-immunoprecipitation of GFP-SEPT7 and Flag-HDAC6 in heterologous cells. (Left) Anti-Flag antibody detected Flag-HDAC6 (and gave nonspecific faint bands in the 1st and 3rd lanes) and anti-GFP antibody detected GFP and GFP-SEPT7. (Right) When Flag- HDAC6 was captured on anti-Flag beads, GFP-SEPT7 was co-immunoprecipitated, but GFP was not (compare the 2nd and 4th lanes. *, IgG light chain). ( e ) Direct binding between the purified, recombinant septin complex and HDAC6 in vitro . Immobilized His-tagged SEPT7/6/2 captured GST-HDAC6 but not GST alone. (Coomassie blue staining). ( f ) Representative results of in situ proximity ligation assay for endogenous SEPT7 and HDAC6 in div2 cerebrocortical neurons. Fluorescent puncta were generated in the somata and neurites only when anti-SEPT7 antibodies and anti-HDAC6 antibodies were present in close proximity. Scale bars, 25 μm. ( g ) Immunoblot showing that SEPT7 depletion did not affect the amount of HDAC6 in div2 cerebrocortical neurons. (Triplicated experiment. NS, P >0.05 by t -test). Error bars denote s.e.m. ( h ) SEPT7 depletion via RNAi (#1, S7KD) did not alter the deacetylating activity of HDAC6. Cell lysates as in ( b ) were subjected to an in vitro assay with a fluorogenic substrate. (Triplicated experiment. NS, P >0.05 by t -test). Error bars denote s.e.m. ( i ) SEPT7 depletion via RNAi (#1, S7KD) caused dissociation of HDAC6 and acetylated α-tubulin. (Left) Cell lysates as in ( b , h ) were immunoblotted for endogenous HDAC6, acetylated α-tubulin and total α-tubulin. Hyperacetylation of α-tubulin in SEPT7-depleted neurons was recapitulated ( cf. ). Each lane contained 50 μg total protein. (Right) Although SEPT7 depletion increased acetylated α-tubulin, its association with HDAC6 was paradoxically reduced. (Triplicated experiments. * P <0.05, ** P <0.01 by t -test). Error bars denote s.e.m.

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Western Blot, Immunoprecipitation, Incubation, Expressing, Binding Assay, Purification, Recombinant, In Vitro, Staining, In Situ, Proximity Ligation Assay, Generated, Activity Assay

Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: ( a ) (Top panels) Representative images of GFP-expressing cerebrocortical neurons at div2 treated with vehicle alone (0.1% DMSO) or with 10 μM tubacin in vehicle. HDAC6 inhibition by tubacin inhibited the growth of both dendrites and axons. (Bottom panels) SEPT7 depletion via RNAi (#1) combined with tubacin treatment did not exhibit an obvious additive effect on neurite morphology. Scale bar, 50 μm. ( b ) Scattergram of total dendrite length and total axon length of pyramidal-like cerebrocortical neurons treated with tubacin and SEPT7-depletion in the four combinations shown in ( a ). The growth of both dendrites and axons was markedly inhibited by tubacin treatment (black closed circles) in comparison with the control (black open circles), which was not enhanced by additional depletion of SEPT7 (red closed circles). ( c ) Statistical analysis of the results shown in ( b ) with plots of the mean values, showing that tubacin treatment significantly and proportionally shortened dendrites and axons. The neurite-shortening effect of tubacin was more potent than, and not enhanced by, SEPT7 depletion. The grey bars represent average±2 s.e.m. of the control samples. ( n =45 × 4. *** P <0.001 by one-way ANOVA with post hoc Tukey). Error bars denote s.e.m. ( d and e ) Tip numbers of axons and dendrites in the above samples were significantly reduced by tubacin alone, by SEPT7 depletion alone, or by the combination of the two. ( n =45 × 4. *** P <0.001 by one-way ANOVA with post hoc Tukey). Error bars denote s.e.m.

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Expressing, Inhibition, Comparison, Control

Journal: Nature Communications

Article Title: Septins promote dendrite and axon development by negatively regulating microtubule stability via HDAC6-mediated deacetylation

doi: 10.1038/ncomms3532

Figure Lengend Snippet: HDAC6 is the major microtubule deacetylase, which promotes microtubule remodelling by counteracting acetyl transferases that stabilize microtubules . This study demonstrated that the direct interaction with the septin complex facilitates the access of HDAC6 to acetylated α-tubulin and/or stabilizes the enzyme-substrate interaction without altering the deacetylation activity of HDAC6. An open question is whether the putative tripartite interaction of the septin complex/HDAC6/acetylated α-tubulin (a subset of the signals in the PLA assay shown in should represent this) occurs on α/β-tubulin heterodimers, protofilaments and/or microtubules. It is also worth testing whether an actomyosin-dependent mechanism, including another HDAC6 substrate cortactin , could contribute to the stagnant neurite growth after septin depletion. Independent of the specific underlying mechanism, the novel molecular network identified in this study has shed new light on the common machinery for the growth of axons and dendrites in vivo and in vitro .

Article Snippet: Sf9-expressed His 6 -tagged SEPT7/6/2 (ref. ) was immobilized on Ni-NTA beads (Qiagen) and incubated with Sf9-expressed GST-tagged human HDAC6 (SignalChem) or GST alone in PBS for 6 h at 4 °C.

Techniques: Histone Deacetylase Assay, Activity Assay, In Vivo, In Vitro