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doi: 10.1242/10.1242/jcs.00148

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Effects of Charcot-Marie-Tooth-linked mutations of the neurofilament light subunit on intermediate filament formation

Department of Pathology, Columbia University College of Physicians & Surgeons, 630 West 168th Street, New York, NY 10032, USA

View larger version (51K): [in a new window]   Fig. 1. Sequence alignment of NFL clones, location of mutations and western-blotting of transfected proteins with anti-NFL antibody. (A) Multiple sequence alignment of the hNFL protein encoded by our hNFL cDNA (hNFL), the previously published hNFL sequence (hNFL P09716), and the published rNFL sequence (rNFL). All the differences in the protein sequences are marked by vertical boxes. Differences in the amino acid sequence between hNFL and hNFL (P09716) are marked with an asterisk (*), while amino acids that differ between hNFL and both hNFL (P09716) and rNFL are indicated with a hash mark (#). (B) Schematic representation of NFL protein and the positions of the mutant and variant amino acids. The head, rod and tail domains are indicated. The rod domain is divided into coils 1A, 1B, 2A and 2B separated by linkers L1, L12 and L2. The CMT2 mutations are: P8R in the head domain and Q333P in the coil 2B. The D469N variant is in the tail domain. (C) Western blots of transfected human (left) and rat (right) NFL proteins. Cytoskeletal extracts of transfected cells were separated by SDS-PAGE and subjected to western blotting with a polyclonal anti-NFL antibody. The antibody recognized a band of the same size in wild-type NFL (wt), variant NFL (D469N), head mutant NFL (P8R), and rod mutant NFL (Q333P). The bar indicates the position of the 64 kDa size marker.

View larger version (31K): [in a new window]   Fig. 2. Co-transfections of wild-type and variant rNFL with rNFM or rNFH in SW13Vim- cells. (A-D) Rat NFM was co-transfected with wild-type rNFL (A,B) or D469N variant rNFL (C,D). The cells were double-labeled with polyclonal anti-NFL antibody (A,C) and monoclonal anti-NFM antibody (B,D). For both wild-type and variant NFL, a filamentous network throughout the cytoplasm was observed that stained with both antibodies. (E-H) Rat NFH was co-transfected with wild-type rNFL (E,F) or D469N variant rNFL (G,H). The cells were double-labeled with polyclonal anti-NFL antibody (E,G) and monoclonal anti-NFH antibody (F,H). A filamentous network was formed with both wild-type and variant rNFL co-transfected with rNFH. In all the subsequent experiments, the variant NFL behaved identically to wild-type NFL. Bar, 25 µm.

View larger version (44K): [in a new window]   Fig. 3. Co-transfections of mutant rNFL with rNFM or rNFH in SW13 Vim- cells. (A-F) Rat NFM was co-transfected with P8R mutant rNFL (A-D) and Q333P mutant rNFL (E,F). The cells were double-labeled with polyclonal anti-NFL antibody (A,C,E) and monoclonal anti-NFM antibody (B,D,F). The P8R mutant rNFL co-transfected with rNFM either formed a bundle of filaments (A,B) or displayed punctate or aggregate staining (C,D). The Q333P mutant rNFL resulted in aggregates that contained rNFM (E,F). (G-L) Rat NFH was co-transfected with P8R mutant rNFL (G-J) and Q333P mutant rNFL (K,L). The cells were double-labeled with polyclonal anti-NFL antibody (G,I,K) and monoclonal anti-NFH antibody (H,J,L). The P8R mutant co-assembled into bundled filaments with rNFH in most cases (G,H), or into more punctate material or aggregates (I,J), whereas the Q333P mutant rNFL resulted only in the formation of aggregates with rNFH (K,L). Bar, 25 µm.

View larger version (29K): [in a new window]   Fig. 4. Transfection of hNFL clones in SW13 Vim- cells. (A-D) Transient transfection results of wild-type (A,B), P8R mutant (C) and Q333P mutant (D) hNFL. Staining was carried out with monoclonal anti-NFL antibody. Wild-type hNFL generally assembled into short filaments (A), but occasionally also formed a homopolymeric filamentous network (B) that was not stained with anti-vimentin antibody (not shown). The P8R and the Q333P hNFL mutants (C,D) were not capable of self-assembly into a filamentous network, but formed aggregates. (E-F) Co-transfections and staining of wild-type hNFL with the P8R mutant hNFL (E) and Q333P mutant hNFL (F) showed that the mutant proteins disrupted the self-assembly of wild-type hNFL. Results similar to those in F were obtained with a bicistronic construct that expressed both wild-type and Q333P mutant hNFL (not shown). Bar, 25 µm.

View larger version (35K): [in a new window]   Fig. 5. Co-transfection of hNFL constructs with hNFM in SW13 Vim- cells. hNFM was co-transfected with either wild-type hNFL (A,B), P8R mutant hNFL (C,D) and Q333P mutant hNFL (E,F). The cells were double-labeled with polyclonal anti-NFL (A,C,E) and monoclonal anti-NFM (B,D,F) antibodies. A filamentous network throughout the cytoplasm was observed for wild-type hNFL and hNFM (A,B). The P8R mutant hNFL formed bundled filaments that stayed on one side of the cell (C,D; cell on the left) or around the nucleus (C,D; cell on the right). The Q333P mutant NFL always formed aggregates with hNFM (E,F). Bar, 25 µm.

View larger version (32K): [in a new window]   Fig. 6. Transient transfection of hNFL constructs in SW13 Vim+ cells. Transfection of wild-type (A,B), P8R mutant (C,D) and Q333P mutant (E-H) hNFL. The cells were double-labeled with monoclonal anti-vimentin (A,C,E,G) and polyclonal anti-NFL (B,D,F,H) antibodies. Both wild-type hNFL (A,B) and D469 hNFL (not shown) incorporated into the endogenous vimentin network. The P8R mutant hNFL protein incorporated into the endogenous vimentin network in most cases (C,D). The co-assembly of the Q333P mutant hNFL with endogenous vimentin was dependent on their relative levels of expression (E-H). At lower levels of expression the mutant Q333P hNFL incorporated into the vimentin network (E,F), while at higher levels of expression it aggregated in the cytoplasm (G,H). Bar, 25 µm.

View larger version (141K): [in a new window]   Fig. 7. Transfection of hNFL does not affect the microtubule network of SW13 Vim- cells. Transient transfection of wild-type hNFL (A), D469N variant hNFL (B), P8R mutant hNFL (C) and Q333P mutant hNFL (D) into SW13 Vim- cells. Cells were co-stained with monoclonal anti-ß-tubulin antibody (green) and polyclonal anti-NFL antibody (red). Shown are the merged images corresponding to staining with both antibodies. Transfection of hNFL constructs did not affect the microtubule network, and NFL and tubulin did not colocalize.

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