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Results: 7

1.
Figure 3

Figure 3. Numb Colocalizes With α-adaptin and TrkB in Leading Processes of Migrating GCPs. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

Phase images (Phase) of GCPs co-cultured with Bergmann glia in vitro (A5, B5, C1, C5 and D1). Broken lines: Bergmann glia. DAPI stain (blue). Scale bar: 5 μm.
(A) Numb colocalizes with α-adaptin in the leading processes of GCPs in the inner EGL in vivo (A1-A4) and GCPs migrating along Begmann glia fibers in response to BDNF in vitro (A5-A8). Arrows indicate colocalized Numb and α-adaptin.
(B) Numb colocalizes with TrkB in the leading processes of GCPs in the inner EGL in vivo (B1-B4) and in GCPs migrating along Bergmann glia in response to BDNF in vitro (B5-B8). Arrows indicate the leading process and the front of the cell where Numb and α-adaptin colocalize.
(C) TrkB (C1-C4) or activated TrkB (p-Trk) (C5-C8) colocalizes with α-adaptin (green) in the leading processes of GCPs migrating along Bergmann glia in response to BDNF. Arrows indicate the leading process and the front of the cell where TrkB (red) and α-adaptin colocalize.
(D) Activated TrkB (red) receptors are localized to the leading process, not the Golgi apparatus (green) of GCPs migrating along Bergmann glia in response to BDNF in vitro. GCPs stained with anti-phospho-Trk (Tyr490) (p-Trk). Arrows indicate the leading process and the front of the cell where activated TrkB was detected.

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
2.
Figure 5

Figure 5. BDNF Gradient Promotes Numb Polarization. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) Purified GCPs were exposed to vehicle (A1-A3), uniform (A4-A6) or gradient BDNF (A7-A9) for 12 hours in vitro, then stained with anti-Numb (green). BDNF source is at the top. DAPI stain (blue). Arrows indicate representative GCPs with Numb polarization. Scale bar: 5 μm.
(B) Analysis of Numb polarization in GCPs in response to BDNF as shown in (A). For each condition (no BDNF, uniform and gradient BDNF), the number of GCPs with Numb polarization toward the BDNF source was normalized to that of GCPs with Numb polarization away from the BDNF source (**, P<0.001; n = 4).
(C) Representative images of Numb polarization in vivo. Cerebellar sections from P7 control (C1-C4) and bdnf−/− mutant mice (C5-C8) were stained with anti-TrkB (red), anti-Numb (green). DAPI stain (blue). Scale bar: 5 μm.
(D) Analysis of Numb polarization in EGLb of control and bdnf−/− mice in (C) as described in Figure S7 (*, P<0.05; n = 4).

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
3.
Figure 2

Figure 2. Numb Interacts With TrkB. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) pcDNA3-TrkB-Flag was co-transfected with empty vector or pcDNA3-myc-Numb into HEK 293T cells. Lysates were precipitated with anti-Flag or anti-Myc, and immunoprecipitates (IPs) were blotted with anti-Myc or anti-Flag. Lysates were blotted for expression control.
(B) Numb interacts with TrkB in GCPs. GCPs were stimulated with BDNF or vehicle for 10 minutes; lysates were precipitated with anti-Numb, then blotted with anti-TrkB. Membrane was stripped and re-probed with anti-Numb.
(C) Schematic of Numb structure and truncated Numb proteins.
(D) Numb interacts with TrkB in developing cerebellum. Immobilized full-length (FL) or truncated Numb on sepharose beads was incubated with cerebellar lysates (P7) and blotted with anti-TrkB.
(E) TrkB kinase activity is required for interaction with Numb. pcDNA3-Myc-Numb was co-transfected with empty vector or pcDNA3-TrkB-Flag wild-type (wt), Y515F or K571M mutant into HEK 293T cells. Lysates were precipitated with anti-Myc or anti-Flag, and blotted with anti-Flag or anti-phosphotyrosine (pY). Membrane was re-probed with anti-Myc or anti-Flag.
(F) Numb directly binds TrkB in vitro. Bacteria-derived GST-Numb (FL) immobilized on sepharose beads was incubated with purified TrkB (FL) or His-TrkB cytoplasmic tail (CT) and blotted with anti-TrkB and anti-His.
(G) ATP enhances TrkB interaction with Numb. GST or GST-TrkB cytoplasmic tail (CT) immobilized on sepharose beads was incubated with purified Numb (FL) in the presence or absence of ATP, and blotted with anti-Numb.

