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MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells - PubMed

  • ️Tue Jan 01 2013

MAP1B regulates microtubule dynamics by sequestering EB1/3 in the cytosol of developing neuronal cells

Elena Tortosa et al. EMBO J. 2013.

Abstract

MAP1B, a structural microtubule (MT)-associated protein highly expressed in developing neurons, plays a key role in neurite and axon extension. However, not all molecular mechanisms by which MAP1B controls MT dynamics during these processes have been revealed. Here, we show that MAP1B interacts directly with EB1 and EB3 (EBs), two core 'microtubule plus-end tracking proteins' (+TIPs), and sequesters them in the cytosol of developing neuronal cells. MAP1B overexpression reduces EBs binding to plus-ends, whereas MAP1B downregulation increases binding of EBs to MTs. These alterations in EBs behaviour lead to changes in MT dynamics, in particular overstabilization and looping, in growth cones of MAP1B-deficient neurons. This contributes to growth cone remodelling and a delay in axon outgrowth. Together, our findings define a new and crucial role of MAP1B as a direct regulator of EBs function and MT dynamics during neurite and axon extension. Our data provide a new layer of MT regulation: a classical MAP, which binds to the MT lattice and not to the end, controls effective concentration of core +TIPs thereby regulating MTs at their plus-ends.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1

Localization of MAP1B and EB1/3 in serum-starved N1E-115 neuroblastoma cells. Confocal images of differentiating N1E-115 cells, either bearing neurites (A, B) or flat (C, D). In (A, C), cells were co-stained with anti-MAP1B (N-t, red) and anti-EB1 (green) and in (B, D), cells were triple stained with anti-MAP1B (N-t, red), anti-EB3 (green) and anti-α-Tyrosinated-tubulin (blue). Scale bars=10 μm.

Figure 2
Figure 2

MAP1B overexpression displaces EBs from MT plus-ends. (A) Confocal pictures of N1E-115 cells transfected with MAP1B-GFP (green) and stained with an anti-EB1 antibody (red). Scale bar=10 μm. Insets show details of EB1 comets in a control (non-transfected) (a) versus an MAP1B-GFP transfected cell (a′). (B) EB1 comets per 100 μm2 in control cells and cells transfected with MAP1B-GFP. (C) Average length of EB1 comets. Error bars in (B, C) are s.e.m. Number of comets measured was n=419 (control) and n=415 (MAP1B-GFP transfected). (D) Average fluorescence intensity profiles of EB1 dashes in control and MAP1B-GFP-transfected cells. (E) Confocal images of N1E-115 cells transfected with MAP1B-GFP (green). Total MTs were visualized with anti-α-tubulin (red) and stable MTs with anti-α-Acetylated-tubulin (blue). (F) Quantification of the average fluorescence intensity (±s.e.m.) of the whole MT network (α-tubulin) and of stable MTs (α-Acetylated-tubulin) in control and MAP1B-GFP-expressing cells. No significant changes in MT density or stability were observed in transfected cells. **P<0.005.

Figure 3
Figure 3

MAP1B stable downregulation enhances binding of EB1/3 to MTs. N1E-115 stable cell lines generated by lentiviral infection with either scramble shRNA (control) or different specific MAP1B-shRNAs (1, 2, or a pool of 1+2+3). Reduction in MAP1B levels was confirmed by immunocytochemistry (A) (anti-MAP1B (N-t, red) and anti β3-tubulin (green)) and western blot (B), in which MAP1B, EB1 and EB3 levels were analysed and β-actin was used as a loading control. (C) Quantification of average number of EB1 comets/100 μm2 (±s.e.m.) in control and MAP1B-knocked down cells. (D) Average comet length (±s.e.m.) of EB1 or EB3-positive MT tails in control and MAP1B-deficient cells. Number of comets measured in each experimental condition was (a) EB1 staining; n=877 (control), n=1176 (shRNA pool); (b) EB3 staining; n=1497 (control), n=1165 (shRNA pool). Number of cells counted were n=26 (control) and n=55 (shRNA pool). (E) Confocal immunofluorescence pictures of control and MAP1B-silenced cells, co-stained with anti-EB1 (green) and anti-α-tubulin (red). Scale bar=10 μm. Details are presented in insets. (F) Average fluorescence intensity profile of EB1 dashes in control and MAP1B-depleted cells. EB1 interaction is enhanced upon MAP1B knockdown. (G) Quantification of the fluorescence intensity of the MT network (α-tubulin) (±s.e.m.) in control cells and in cells deficient in either MAP1B or tau. (H) Confocal pictures of control (scramble), MAP1B-depleted and tau-depleted cells, stained with anti-EB3. EB3-positive comets and segments are shown at higher magnification in insets. Scale bar=10 μm. Arrowheads in (E, H) point to MT segments highlighted by EB1 (E) or EB3 (H). ***P<0.0005. Source data for this figure is available on the online supplementary information page.

