Microtubule-mediated transport of the tumor-suppressor protein Merlin and its mutants

Abstract

The neurofibromatosis type 2 (NF2) tumor-suppressor protein Merlin is a member of the ERM family of proteins that links the cytoskeleton to the plasma membrane. In humans, mutations in the NF2 gene cause neurofibromatosis type-2 (NF2), a cancer syndrome characterized by the development of tumors of the nervous system. Previous reports have suggested that the subcellular distribution of Merlin is critical to its function, and that several NF2 mutants that lack tumor-suppressor activity present improper localization. Here we used a Drosophila cell culture model to study the distribution and mechanism of intracellular transport of Merlin and its mutants. We found that Drosophila Merlin formed cytoplasmic particles that move bidirectionally along microtubules. A single NF2-causing amino acid substitution in the FERM domain dramatically inhibited Merlin particle movement. Surprisingly, the presence of this immotile Merlin mutant also inhibited trafficking of the WT protein. Analysis of the movement of WT protein using RNAi and pull-downs showed that Merlin particles are associated with and moved by microtubule motors (kinesin-1 and cytoplasmic dynein), and that binding of motors and movement is regulated by Merlin phosphorylation. Inhibition of Merlin transport by expression of the dominant-negative mutant or depletion of kinesin-1 results in increased nuclear accumulation of the transcriptional coactivator Yorkie. These results demonstrate the requirement of microtubule-dependent transport for Merlin function.

Fig. 1.

Distribution of Merlin and endocytic vesicles in Drosophila S2 cells. (A–D) Drosophila S2 cells plated in Con A without or with Cyto-D treatment; and expressing either endogenous Mer^wt (A and C) or Mer^GFP under a heat-shock promoter (B and D). (A and B) Spread S2 cells were fixed and stained with anti-Merlin antibody. Merlin particles are found in the proximity of cell membrane (Inset) and in the perinuclear region (arrow). The distribution is identical for both Mer^wt (A) and Mer^GFP (B). (C and D) Cyto-D Mer^GFP S2 cells form processes that are filled with particles. This distribution is identical for both Mer^wt (C) and Mer^GFP (D). (E–G) Mer^GFP cells incubated with rhodamine-dextran and analyzed by fluorescence microscopy. (E) DIC image of S2 cells plated in Con A. (F) Distribution of Mer^GFP particles. (G) Rhodamine-dextran–labeled endosomes. See Movie S1.

Fig. 2.

Movement of Mer^GFP particles along processes of S2 cells. (A and B) Mer^GFP particles move bidirectionally in the processes of Cyto-D–treated S2 cells (Movie S2). Frames in B correspond to the boxed area in A. Time 0 indicates time-lapse start point (1 h after heat shock), and particle plus and minus end movement is shown in the first and second columns respectively (white arrowheads). (C) Histogram of velocities for Mer^GFP particles shows unbiased bidirectional movement.

Fig. 3.

An NF2 mutant of Merlin, Mer(K70E)^GFP, is defective in intracellular transport. (A and B) Cyto-D–treated S2 cells stably expressing Mer^GFP (A) or Mer^mCherry (B) (Movie S4). (A′ and B′) Kymographs of the boxed areas showing particles moving bidirectionally along processes. (C) Expression of the NF2-mutant Mer(K70E)^GFP in S2 cells. (C′) Kymographs of the boxed areas showing absence of movement (Movie S3). (D–F) Coexpression of Mer^mCherry (D) and Mer(K70E)^GFP (E) in S2 cells. (F and F′) Colocalization of the mutant and the WT protein. (D′ and E′) Kymographs of the boxed areas. (G) Pull-down using GBP. Input, crude cell extract from WT cells (untransfected) and cells expressing Mer^GFP or Mer(K70E)^GFP after heat shock. GBP, pull-down from WT extracts (untransfected) or extracts from S2 cells expressing Mer^GFP or Mer(K70E)^GFP. Blots were probed with antibodies against Merlin.

Fig. 4.

Expression of Merlin mutant affects Yorkie distribution. (A) From left to right, expression of Yki^mCherry alone, coexpression with Mer^GFP, Merlin RNAi, coexpression with Mer(K70E)^GFP, and KHC RNAi. (B) Quantification of Yorkie fluorescence in the nucleus. Error bar indicates SD (ANOVA). P = 0.01.

Fig. 5.

Bidirectional movement of Merlin clusters depends on kinesin-1, dynein motors, and adaptor proteins KLC and dynactin. (A) Frequencies of plus-end (white bars) and minus-end (black bars) movements of Mer^GFP particles. Bars represent the percentage of vectors of length >0.35 μm. Depletion of either KHC or DHC was sufficient to inhibit bidirectional movement of Merlin particles. (B) Kymographs of Mer^GFP particles moving in the processes. RNAi targets are listed above the corresponding panels. Error bar indicates SD (ANOVA). P = 0.005.

Fig. 6.

Merlin forms a complex with kinesin-1 and dynein motors. (A) Immunoprecipitation of endogenous Merlin from WT S2 cell extracts with anti-Merlin antibody. Input, crude cell extract; IgG, control IgG preimmune serum; anti-Mer, anti-Merlin antibody. (B) Pull-down using GBP. Input, crude cell extract from WT cells (untransfected) and cells expressing Mer^GFP or Mer(K70E)^GFP after heat shock; GBP, pull-down from WT extracts (untransfected) or extracts from S2 cells expressing Mer^GFP or Mer(K70E)^GFP. Blots were probed with antibodies against kinesin (KHC) or dynein (DHC).

Fig. 7.

Thr-616 phosphorylation regulates Merlin bidirectional transport and motor association. (A and A′) S2 cells expressing Mer^GFP particles move bidirectionally in processes (Inset) as shown by kymograph analysis (A′). (B and B′) Expression of Mer(T616A)^GFP, a nonphosphorylatable mutant, produces particles that move bidirectionally (Inset and B′), but mostly accumulate at the perinuclear region (Movie S7). (C and C′) Expression of Mer(T616D)^GFP, a phosphomimetic mutant, generates small particles that lack bidirectional movement (C′ and Movie S8) and are located at the cortex (Inset). (D) Comparison of particle distributions in Mer^GFP and its phosphomutants. The bar graph displays the percentage of particles found in the processes. (E) Immunoprecipitation of cell extracts with anti-GFP antibody. Input, crude cell extract; Mer^GFP, IP from extracts of cells expressing Merlin-GFP after heat shock; induction, IP from extracts from Mer^GFP cells before heat shock (control); T616D^GFP, IP from extracts from cells expressing Mer(T616D)^GFP; T616A^GFP, IP from extracts from cells expressing Mer(T616A)^GFP. Blots were done with anti-dynein and anti–kinesin-1 antibodies. The bottom part of the gel was stained with Coomassie blue for loading control (load).