Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr and immuno-stained with GT335, anti-acetylated tubulin and anti-Arl13b antibodies. ≥90 cells per sample were analyzed in three independent experiments. Error bars, S.D. ***p ≤ 0.001, **** p ≤ 0.0001. B ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immuno-stained with antibodies against GT335, Arl13b, Myo-Va, Cep89, Rab8a, Rabin8, IFT88, Cep290 and RPGRIP1L. Scale bar, 1µm. C ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A and B and visualized by staining with GT335 and antibodies against CP110. Scale bar, 1µm. N ≥ 90 cells per sample were analyzed in three independent experiments. Error bars, S.D. D ) sgRNA control and TBC1D19 knockout cells were serum starved for 24 hr, and immunostained with antibodies against Rab34 and Arl13b. Scale bar, 1µm. E ) sgRNA control and TBC1D19 knockout cells were serum starved as in panels A-C and immunostained with antibodies against GT335 and Arl13b. Scale bar, 1µm. F ) Primary cilia in sgRNA control and TBC1D19 knockout cells were examined by transmission electron microscopy (TEM) after 24 hr of serum starvation. Images of TBC1D19 knockout cilia from two consecutive sections are shown.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Staining, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: The tubulin poly-glutamylase complex, TPGC, is required for phosphatidyl inositol homeostasis and cilium assembly and maintenance

doi: 10.1101/2025.03.03.641315

Figure Lengend Snippet: A ) Control (sgCTL) and TBC1D19 knockout cells were treated with non-targeting control or CCP1 siRNA for 48 hr and serum starved for 24 hr, and extracts were subjected to immunoblotting with the indicated antibodies. B ) TBC1D19 knockout cells were infected with lentivirus expressing Flag-map4m or Flag-map4m-TTLL1 for 72 hr, serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. C ) Control (sgCTL), TBC1D19 knockout cells, and TBC1D19 knockout cells stably expressing TTLL5-EYFP or TTLL6-EYFP were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. D ) Control (sgCTL), TBC1D19 -/- , C11ORF49 -/- , and TTLL1 -/- cells were serum starved for 24 hr and subjected to immunoblotting with the indicated antibodies. E ) sgCTL, C11ORF49 -/- , and LRRC49 -/- cells were serum starved for 24 hr, immuno-stained with antibodies against acetylated tubulin and INPP5E. N ≥ 50 cilia were counted per sample in two independent experiments. Error bars, S.D. ns, not significant. F ) 293T cells were co-transfected with GFP or GFP-TBC1D19 with mCherry-Arl13b for 32 hr and serum starved for 16 hr. Lysates were subjected to immunoprecipitation with GFP-trap beads and immuno-blotted with antibodies against GFP and mCherry.

Article Snippet: Mouse anti-α-tubulin (1:10000 for WB 66031-1-Ig), rabbit anti-Arl13b (1:2000 for IF and 1:1000 for WB, 17711-1-AP), rabbit anti-Rab8a (1:50 for IF, 55296-1-AP), rabbit anti-IFT88 (1:500 for IF, 13967-1-AP), rabbit anti-INPP5E (1:1000 for WB and 1:1000 for IF, 17797-1-AP), mouse anti-β-actin (1:10000 for WB, 66009-1-Ig PT), mouse anti-GAPDH (1:10000 for WB, 60004-1-Ig), rabbit ant-CCP1 (1:1000 for WB, 14067-1-AP), Rabbit anti-TBC1D19 (1:1000 for WB, 21085-1-AP) and rabbit anti-Tulp3 (1:1000 for WB, 1:150 for IF, 13637-1-AP), RPGRIP1L (1:150 for IF, 55160-1-AP) were from Proteintech.