Figure 4
Figure 4

MAP1B interacts directly with EB1 and EB3. (A) N1E-115 cells were transfected with GFP (control) or GFP-tagged EB1 or EB3, either at their N- or C-terminal regions. Cells were serum starved overnight and co-IP assays were performed with an antibody against MAP1B (N-t). Expression of each construct was confirmed by western blot using anti-GFP (Inputs), and IP of MAP1B was corroborated with anti-MAP1B (lower blot). Co-IP of GFP-tagged EBs with MAP1B in control or Nocodazole-treated cells (10 μM, 20 min) was confirmed by western blot using an anti-GFP antibody. (B) Co-IP of endogenous MAP1B and EB3 proteins from E18 mouse brain lysates. (C) MAP1B from E18 mouse brain lysates was pulled down with GST-EB1 and GST-EB3 but not with GST (control). (D) In vitro pull-down assays of MAP1B-N-t (1–508)-6x-His with either GST or GST-EBs. The MAP1B-N-t (1–508) fragment interacts directly with GST-EBs but not with GST. Expression of each construct was confirmed and shown by Ponceau staining of the nitrocellulose membrane (C) or by Coomassie staining of the acrylamide gel (D) (n=3 in each case). Source data for this figure is available on the online supplementary information page.

Figure 5
Figure 5

EB3 mobility in distal neurites is regulated by MAP1B. Analysis of slow FRAP in control (scramble) and stably MAP1B-depleted (MAP1B-shRNA pool) N1E-115 cells. Cells were transfected with EB3-GFP, serum starved overnight, and either treated with vehicle (DMSO 0.1%, 1 h) or Nocodazole (10 nM, 1 h) and subjected to FRAP in distal neurites. Data collected from three different sets of experiments (∼8–15 cells/experiment) were normalized and fitted with a two-phase association equation. (A) k (1/s) corresponding to the slow phase of fluorescence recovery in vehicle-treated and Nocodazole-treated cells. (B) Curves of averaged actual FRAP data in control and MAP1B-depleted cells, upon Nocodazole treatment. (C) Representative example of time lapses of FRAP in distal neurites of control and MAP1B-silenced cells, treated with Nocodazole. *P<0.05.

Figure 6
Figure 6

MAP1B regulates EB3 dynamics at MT plus-ends. EB3-GFP was transfected into control (scramble) or MAP1B-depleted (with shRNA2 or a pool of shRNAs 1–3) neuroblastoma cells. Cells were serum starved overnight and EB3-GFP displacements were analysed. Examples of still images of control and knocked down cells are shown in (A) (Snapshot). Pictures were taken every 2 s for 30 frames. Z-maximal projections of the whole time-lapse series are shown in (A). Scale bar=10 μm. Details are shown in insets. A fragment of a representative time lapse of EB3-GFP-comet tracking (0–16 s) is shown for control and MAP1B-depleted cells in small insets under the main pictures (A). Arrows follow displacements of individual comets in the selected frames. EB3-GFP comets were manually tracked and different parameters were measured in control and MAP1B-silenced cells. (B) EB3-GFP comet length (μm). (C) Number of comets per 100 μm2. In (B, C), number of comets measured was n=382 (control), n=303 (MAP1B-shRNA2) and n=150 (MAP1B-shRNA pool). (D) MT growth speed (μm/s). Number of EB3-GFP comets tracked in each case was n=187 (control), n=189 (MAP1B-shRNA2) and n=77 comets (MAP1B-shRNApool). (E) Duration of MT growth events (s). Average values±s.e.m. are represented. *P<0.05, **P<0.005, ***P<0.0005.

