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First published online 2 August 2005
doi: 10.1242/jcs.02510

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Microtubule actin crosslinking factor 1b: a novel plakin that localizes to the Golgi complex

¹ Department of Pathology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA
² Institute of Fundamental Sciences, Massey University, Private Bag 11 222, Palmerston North, New Zealand

View larger version (21K): [in a new window]   Fig. 1. MACF1 gene and protein isoforms. (A) Organization of mouse Macf1 gene. Exons encoding the following domains are illustrated: CH domains, plakin domain, plakin repeats domain (PRD), spectrin repeats, EF hands (EF) and Gas2-related (GAR) domain. Alternatively spliced first exons that are responsible for generating isoform 1 (Iso1), isoform 2 (Iso2) and isoform 3 (Iso3) are in gray. The ATG start site of isoform 1 has not been identified. The region that undergoes alternative splicing to generate MACF1a and MACF1b is enlarged. (B) Domain organization of MACF1 isoforms. MACF1b contains two A PRDs (labeled A), one C PRD (labeled C), one incomplete B (labeled b) and one incomplete A (labeled a) PRD. Below the schematic drawing of MACF1b, bold lines indicate cDNA clones (R1, R2, R3, R4 and R5) and fine lines represent genomic PCR products (G1, G2 and G3). Clone names are shown above each line for cDNA clones and on the right for genomic products. The slashed bar (NB1) indicates the position of the probe for both MACF1a and MACF1b used for the northern blot; the gray bar (NB2), the position of the MACF1b specific probe used for the northern blot; the black bar, the region of MACF1b that was used for generating the CU149 antibody; white bar, the region that was used to generate the CU119 antibody (for all MACF1 isoforms).

View larger version (49K): [in a new window]   Fig. 2. Northern blot analysis of MACF1. Expression of MACF1 (MACF1a/b) in adult mouse tissues. A multiple tissue blot was hybridized with 32P-labelled cDNA probes that recognized the plakin domain of both MACF1a and MACF1b (NB1 in Fig. 1B) or the PRD region of MACF1b (NB2 in Fig. 1B). Ethidium bromide stained 28S ribosomal RNAs were used as the loading control.

View larger version (14K): [in a new window]   Fig. 3. Western blotting with anti-MACF1b antibody. (A) Analysis of rabbit polyclonal CU149 antibody. Cell lysate (80 µg) collected from pFLAG-MACF1b-PRD-transfected COS7 cells was subjected to western blotting with mouse monoclonal anti-FLAG antibody (M2), CU149 (149), and preimmune serum of the rabbit that generated CU149 (pres). CU149 and M2 both recognized FLAG-MACF1b-PRD protein (FMP) that was not detected by the preimmune serum. A molecular size standard (kDa) is indicated on the left. (B) Detection of MACF1b in HaCaT cells. HaCaT cell lysate was subjected to immunoblotting with CU149 (MACF1b). Only one slow mobility band was detected. (C) Specificity of CU149 antibody to MACF1b. H460 cells were transfected with MACF1 siRNA vector (MACF siRNA) that was designed to knockdown MACF1a/b, or control vector (Control) for 72 hours. Cell lysates were probed for either MACF1b (top panel) or ß-tubulin (loading control; lower panel). MACF1b protein in cells transfected with MACF siRNA vector was reduced significantly compared to cells transfected with the control vector. (D) Detection of MACF1b proteins in the enriched Golgi fraction. H460 cell lysate was subjected to sucrose step gradient centrifugation, Golgi and ER fractions were isolated, resolved in SDS-PAGE, transferred to PVDF membranes, and probed with CU149 antibody (MACF1b; top panel), anti-p115 antibody (p115; middle panel), and anti-FTCD antibody (FTCD; lower panel). Crude: crude lysate. CU149 only recognizes MACF1b proteins in enriched Golgi fraction of H460 cells. A molecular size standard (233 kDa) is indicated.

