Microtubule depolymerization in Caenorhabditis elegans touch receptor neurons reduces gene expression through a p38 MAPK pathway

Abstract

Microtubules are integral to neuronal development and function. They endow cells with polarity, shape, and structure, and their extensive surface area provides substrates for intracellular trafficking and scaffolds for signaling molecules. Consequently, microtubule polymerization dynamics affect not only structural features of the cell but also the subcellular localization of proteins that can trigger intracellular signaling events. In the nematode Caenorhabditis elegans, the processes of touch receptor neurons are filled with a bundle of specialized large-diameter microtubules. We find that conditions that disrupt these microtubules (loss of either the MEC-7 β-tubulin or MEC-12 α-tubulin or growth in 1 mM colchicine) cause a general reduction in touch receptor neuron (TRN) protein levels. This reduction requires a p38 MAPK pathway (DLK-1, MKK-4, and PMK-3) and the transcription factor CEBP-1. Cells may use this feedback pathway that couples microtubule state and MAPK activation to regulate cellular functions.

Fig. 1.

Protein levels are reduced in TRNs when microtubules are disrupted. (A) Fluorescence in P_mec-3gfp-expressing ALM neurons in L1 larvae, L4 larvae, and adults 48 h after hatching is reduced by mutation of mec-7 or mec-12 or growth in 1 mM colchicine. The mec-12(e1605) is an exception. (B) MEC-18 immunoreactivity is similarly reduced in adult ALM neurons. (C) Similar conditions reduce fluorescence from P_unc-119gfp in the ALM and AVM TRNs but not in other cells (e.g., SDQ and nerve ring neurons). Nerve ring (Left); anterior midbody (Right). (Scale bars, 20 μm.)

Fig. 2.

Mutations in several genes result in the colchicine-resistant expression (Cre) of short-lived PRAJA::GFP. (A) Colchicine abolishes TRN fluorescence in wild-type animals expressing uIs44 (P_mec-18praja::gfp) in a time-dependent manner. (B) Mutants defective in dlk-1 and cebp-1 retain TRN fluorescence when grown on colchicine. (C) Mutants defective in mkk-4 and pmk-3 also display Cre phenotypes, although it is incomplete in mkk-4 animals. An ALM neuron is shown in all panels; similar changes were seen for the other TRNs. (Scale bars, 20 μm.)

Fig. 3.

Extent of the Cre phenotype in dlk-1, cebp-1, mkk-4, and pmk-3 adults. The Cre phenotype was measured as the number of fluorescing TRNs (maximum six) seen in uIs44-expressing adults that had been grown on 1 mM colchicine. (A) Although completely penetrant in all four mutant backgrounds, expressivity is incomplete in mkk-4 mutants. (B) dlk-1(u818) and ceb-1(u819) Cre phenotypes can be rescued by expressing a wild-type copy of the gene under the TRN-specific mec-18 promoter (P_mec-18). The mean ± SEM is indicated for n ≥ 20 animals.

Fig. 4.

Mutations in genes for p38 MAPK pathway proteins and cebp-1 suppress the reduction of endogenous MEC-18 protein caused by microtubule defects. MEC-18 immunoreactivity in ALM neurons is shown for (A) colchicine-treated animals, (B) mec-7 mutants, and (C) mec-12 mutants. mec-7(0) denotes null allele; dlk-1 double mutants were generated with mec-7(u440); cebp-1 and pmk-3 were generated with mec-7(u443). (Scale bar, 20 μm.)

Fig. 5.

Model for microtubule-based regulation of gene expression. Interactions are derived from data in this paper and others (4, 8, 12, 16, 35). Direct positive (→) and negative (⊣) regulation is indicated by solid lines. Dashed lines indicate parts of the pathway that may be indirect. The MEC-3 transcription factor is also required for maintenance of its own expression (15). The question mark denotes an unidentified MAPKK believed to be partially redundant with MKK-4.