Figure 7
Figure 7

MAP1B negatively regulates binding of EBs to MT plus-ends in developing neurons. Embryonic wild-type (wt) and Map1b−/− primary hippocampal neurons were cultured for 1 day, fixed and stained for MAP1B and/or different +TIPs. (A) Confocal images show wt neurons stained with anti-MAP1B (N-t, red) and anti-EB3 (green). Insets show details of the axon (a) and growth cone (a′). (B) Confocal pictures of wt and Map1b−/− neurons stained with antibodies against EB1 (red) and CLIPs (green). Picture of wt neuron is a composite of two images of the same cell. Insets show details of growth cones of wt (b) and Map1b−/− (b′) neurons. Scale bars=10 μm. (C) Immunofluorescence pictures of growth cones from wt and MAP1B-deficient neurons stained with anti-EB3. Note that EB3 localizes at MT segments in the Map1b−/− growth cone. (D) Average length of MT comets highlighted by CLIPs, EB1 or EB3 in wt versus Map1b−/− neurons. Quantification was performed in different cell compartments (axon, cell body and axonal growth cones). Comets highlighted by different +TIPs were longer in MAP1B-deficient neurons. Number of comets measured per staining and genotype was for CLIPs, n=1043 (wt) and n=1446 (Map1b−/−); for EB1, n=2285 (wt) and n=1999 (Map1b−/−) and for EB3, n=70 (control) and n=144 (Map1b−/− neurons). (E) Average fluorescence intensity patterns of EB3 comets in growth cones of wt and Map1b−/− neurons. **P<0.005, ***P<0.0005.

Figure 8
Figure 8

MAP1B controls MT dynamics during axon extension by regulating EB3 function in axons and growth cones. Embryonic wt and Map1b−/− primary hippocampal neurons were cultured and transfected with EB3-GFP 4 h after plating. Twenty-four hours later, cells were recorded by time-lapse confocal microscopy and EB3-GFP displacements were followed and analysed. Pictures were taken every second (1 s) for 120 s. (A) Representative examples of growth cones from wt (A) and Map1b−/− neurons (B). Snapshots show still images in each case and Z-Max Projections represent the maximal projection of the 120 images taken. Inverted grey scale is shown to visualize EB3-GFP comets and trajectories more clearly. Insets with details of EB3-GFP comets displacements are shown in (A, B). Quantifications of different parameters related to EB3-GFP displacements were performed in wt and Map1b−/− neurons. We focused on axons and axonal growth cones, with most EB3-GFP comets moving anterogradely. (C) Quantification of the direction of EB3-GFP comet displacements in growth cones. MAP1B-deficient neurons present an increase in the number of comets moving backwards from the leading edge of the growth cone. Number of comets measured per genotype was n=204 (wt) and n=102 (Map1b−/−). Number of neurons taken was n=10 (wt) and n=12 (Map1b−/−). (D) MT growth speed in axons and growth cones (GC). (E) Number of pauses counted per 100 displacements of EB3-GFP comets in axons and GC. (F) Average duration of EB3-GFP comet pauses in axons and GC. (G) Quantification of the different behaviours of comets that undergo pauses in growth cones. After having paused, EB3-GFP comets continued moving (MTs that resumed growing), remained paused (pausing MTs) or disappeared (shrinking MTs). (H) Time EB3-GFP comets were followed in axons and GC (duration of MT growth events). (I) Distribution of duration of MT growth events in axons and GC (from 0–5 to >30 s). Average values±s.e.m. are represented. *P<0.05, ***P<0.0005.

Figure 9
Figure 9

Model of regulation of EBs localization by MAP1B during neurite/axon outgrowth. In control/wt cells, MAP1B negatively controls binding of EB1/3 to MTs in extending neurites/axons and growth cones, thereby regulating MT dynamics and growth cone advance during neuritogenesis and axonogenesis. In MAP1B deficient neuronal cells, interaction of EB1/EB3 to MTs is enhanced, leading to MT overstabilization and looping.

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