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The projection domain of MAP2b regulates microtubule protrusion and process formation in Sf9 cells

Dave Bélanger^1,*, Carole Abi Farah^1,*, Minh Dang Nguyen², Michel Lauzon¹, Sylvie Cornibert¹ and Nicole Leclerc^1,

¹ Département de pathologie et biologie cellulaire, Université de Montréal, Montréal, Québec, Canada, H3T 1J8
² Montreal General Hospital Research Institute in Neuroscience, McGill University, Montréal, Canada, H3G 1A4
^* These authors contributed equally to the experimental work in this study

View larger version (18K): [in a new window]   Fig. 1. Baculoviral constructs of MAP2 expressed in Sf9 cells. (A) The three open boxes represent the three repeat sequences involved in the microtubule-binding activity of MAP2. The highlighted parts of the sequence correspond to the highly conserved regions. The percentage of cells with processes is indicated for each MAP2 construct. For details on the morphological analysis see Materials and Methods. (B) Sf9 cells were infected with the recombinant baculovirus containing the truncated forms of MAP2 at an m.o.i. of 5.0. At 72 hours post-infection, the cells were lysed and 30 µg of cell lysates was separated on SDS-PAGE (7.5%). The proteins were transferred to a nitrocellulose membrane and the expression of the constructs was revealed using the anti-MAP2 antibody HM2, except for the construct corresponding to the microtubule-binding domain (Mt), which was revealed with the antibody 46.1. The molecular mass of the standards are indicated on the left: myosin (209 kDa), ß-galactosidase (134 kDa), bovine serum albumin (84 kDa), carbonic anhydrase (40 kDa), soybean trypsin inhibitor (32 kDa) and lysozyme (19 kDa). In lane MAP2b, MAP2b-1 and Prob, the multiple bands are caused by the protein degradation that occasionally occurs during preparation of cell lysates.

View larger version (45K): [in a new window]   Fig. 2. Micrographs illustrating the different patterns of process formation induced by the expression of the MAP2b and MAP2c truncated forms in Sf9 cells. All truncated forms and full-length MAP2b and MAP2c were revealed with the anti-MAP2 antibody HM2, except for Mt, which was revealed with the antibody 46.1. Each protein was distributed uniformly in the cell body and along the processes. MAP2c and Mt induced the highest percentage of cells with multiple processes whereas MAP2b, MAP2b-1, MAP2b-2 and MAP2b-3 induced the formation of a unique process. Prob and Proc did not induce process outgrowth. Bar, 20 µm.

View larger version (28K): [in a new window]   Fig. 3. Histograms illustrating the quantitative analysis of process formation for each MAP2 construct. For cells expressing either a truncated or full-length MAP2c and MAP2b that had processes, the percentage of cells having one, two, or more than two processes per cell was analyzed. Three sets of experiments were analyzed and 150 cells were measured for each protein in each set of experiment. The distribution of cells having one, two, or more than two processes was analyzed by chi-square tests. Chi-square tests were performed to compare the pattern of process formation of the different MAP2 constructs. For all measurements, comparisons between the constructs were significant with a P<0.0033. The brackets indicate the constructs that are not statistically different.

View larger version (10K): [in a new window]   Fig. 4. Histogram illustrating the mean process length induced by the expression of the MAP2 truncated forms in Sf9 cells. Three sets of 50 cells were analyzed for each construct. The data are shown as mean±s.e.m. The length of process was analyzed by one-way ANOVA followed by the Sheffe test. The brackets indicate the constructs that are not statistically different (P>0.05).

View larger version (23K): [in a new window]   Fig. 5. Micrographs showing the distribution of microtubules and F-actin in Sf9 cells expressing the truncated forms of MAP2. The analysis was done by confocal microscopy. Cells were fixed at 72 hours post-infection and double-stained with an anti--tubulin antibody (DM1A, Sigma) and rhodamine-phalloidin (Molecular Probes) to reveal F-actin. Bar, 20 µm.

View larger version (30K): [in a new window]   Fig. 6. Micrographs showing the distribution of F-actin in cells expressing Prob and Proc. Cells were fixed at 72 hours post-infection and double-stained with an anti-His antibody, to reveal Prob and Proc, and rhodamine-phalloidin to visualize F-actin. Bar, 10 µm.

