1. Volume 90, Issue 3, Pages 551-563 (May 2016)
Phosphorylation of β-Tubulin by the Down Syndrome Kinase, Minibrain/DYRK1a, Regulates Microtubule Dynamics and Dendrite Morphogenesis 
Kassandra M. Ori-McKenney, Richard J. McKenney, Hector H. Huang, Tun Li, Shan Meltzer, Lily Yeh Jan, Ronald D. Vale, Arun P. Wiita, Yuh Nung Jan 
Neuron 
Volume 90, Issue 3, Pages (May 2016)
DOI: /j.neuron
Copyright © 2016 Elsevier Inc. Terms and Conditions

2. Figure 1
Drosophila melanogaster Class IV and Class III DA Neurons Develop Distinct Terminal Branch Morphologies with Different Cytoskeletal Compositions
(A) Dendrite morphologies of class IV and class III wild-type neurons, visualized by UAS-CD4-tdGFP expressed by ppk-Gal4 and 1912-Gal4 for class IV and class III, respectively (scale bars, 30 μm). Enlarged regions illustrate the differences in terminal branch length and actin composition between class IV and class III terminal branches (scale bars, 10 μm). UAS-CD4-tdGFP and UAS-LifeAct-tdTOM were expressed in class IV and class III DA neurons by ppk-Gal4 and 1912-Gal4, respectively.
(B) Quantification of the percent of terminal branches that contain dynamic MTs, as evidenced by EB1 entry into a terminal branch, for class III DA neurons versus class IV DA neurons (p < ; n = 7 neurons for class IV and 23 neurons for class III DA neurons). UAS-EB1-GFP was expressed in class IV and class III DA neurons by ppk-Gal4 and Gal , respectively.
(C) Quantification of the number of EB1 comets that grow along the primary branches of class IV and class III DA neurons (p = 0.10; n = 11 neurons for class IV and 6 neurons for class III DA neurons).
(D) Quantification of terminal branch length in class III and IV DA neurons (p < ; n = 3 neurons per genotype). All graphs are box plots with min to max whiskers, which include all data points.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

3. Figure 2
MNB/DYRK1a Kinase Mutants Perturb the Dendritic Architecture of Class III DA Neurons
(A) Dendrite morphologies of class III wild-type neurons compared with those of class III mnb3 and mnb1 mutant DA neurons, 1912-Gal4 > UAS-mnb-RNAi neurons, 1912-Gal4 > UAS-mnb OE neurons, and mnb1 mutant neurons rescued with 1912-Gal4 > UAS-MNB. Neurons were visualized using UAS-CD4-tdGFP expressed by 1912-Gal4 (scale bars, 30 μm). Enlarged pictures show the difference in terminal branch length between each genotype (scale bars, 10 μm).
(B) Quantification of the average terminal branch length for each genotype (one star indicates p < 0.05 and two stars indicate p < 0.005; n = 3 neurons per genotype). Graph is a scatterplot of all data points with the line indicating the mean.
(C) Quantification of the total neuronal branch length for each genotype (one star indicates p < 0.05 and two stars indicate p < 0.005; for mnb rescue, p = 0.56; n = 3 neurons per genotype). Graph is a box plot with min to max whiskers, which include all data points.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

4. Figure 3
MNB Regulates Microtubule Dynamics within Both the Terminal and Primary Branches of Class III DA Neurons In Vivo
(A) Movie montage depicts the movement of EB1-GFP comets within the terminal branches of an mnb1 mutant neuron (scale bars, 2 μm). Blue and red arrows indicate the starting and ending positions of two comets growing into a particular terminal branch. Due to bleaching over time, the contrast of the last four panels is increased compared to the first two montage panels in order to see the EB1 comets.
(B) Quantification of the percent of terminal branches that contain dynamic MTs, as evidenced by EB1 entry into a terminal branch, for each genotype (two stars indicate p < ; for mnb OE, p = 0.52; n = 23, 7, 7, 7, and 10 neurons for wild-type, mnb3, mnb1, mnb RNAi, and mnb OE, respectively).
(C) Quantification of the number of EB1 comets in the primary branches of class III DA neurons for each genotype (two stars indicate p < ; for mnb3, p = 0.66; for mnb1, p = 0.18; for mnb RNAi, p = 0.86; n = 23, 6, 12, 7, and 22 neurons for wild-type, mnb3, mnb1, mnb RNAi, and mnb OE, respectively). All graphs are box plots with min to max whiskers, which include all data points.
(D) Kymographs showing the number of EB1-GFP comets growing along the primary branches of wild-type, mnb3, mnb1, and mnb OE neurons (scale bars, 2.5 μm [x axis] and 30 s [y axis]). UAS-EB1-GFP was expressed in class III DA neurons by Gal
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

