Figure 4.

Defective Microtubule Polymerization in Subjects with Homozygous/Heterozygous TBCE c.464T>A Missense Change and in pmn/pmn Mice

(A) Western blot (WB) analyses showing total (T), soluble (S), and polymerized (P) α-tubulin (above), detyrosinated tubulin (middle), and β-actin (below) in fibroblasts from affected individuals (1544334, 2518864) and in control cells (Ctrl1, Ctrl2). Diagrams (right) show reduced fractions of polymerized α-tubulin and detyrosinated tubulin in fibroblasts of affected subjects. In both comparisons, differences were statistically significant by Mann-Whitney test (p < 0.01).

(B) WB analyses showing reduced amount of polymerized α-tubulin (P) in fibroblasts from pmn/pmn mice compared to control fibroblasts (wt). Differences were statistically significant by Mann-Whitney test (p < 0.01).

(C) Immunofluorescence analysis documenting loss of microtubules containing detyrosinated tubulin (detyr-Tub) in fibroblasts of affected subjects. Note that detyrosinated microtubules (red) in control fibroblasts are closely associated with the GM130-stained Golgi apparatus (green), while the Golgi apparatus in fibroblasts carrying mutated TBCE alleles appears to have lost compaction. Nuclei are DAPI stained (blue). Scale bar represents 10 μm.

(D) Immunofluorescence analyses showing reduced re-polymerization of microtubules (labeled for α-tubulin, red) after nocodazole treatment in fibroblasts from subjects 1544334 and 2518864, as compared to control cells (Ctrl). Nuclei are labeled with DAPI (blue). Scale bar represents 20 μm.