View larger version (37K): [in a new window]   Fig. 4. Characterization of anti-MACF1 (CU119) antibody. (A) Detection of the full-length plakin domain of MACF1 proteins by CU119 antibody. COS7 cells were transfected with plasmid pFLAG-MACF1-plakin and cell lysate was resolved by SDS-PAGE. The transblot was then probed with mouse monoclonal anti-FLAG antibody (M2) and anti-MACF1-plakin antibody (CU119). The expressed full-length MACF1-Plakin protein (FL-plakin) can be detected by both M2 and CU119 antibodies, but not in mock-transfected control (C). (B) Detection of endogenous MACF1 proteins by CU119 antibody in CAD cells. Neuronal CAD cells were lysed and resolved by SDS-PAGE, and the transblot was probed with CU119 antibody. MACF1 proteins can be detected in CAD cells (lane 2), and this signal can be completely abolished by pretreatment of CU119 antibody with antigen (lane 1). (C) Specificity of CU119 antibody revealed by the detection of MACF1 proteins in siRNA-treated cells. H460 cells were transfected with MACF siRNA vector (MACF-siRNA) that was designed to knockdown MACF1, or control vector (Control) for 72 hours. After SDS-PAGE, the transblot was probed for either MACF1 (top panel) or ß-tubulin (loading control; lower panel). MACF1 proteins in cells transfected with MACF siRNA vector (MACF siRNA) were reduced compared to cells transfected with the control vector (Control). (D) Localization of MACF1 in H460 cells. H460 cells were stained for MACF1 (left panel; CU119 antibody that recognizes all isoforms; green) and ß-tubulin (middle panel; red). MACF1 protein was found around the nuclei, dotted in the cytoplasm and decorating microtubules (right panel; merged picture).

View larger version (90K): [in a new window]   Fig. 5. Localization of MACF1b to the Golgi complex in lung cell lines. H460 (A,B) and A549 (C,D) cells were stained with antibodies against MACF1b (A,C) and GM130 (B,D). MACF1b staining was of a widespread vesicular pattern with strong labeling near the Golgi complex. Bar, 20 µm.

View larger version (115K): [in a new window]   Fig. 6. Localization of MACF1b proteins, microtubule elements and actin microfilaments in H460 cells. H460 cells were stained for MACF1b (A,C), ß-tubulin (B) or actin (D). In addition to Golgi staining, staining of MACF1b proteins was strong around the nucleus and a dotted pattern in the cytoplasm that partially decorated the microtubule network. Bar, 20 µm.

View larger version (53K): [in a new window]   Fig. 7. Displacement of MACF1b in H460 cells treated with drugs that disrupt microtubules, Golgi or actin. (A-D) MACF1b colocalizes with the displaced Golgi ministacks in H460 cells treated with nocodazole and brefeldin A. H460 cells were treated with nocodazole [NZ] for 1 hour (A,B) or brefeldin A [BFA] for 1hour (C,D) and stained for MACF1b (A,C) or GM130 (B,D). MACF1b proteins colocalized with the dispersed Golgi ministacks in the cytoplasm after nocodazole or brefeldin A treatments. (E,F) Localization of MACF1b in H460 cells after cytochalasin D treatment [Cyt.D] for 1 hour. Cells were then stained for MACF1b (E) or GM130 (F). MACF1b remained on the Golgi. Bar, 20 µm.

View larger version (48K): [in a new window]   Fig. 8. Targeting of MACF1b-PRD to the Golgi complex in COS7 cells. (A) Illustration showing MACF1b PRD and cDNA clones that were used to generate HA-tagged MACF1b PRD constructs. FL, all PRD subdomains; N, two A sundomains; C1, C subdomains and the two incomplete B and A subdomains; C2, C-terminal region that did not include any PRDs. (B) Expression of HA-tagged MACF1b-PRD proteins in COS7 cells. COS7 cells that were transfected with various cDNA constructs (FL, N, C1, C2) were collected for western blot analysis with anti-HA antibody. Mk, mock-transfected cells. Bars on left indicate molecular mass standards: from top to bottom (kDa), 181.8, 115.5, 82.2, 64.2, 48.4 and 37.1. (C-H) Immunostaining of cells that were transfected with different cDNA clones of MACF1b-PRD. COS7 cells were transfected with cDNA constructs FL (C,D), N (E-H), and immunostained with antibodies against HA-tag (C,E,G) or GM130 (D,F,H). Bar, 20 µm.

View larger version (70K): [in a new window]   Fig. 9. Knockdown of MACF1b resulted in the disassembly and dispersion of the Golgi complex in H460 cells. H460 cells transfected with control vector (Control; A and B, E and F) or MACF-siRNA vector (MACF-siRNA; C and D, G and H) were fixed and stained for MACF1b (B,D), GM130 (F,H), and GFP (A,C,E,G). The siRNA plasmid contained a GFP cassette to identify the transfected cells. Note that MACF1b staining in cells treated with MACF1-siRNA (GFP positive) was greatly reduced (compare D with B) and the Golgi complex was disassembled (asterisks in D and H). Arrowhead in D indicates a cell that was not transfected with the MACF-siRNA vector (GFP negative) and displayed an undisrupted Golgi pattern. Bar, 20 µm.