View larger version (229K): [in a new window]   Fig. 7. Electron micrographs illustrating longitudinal sections of (a) MAP2b, cell body, (b) MAP2c, cell body, (c) MAP2c, process, (d) MAP2b-1, process, (e) MAP2b-2, cell body, (f) MAP2b-2, process, (g) MAP2b-3, cell body, (h) MAP2b-3, process, (i) Mt, process, (i) Mt, process. Bars, 0.5 µm (c,f,h,i); 0.25 µm (a,b,e,g); 0.5 µm (d).

View larger version (60K): [in a new window]   Fig. 8. Purification of microtubules from Sf9 cells expressing the truncated forms of MAP2 and full-length MAP2b and MAP2c. Microtubules were prepared 64 hours following the infection as described in Materials and Methods. (A) The purity of microtubules was verified by Coomassie staining; 2 µg of microtubules were loaded per lane. Coomassie staining revealed microtubules as well as their associated protein. The numbers indicate MAP2b, MAP2c, MAP2b-1, MAP2b-2, MAP2b-3 and Mt, respectively. The molecular weight standards are (top to bottom): myosin (203 kDa), ß-galactosidase (135 kDa), bovin serum albumin (86 kDa) and carbonic anhydrase (42 kDa). On a 7.5% acrylamide gel, Mt and tubulin migrate at the same level and thus it is not possible to visualize Mt. However, Mt was revealed by dot blotting using the 46.1 antibody. (B) To verify the level of tubulin, proteins were transferred on a nitrocellulose membrane. The membrane was revealed with an anti-tubulin monoclonal antibody.

View larger version (35K): [in a new window]   Fig. 9. Sf9 cells co-infected with recombinant baculovirus containing Prob and Mt. Prob/Mt co-infected cells were double-stained with a polyclonal antibody anti-MAP2 (A), to visualize the expression of Prob, and the monoclonal antibody 46.1, to reveal the expression of Mt (D). Prob/Mt co-infected cells were also double-stained with the polyclonal antibody anti-MAP2 and a monoclonal antibody against -tubulin to visualize the formation of microtubules by Mt. Cells expressing Prob were double-stained with the polyclonal antibody anti-MAP2 and a monoclonal antibody against -tubulin to show that Prob does not induce the formation of microtubules in Sf9 cells. Bar, 40 µm.

View larger version (19K): [in a new window]   Fig. 10. Quantitative morphological analysis of Sf9 cells co-expressing the constructs Prob and Mt. A viral m.o.i. of 4 was used for each virus. (A) In Prob/Mt-expressing cells, the percentage of cells with processes was significantly lower than that of Mt-expressing cells. However, it was not the case in Proc/Mt-expressing cells (P<0.0166). (B) The percentage of cells presenting one, two, or more than two processes was quantified in Sf9 cells co-expressing Prob and Mt and having processes. The number of processes per Prob/Mt-expressing cell was statistically different from that of Mt-expressing cell. P<0.0166. The horizontal lines indicate the constructs that are statistically different. The reproducibility of the data from one experiment to another was analyzed using a chi-square test. Chi-square tests were also used to compare the number of processes per cell induced by the different MAP2 constructs.

View larger version (37K): [in a new window]   Fig. 11. Prob and Mt interact in Sf9 cells. Cells were co-infected with recombinant baculovirus containing Prob and Mt. At 72 hours post-infection, cells were lysed. Total cell lysates were immunoprecipitated with either the anti-MAP2 antibody, or with AP20 or HM2, which recognize Prob. The presence of Mt in the immunoprecipitates was analyzed by immunoblotting using the antibody 46.1. In lane 1, no antibody was added to the cell lysates prepared from Prob/Mt-expressing cells. In lane 2, the membrane was stained with AP20 to reveal the presence of Prob in the immunoprecipitates. In lane 3, the membrane probed with AP20 was stripped and stained with the antibody 46.1 to reveal the presence of Mt in the immunoprecipitates (arrowhead). In lane 4, Mt was immunoprecipitated using the antibody 46.1 from cells expressing only Mt. In lane 5, no antibody was added to the cell lysates prepared from Prob-expressing cells. In lane 6, the membrane was stained with AP20 to reveal the presence of Prob in the immunoprecipitates. In lane 7, the membrane probed with AP20 was stripped and stained with the antibody 46.1 to reveal the presence of Mt in the immunoprecipitates. No band corresponding to Mt was found in the immunoprecipitates. The arrow indicates the immunoglobulins.

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