Techniques: Control, Knock-Out, Western Blot, Infection, Expressing, Stable Transfection, Staining, Transfection, Immunoprecipitation

Journal: Molecular Neurodegeneration

Article Title: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

doi: 10.1186/s13024-018-0235-y

Figure Lengend Snippet: Pathogenic LRRK2 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous Rab8a. a , b Examples of non-transfected HEK293T cells (ctrl) or cells transfected with either wildtype or pathogenic LRRK2, or with kinase-dead pathogenic LRRK2 as indicated, and stained with γ-tubulin antibody, Rab8a antibody (rabbit polyclonal Rab8a antibody for panel a, sheep polyclonal antibody for panel b ), and TOPRO. Scale bar, 5 μm. c Quantification of the percentage of cells displaying pericentrosomal Rab8a staining in either non-transfected cells (ctrl), or pathogenic LRRK2-transfected cells as indicated, either in the absence or presence of GSK2578215A (GSK) (500 nM, 1 h). Bars represent mean ± s.e.m., ( n = 3 independent experiments); ****, p < 0.001; *, p < 0.05. d Cells were transfected with the indicated constructs, and extracts blotted for GFP-tagged LRRK2, endogenous Rab8a, and tubulin as loading control. e Cells were either left untreated or incubated with 500 nM GSK2578215A for 1 h as indicated, and extracts analyzed for phosphorylated (p-S935) or total (GFP) LRRK2. f Quantification of S935 dephosphorylation in cells expressing wildtype or mutant LRRK2 as indicated, in either the absence or presence of 500 nM GSK2578215A for 1 h. Bars represent mean ± s.e.m. ( n = 3); ***, p < 0.005; **, p < 0.01

Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400).

Techniques: Transfection, Staining, Construct, Incubation, De-Phosphorylation Assay, Expressing, Mutagenesis

Journal: Molecular Neurodegeneration

Article Title: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

doi: 10.1186/s13024-018-0235-y

Figure Lengend Snippet: Pathogenic LRRK2 causes kinase-dependent pericentrosomal/centrosomal accumulation of endogenous phospho-Rab8a. a , b Cells were transfected with pathogenic LRRK2 or with kinase-dead pathogenic LRRK2 as indicated, and stained using an anti-phospho-T72-Rab8a antibody preabsorbed either with dephospho-peptide (p-Rab8a) or with phospho-peptide (p-Rab8a + pp), or with an anti-phospho-T72-Rab8a antibody preabsorbed with dephospho-peptide upon incubation of cells with 100 nM MLi2 for 60 min prior to immunocytochemistry as indicated. Scale bar, 5 μm. c Quantification of the percentage of non-transfected or transfected cells displaying phospho-Rab8a staining colocalizing with centrosomes within a 3 μm diameter circle in either the absence or presence of antibody preabsorption with peptides or pretreatment of cells with MLi2 as described above. Around 50 cells were quantified per condition per experiment. Bars represent mean ± s.e.m., ( n = 3 independent experiments); ****, p < 0.001. d Quantification of the percentage of non-transfected or transfected cells displaying phospho-Rab8a staining colocalizing with centrosomes as described above. Around 50 cells were quantified per condition per experiment. Bars represent mean ± s.e.m., ( n = 3 independent experiments); **, p < 0.01

Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400).