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
4.
Figure 1

Figure 1. Numb Is Required For GCP Migration. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) GCP migration is impaired in conditional mutant mice. Dividing GCPs in P10 conditional mutants (Math1-Cre/Numbflx/flx/Numblflx/flx) and control littermates (Numb flx/flx/Numblflx/flx) were labeled with BrdU. Cerebellar sections were stained with anti-BrdU (red) and DAPI (blue) at indicated times post-injection. EGL, external germinal layer; ML, molecular layer; PCL, Purkinje cell layer; IGL, internal granule cell layer. Scale bar: 50 μm.
(B) Migrated granule cells in IGL. At 36, 72 and 96 hours post-BrdU labeling, the number of migrated granule cells in the IGL is reduced in conditional mutant mice (*P<0.05).
(C) Migrating granule cells in the ML. At 72 and 96 hours post-BrdU labeling, more migrating granule cells remain in the ML of conditional mutant mice (*, P<0.05).
(D) Conditional deletion of Numb/Numbl impairs BDNF-induced GCP chemotaxis in vitro. GCPs from P6 cerebella of conditional mutant mice (Math1-Cre/Numbflx/flx/Numblflx/flx) or control littermates (Numbflx/flx/Numblflx/flx) were placed in the upper chamber of Transwell apparatus; cells that migrated to the lower side of the membrane after 18 hours were counted. For each experiment, the number of migrating cells was normalized to that of migrating GCPs from wild-type mice in the absence of BDNF (*, P< 0.05; **, P< 0.05 and ***, P< 0.001; n=4).
See also Figure S1-6.

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
5.
Figure 4

Figure 4. Numb Deletion Impairs BDNF-induced TrkB Endocytosis and Polarization. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) Conditional deletion of Numb/Numbl inhibits BDNF-induced TrkB endocytosis in GCPs. GCPs from conditional Numb/Numbl or control littermate mice were treated with BDNF as indicated. Surface or total TrkB level was measured by staining with anti-TrkB as described in Methods. Relative surface TrkB for each time point was calculated by dividing the value of surface TrkB signal by that of total TrkB signal, and normalized to relative surface TrkB in that experiment at time 0 (*, P<0.05; n=4).
(B) Time course of BDNF-stimulated TrkB internalization measured by biotinylation of cell surface molecules and detection of internalized TrkB by blot with anti-TrkB. BDNF induces rapid TrkB internalization and this continues through 2 hours.
(C) Knockdown of Numb attenuates BDNF-induced TrkB internalization. GCPs infected with Numb-specific shRNA or control shRNA lentivirus were cultured and treated with BDNF for 30 minutes on ice, and labeled with biotin for 30 minutes. Biotinylated cells were incubated at 37 °C or on ice for 2 hours. Biotin moieties on the cell surface were removed with reduced glutathione. Cells were lysed and precipitated with streptavidin sepharose. Internalized biotinylated-surface TrkBs were blotted with anti-TrkB. Aliquots of lysates were blotted with anti-Numb. Data shown represent one of three independent experiments.
(D) Conditional deletion of Numb/Numbl impairs TrkB polarization. Cerebellar sections from P7 conditional mice (Math1-Cre/ Numbflx/flx/Numblflx/flx) (D4-D6) and control littermates (Numbflx/flx/Numblflx/flx) (D1-D3) were stained with anti-TrkB (red). DAPI stain (blue). Scale bar: 5 μm.
(E) Analysis of TrkB polarization in the inner EGL (EGLb) of Math1-Cre/Numbflx/flx/Numblflx/flx mutant and control littermate mice as described in Figure S7B (*, P<0.05; n = 8).
See also Figure S7.