5. Figure 4
Drosophila MNB Binds Microtubules through a Conserved Basic Cluster In Vitro
(A) Coomassie brilliant blue (CBB)-stained SDS-Polyacrylamide gel electrophoresis (SDS-PAGE) of purified recombinant GFP-tagged MNB kinase (left), and an immunoblot of MNB protein detected by an antibody against phosphotyrosine (right).
(B) TIRF-M image showing GFP-MNB (50 nM, green) binds along the lattice of taxol-stabilized MTs (red) in vitro (scale bar, 2.5 μm).
(C) CBB-stained SDS-PAGE shows the binding behavior of GFP-MNB in the presence of increasing concentrations of taxol-stabilized MTs. Results from five separate experiments were plotted and fit to a Michaelis-Menten equation (right) producing a Km of 0.96 ± 0.10 μM (mean ± SD).
(D) GFP-MNB (green) binds normal taxol-stabilized MTs (red) but does not bind subtilisin-treated MTs that lack the CTTs (blue) in two different panels. Scale bar, 2.5 μm.
(E) Sequence alignment reveals that Drosophila MNB-E contains a basic cluster (orange, residues 118–122) that is highly conserved. We mutated each of these basic residues to acidic residues to produce a basic-to-acidic mutant (B > A) MNB. Sequence accession numbers are as follows: Uniprot: Q13627, I3L9V1, P49657, Q2TAE3, Q9XTF3, and Q09690 for H. sapiens, S. scrofa, D. melanogaster, X. laevis, C. elegans, and S. pombe, respectively.
(F) CBB-stained SDS-PAGE of purified recombinant GFP-B > A-MNB kinase.
(G) TIRF-M imaging of GFP-B > A-MNB (50 nM, green) reveals only weak binding to taxol-stabilized MTs (red) in vitro (scale bar, 3 μm).
(H) CBB-stained SDS-PAGE gels show the binding behavior of recombinant GFP-B > A-MNB in the presence of increasing concentrations of taxol-stabilized MTs. Results of three separate cosedimentation experiments are plotted and fit to the Michaelis-Menten equation (right) producing a Km of 4.46 ± 2.62 μM (mean ± SD), which is significantly different from that of wild-type MNB (p = 0.019).
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

6. Figure 5
Mnb Inhibits Microtubule Polymerization In Vitro by Phosphorylating β-Tubulin at Serine 172
(A) Tubulin polymerization determined via turbidity (at 350 nm) in the absence of MNB (dark blue), in the presence of MNB (pink), and in the presence of MNB without ATP (light blue).
(B) Tubulin polymerization determined via turbidity in the presence of either the MNB1 (A191T) mutant MNB (pink/purple), or the B > A mutant MNB (orange) (p = and for MNB1 and B > A MNB versus wild-type MNB). All turbidity assays were conducted at 25°C in BRB80 buffer with 25 μM tubulin, 5 μM GFP-MNB, 1 mM GTP, and 1 mM ATP, unless otherwise noted. Means ± SD are plotted from at least n = 3 experiments per condition.
(C) Sequence analysis of β-tubulin shows two potential MNB phosphorylation sites: serine 172 (purple) and threonine 219 (green), both of which are highly conserved. Sequence accession numbers are as follows: Uniprot: P07437, Q767L7, Q24560, Q91575, P12456, and P05219 for H. sapiens, S. scrofa, D. melanogaster, X. laevis, C. elegans, and S. pombe, respectively.
(D) Immunoblots of total MNB protein and autophosphorylated MNB protein detected by antibodies against the strep-tag and phosphotyrosine, respectively, show that wild-type and B > A MNB recombinant proteins are active kinases, while MNB1 is not.
(E) Immunoblots of in vitro kinase assays with samples taken at 0 min and 60 min after incubation of 500 nM tubulin and 1 mM ATP alone, or with 500 nM wild-type, MNB1, or B > A MNB at 25°C. MNB proteins were detected by an anti-strep antibody, β-tubulin phosphorylated at serine 172 was detected by a phosphospecific antibody, and total tubulin was detected by an anti-alpha-tubulin antibody.
(F) Extracted ion chromatogram from MS1 filtering of LC-MS/MS (liquid chromatography-tandem mass spectrometry) data shows the unmodified peptide, IMNTFSVMPSPK, containing Ser172 (earlier retention time peak) and the phosphorylated Ser172 peptide (later retention time and inset). The MS1 intensity is normalized to the peak of the unmodified peptide identified in each sample. The phosphorylated Ser172 peptide appears in the presence of wild-type MNB protein, but does not appear in the presence of the kinase-dead mutant, MNB1, or in the absence of the kinase. See also Table S1.
(G) Immunohistochemical staining of wild-type, mnb RNAi, and mnb OE larval fillets with anti-phospho-S172 and anti-GFP (against the membrane marker, CD4). Phospho-S172 is normally present at the base of terminal branches in wild-type class III neurons (white arrows), but expression decreases in mnb RNAi neurons and increases in mnb overexpressing neurons. Scale bars, 10 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

