The multipurpose 15-protofilament microtubules in C. elegans have specific roles in mechanosensation

Abstract

Because microtubules perform many essential functions in neurons, delineating unique roles attributable to these organelles presents a formidable challenge. Microtubules endow neurons with shape and structure and are required for developmental processes including neurite outgrowth, intracellular transport, and synapse formation and plasticity; microtubules in sensory neurons may be required for the above processes in addition to a specific sensory function. In Caenorhabditis elegans, six touch receptor neurons (TRNs) sense gentle touch and uniquely contain 15-protofilament microtubules. Disruption of these microtubules by loss of either the MEC-7 beta-tubulin or MEC-12 alpha-tubulin or by growth in 1 mM colchicine causes touch insensitivity, altered distribution of the touch transduction channel, and a general reduction in protein levels. We show that the effect on touch sensitivity can be separated from the others; microtubule depolymerization in mature TRNs causes touch insensitivity but does not result in protein distribution and production defects. In addition, the mec-12(e1605) mutation selectively causes touch insensitivity without affecting microtubule formation and other cellular processes. Touching e1605 animals produces a reduced mechanoreceptor current that inactivates more rapidly than in wild-type, suggesting a specific role of the microtubules in mechanotransduction.

Figure 1. TRN microtubules are required for intracellular transport

MEC-2 puncta distribution in the process of the ALM TRN in (A) wild type, (B) three mec-7 mutants, (C) two mec-12 mutants, and (D) an animal grown in 1 mM colchicine for several generations. Scale bars = 20 µm.

Figure 2. Depolymerization of microtubules in adults causes touch insensitivity but does not affect protein localization or accumulation

(A) Late colchicine treatment (black bars) reduces touch sensitivity of bus-17 adults compared to untreated animals (white bars). The mean ± S.E.M. is indicated; n=20. *Difference from wild type is statistically significant, p < 0.05. (B) Co-immunostaining against MEC-2 and anti-acetylated a-tubulin in ALM neurons 48 hrs after adults were transferred to colchicine-containing or control plates. (C) Co-immunostaining against MEC-18 and anti-acetylated a-tubulin in ALM neurons under same conditions. Scale bars = 20 µm.

Figure 3. The mec-12(e1605) mutation does not affect microtubules formation, protein distribution, or protein levels

Immunostaining against (A) acetylated α-tubulin, (B) MEC-2, and (C) MEC-18 in ALM neurons of wild type, mec-12 (e1607), and mec-12(e1605) adult animals. Scale bars = 20 µm.

Figure 4. Mutations in tubulin subunits reduce mechanoreceptor currents (MRC)

(A) Representative MRC traces from stimulated TRNs in wild type and tubulin mutant backgrounds. (B) Comparison of peak MRC amplitudes. The mean ± S.E.M is indicated; the number of animals tested is given in parenthesis above bar. *Difference from wild type is statistically significant, p < 0.05. **Significant difference between putative null alleles and mec-12(e1605), p<0.05. † Average of data from uls30 control animals (this study) and uIs31 control animals [9].†† ata from [9]. (C) – (E) Boltzman curves were fit to normalized maximum current responses as a function of stimulus pressure for wild-type and mutant PLM neurons. Data from different cells are depicted with different symbols. (C) Wild type; new data as well as data from [9] were included. Best fit parameters were P_½ = 4.6 ± 0.2 nN/µm², P_sIope = 1.7 ± 0.08 nN/µm, n = 13. (D) mec-12(e1605); best fit parameters were P_½= 3.6 ± 0.5 nN/fim, P_sIope = 1.5 ± 0.4 nN/µm, n = 3. (E) mec-7(u142) from [9]; best fit parameters were P_½ = 10.9 ± 0.8 nN/µm², P^sIope = 3.3 ± 0.8 nN/µm², n = 3. (F) The curves are plotted together for comparison, mec-7(u142) is significantly different (p < 0.001) from both wild type and mec-12(e1605) using the F-test.0