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
6.
Figure 6

Figure 6. Numb Interacts With aPKC to Promote GCP Chemotaxis. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) PKCζ interacts with Numb. 293T cells were transfected with pcDNA3-myc-Numb plasmids, and lysates were precipitated with control IgG or anti-Myc then blotted with anti-PKCζ. Membrane was reprobed with anti-Numb.
(B) PKCζ interacts with Numb in cerebellar GCPs. GCP lysates (P7) were immunoprecipitated with anti-PKCζ or control IgG then blotted with anti-Numb. Membrane was reprobed with anti-PKCζ.
(C) BDNF stimulation promotes PKCζ interaction with Numb in GCPs. GCPs from P7 mice were treated with vehicle or BDNF for 10 minutes; lysates were precipitated and blotted with indicated antibodies.
(D) BDNF deletion impairs PKCζ interaction with Numb. Lysates from P7 wild type and bdnf−/− cerebellum were precipitated with anti-Numb followed by blot with anti-PKCζ and anti-Numb.
(E) Numb binds active PKCζ. GST-Numb or GST immobilized on sepharose beads was incubated with purified His-tagged active PKCζ at 30 °C for 1 hour. Bound protein complex was washed and analyzed by blot with anti-His and anti-Numb, respectively. First lane, His-PKCζ alone.
(F) Time course of PKCζ activation by BDNF. GCPs from P7 were stimulated with BDNF for indicated times; lysates were blotted as indicated.
(G) Inhibition of TrkB kinase blocks BDNF-induced PKCζ activation. GCPs were treated with K252a for 30 minutes prior to BDNF stimulation; lysates blotted as indicated.
(H) Numb is required for BDNF-induced aPKC activation. GCPs from P7 Math1-Cre/Numbflx/flx/Numblflx/flx or control littermates (Numbflx/flx/Numblflx/flx) were treated with BDNF or vehicle. Lysates were blotted with indicated antibodies.
(I) Inhibition of PKCζ activity impairs BDNF-induced GCP chemotaxis. GCPs from P6 were treated with 10 μM chelerythrine (CHT) or vehicle and a BDNF chemotaxis assay was performed using Boyden chamber (*, P<0.05; **, P< 0.001; n=3).
(J) Inhibition of aPKC activity by CHT prevents BDNF-induced Numb polarization in GCPs. For each condition (uniform and gradient BDNF), the number of GCPs with Numb polarization toward the plug (BDNF source) was normalized to that of GCPs with Numb polarization away from the plug (**, P<0.001; n = 3).
(K) Inhibition of aPKC activity by CHT prevents BDNF-induced TrkB polarization in GCPs. For each condition (uniform and gradient BDNF), the number of GCPs with TrkB polarization toward the agarose plug (BDNF source) was normalized to that of GCPs with TrkB polarization away from the agarose plug (**, P<0.01; n = 3).

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.
7.
Figure 7

Figure 7. Numb Is A Substrate of aPKC. From: Numb links extracellular cues to intracellular polarity machinery to promote chemotaxis.

(A) PKCζ phosphorylates Numb in a dose-dependent manner. Purified GST-Numb immobilized on glutathione sepharose beads was incubated with purified His-PKCζ. Bound protein complex was detected by blot with a monoclonal antibody phospho-Ser/Thr (MPM2) or antibody against phospho-Numb (p-Numb (S284)).
(B) Specificity of anti-phospho-Numb (S284). HEK 293T cells were transfected with plasmids expressing wt Numb or Numb mutant (S284A). Lysates were precipitated with anti-Numb. Numb IPs were incubated with or without active PKCζ, and blotted with anti-p-Numb (S284) and anti-Numb, respectively.
(C) BDNF stimulates Numb phosphorylation by PKCζ in vivo. GCPs from P7 mice were treated with 50 ng/ml BDNF for 5 minutes in the presence or absence of phosphatase inhibitor calyculin-A and/or 10 μM PKC inhibitor chelerythrine. Lysates were blotted with anti-p-Numb (S284) and anti-Numb, respectively.
(D) Phosphorylated Numb (green) colocalizes with TrkB (red) in the leading processes of GCPs along Bergmann glia in response to BDNF. Staining with p-Numb (S284) antibody and TrkB antibody. DAPI stains nuclei (blue). Scale bar: 5 μm
(E) Numb mutations at PKCζ phosphorylation sites reduce Numb interaction with TrkB. HEK 293T cells co-transfected with plasmid expressing TrkB-GFP and plasmid expressing wild type Numb (wt) or Numb mutant for all three aPKC phosphorylation sites (mut), and treated with vehicle or BDNF. Lysates precipitated with anti-GFP and blotted with anti-Numb and anti-GFP, respectively.
(F) Model for Numb function in cell migration. In response to a BDNF gradient, Numb associates with activated TrkB to regulate receptor endocytosis and recruit aPKC. Activated aPKC then phosphorylates Numb. Phosphorylation of Numb serves in a feed-forward loop to potentiate binding of Numb to TrkB and thereby fosters formation of TrkB/Numb endosomal complexes. Endocytosis is required for Tiam1-mediated Rac activation and localization of signaling endosomes to leading processes and/or for recycling TrkB to plasma membrane in leading processes.

Pengcheng Zhou, et al. Dev Cell. ;20(5):610-622.

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