7. Figure 6
Mammalian DYRK1a Exhibits a Conserved Mechanism of Action on Tubulin
(A) CBB-stained SDS-PAGE of purified recombinant GFP-tagged DYRK1a kinase (left), and an immunoblot of DYRK1a protein detected by an antibody against phosphotyrosine (right) to show that the recombinant GFP-DYRK1a kinase is active and able to autophosphorylate its own tyrosine residues.
(B) TIRF-M reveals GFP-DYRK1a (green) binds along the lattice of taxol-stabilized MTs (red) in vitro in two different panels (scale bar, 2.5 μm).
(C) Immunoblots of in vitro kinase assays with samples taken at 0 min and 60 min after incubation of 500 nM tubulin and 1 mM ATP alone, or with 500 nM GFP-DYRK1a at 25°C. DYRK1a protein was detected by an anti-strep antibody, β-tubulin phosphorylated at serine 172 was detected by a phosphospecific antibody, and total tubulin was detected by an anti-alpha-tubulin antibody.
(D) Tubulin polymerization determined via turbidity (at 350 nm) in the absence of DYRK1a (dark blue), in the presence of DYRK1a (pink), and in the presence of DYRK1a without ATP (light blue). All turbidity assays were conducted at 37°C in BRB80 buffer with 25 μM tubulin, 5 μM GFP-DYRK1a, 1 mM GTP, and 1 mM ATP, unless otherwise noted. Means ± SD are plotted from at least n = 3 experiments per condition.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions

8. Figure 7
Mnb Mutant Class III DA Neurons Exhibit a Reduced Mechanosensitive Response Due to Altered Morphology and Abnormal Localization of NompC
(A) Quantification of the larval behavioral response to gentle touch reveals defects in the mnb mutant and RNAi animals, as well as in the mnb-OE animals (two stars indicate p < ; for mnb1 rescued with 1912-Gal4>UAS-mnb, p = 1.00; n = 24, 19, 19, 20, 19, and 20 larvae for wild-type, mnb3, mnb1, mnb RNAi, mnb OE, and mnb rescue, respectively). Graph is a box plot with min to max whiskers, which include all data points.
(B) Extracellular electrophysiological recordings from class III DA neurons expressing mnb-RNAi and mnb OE indicate an alteration in the number of action potentials (APs) fired per second for a mechanical stimulus of a particular intensity compared with baseline (Δno. APs) (two stars indicate p < 0.005; n = 8, 9, and 7 larvae for wild-type, mnb RNAi, and mnb OE, respectively).
(C) Representative recording traces from wild-type, mnb OE, and mnb-RNAi neurons for a stimulus of 20 μm intensity.
(D) Immunohistochemical staining of larval fillets expressing UAS-cd4-tdGFP specifically in class III DA neurons (driven by 1912-Gal4) with antibodies against GFP reveals that the axon terminals of wild-type, mnb RNAi, and mnb OE class III DA neurons appear similar. Scale bars, 30 μm.
(E) Immunohistochemical staining of wild-type, mnb OE, and mnb RNAi larval fillets with anti-nompC and anti-HRP reveals altered patterns of NompC localization. NompC fills the terminal branches of wild-type and mnb OE neurons. In mnb RNAi neurons, NompC is apparent in the proximal regions of the terminal branches, but is not localized throughout the entire terminal branch (white arrows). Scale bars, 5 μm.
Neuron  , DOI: ( /j.neuron )
Copyright © 2016 Elsevier Inc. Terms and Conditions