| |
Fig. 5. Tau knockdown selectively disrupted the structural integrity of microtubules containing a neuronal class II b-tubulin. Newly differentiating, embryonic
cultured spinal cord neurons containing tau antisense morpholino oligonucleotide (tau MO; B1–3, D2, E2, F2, G2) or control MO (A1–2, D1, E1, F1, G1) were
stained for the neuron-specific b-tubulin II isotype (Nb-tubulin; Xenopus gene symbol, tubb2b), ubiquitous a- and b-tubulins, tyrosinated (Tyr-), and acetylated
(Ac-) a-tubulins, respectively. (A1–B3) In contrast to the generally filamentous structures seen in the axons and growth cones (A2) of control MO neurons
stained for Nb-tubulin, significantly more short, discontinuous fragments (arrowheads in B1 and 2) were seen in the axons and growth cones (B3) of tau MO
neurons. (C) Quantitation of the number ( SEM) of discontinuous, fragmented microtubules (puncta) stained by the Nb-tubulin antibody along the neuritic
shaft (left; /100 lm) and within growth cones (right; /100 lm2) of control and tau MO cultured neurons (three cultures each). **Significantly different from
control, as determined by t-test; P < 0.01. (D1–G2) As determined by immunostaining, neither the levels of expression nor distributions of ubiquitous b-tubulins
(D1 and 2), ubiquitous a-tubulins, Tyr-tubulin (F1 and 2) and Ac-tubulin (G1 and 2) differed between tau MO and control MO cultured neurons. Scale bars
(20 lm in all panels) in (A2), (B1), (E1), (F1) and (G1) apply to (B3), (B2), (E2), (F2) and (G2), respectively. Neurons were imaged by conventional epifluorescence
with a 1009 objective (NA 1.4). (H) Quantitation of the relative fluorescence intensity (RFI; see Statistical analyses in Materials and methods for
details) within neurites for each of the tubulin antibodies in (D1–G2) revealed no significant differences in tau MO neurons relative to that of control MO neurons
(RFI = 1.0; t-test, three cultures each). |