Techniques: Transfection, Staining, Incubation, Immunocytochemistry

Journal: Molecular Neurodegeneration

Article Title: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

doi: 10.1186/s13024-018-0235-y

Figure Lengend Snippet: Expression of wildtype but not phosphorylation-deficient Rab8a causes centrosomal accumulation of phospho-Rab8a and centrosome cohesion deficits in wildtype LRRK2-expressing SH-SY5Y cells. a Example of SH-SY5Y cells stably expressing flag-tagged wildtype LRRK2, and transfected with mRFP-tagged wildtype or T72A-mutant Rab8a as indicated, stained with an anti-phospho-T72-Rab8a antibody preabsorbed with dephosphopeptide, for pericentrin and DAPI. Where indicated, cells were treated with 100 nM MLi2 for 2 h before immunocytochemistry. Note that phospho-Rab8 can be detected both in cells with one centrosome (first panel) as well as in cells with duplicated centrosomes (second panel). Scale bar, 10 μm. b Same as in a , but SH-SY5Y cells stably expressing flag-tagged G2019S mutant LRRK2. Scale bar, 10 μm. c Quantification of mean fluorescence intensity of phospho-Rab8a signal as described in Methods in cells either stably expressing wildtype (wt) or G2019S mutant LRRK2, transfected with RFP-tagged Rab8a or T72-mutant Rab8a, and treated with 100 nM MLi2 for 2 h before immunocytochemistry as indicated. Bars represent mean ± s.e.m., ( n = 3 independent experiments); ****, p < 0.001; ***, p < 0.005. d Example of non-differentiated SH-SY5Y cells stably expressing GFP, flag-tagged wildtype or G2019S-mutant LRRK2 as indicated, and transfected with Rab8a or phosphorylation-deficient Rab8a (Rab8a-T72A) as indicated, and stained for pericentrin and TOPRO. Scare bar, 10 μm. e Quantification of the split centrosome phenotype in SH-SY5Y cells from the type of experiments depicted in a . Bars represent mean ± s.e.m. ( n = 3 experiments); *, p < 0.05. f SH-SY5Y cells stably expressing GFP, flag-tagged wildtype or G2019S-mutant LRRK2 as indicated were transfected with mRFP-tagged wildtype or phosphorylation-deficient Rab8a (Rab8a-T72A) as indicated, and extracts blotted for Rab8a levels and GAPDH as loading control

Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400).

Techniques: Expressing, Stable Transfection, Transfection, Mutagenesis, Staining, Immunocytochemistry, Fluorescence

Journal: Molecular Neurodegeneration

Article Title: Parkinson disease-associated mutations in LRRK2 cause centrosomal defects via Rab8a phosphorylation

doi: 10.1186/s13024-018-0235-y

Figure Lengend Snippet: Knockdown of Rab8a significantly reverses the centrosomal deficits mediated by pathogenic LRRK2. a Representative Western blot of extracts from control cells (ctrl), or cells transfected with wildtype (wt) or Y1699C-mutant LRRK2, along with either ctrl-siRNA or Rab8a-siRNA as indicated, and blotted against Rab8a or tubulin as loading control. b Quantification of the type of experiments depicted in a , with levels of Rab8a normalized to tubulin and to Rab8a levels in the presence of ctrl-siRNA. Bars represent mean ± s.e.m. ( n = 3–5 independent experiments); * p < 0.05. c Example of cells co-transfected with GFP-tagged pathogenic LRRK2 and either ctrl-siRNA or Rab8a-siRNA as indicated, and stained with pericentrin antibody and DAPI. Scale bar, 10 μm. d Quantification of the split centrosome phenotype in control cells transfected with either ctrl-siRNA or Rab8a-siRNA, or in cells co-transfected with wildtype or mutant LRRK2 as indicated. Bars represent mean ± s.e.m. ( n = 3–5 independent experiments); **** p < 0.001; * p < 0.05. e Western blot of cell extracts transfected with either ctrl-siRNA or Rab8a-siRNA as indicated, and four hours later cotransfected with mutant LRRK2 and mRFP-tagged Rab8a, Rab8a-T72A, or siRNA-resistant versions thereof (res), and blotted against Rab8a and GAPDH as loading control. f Quantification of the split centrosome phenotype in the presence of ctrl-siRNA or Rab8a-siRNA, and in the presence of pathogenic mutant LRRK2 and mRFP-tagged Rab8a, Rab8a-T72A or siRNA-resistant versions thereof (res) as indicated. Bars represent mean ± s.e.m. ( n = 3 independent experiments); *** p < 0.005; ** p < 0.01. g Quantification of centrosome reorientation in cells stably expressing flag-tagged wildtype or G2019S-mutant LRRK2 together with RFP-tagged wildtype or phospho-deficient T72A Rab8a 4 h after generating the wound ( t = 4 h). Random orientation is expected to be 25%. n > 50 cells were quantified for each condition in each experiment. Bars represent mean ± s.e.m. ( n = 3 independent experiments); **, p < 0.01; *, p < 0.05

Article Snippet: Primary antibodies included rabbit polyclonal anti-pericentrin (Abcam, ab4448, 1:1000), mouse monoclonal anti-pericentrin (Abcam, ab28144, 1:1000), mouse monoclonal anti-γ-tubulin (Abcam, ab11316, 1:1000), mouse monoclonal anti-c-Myc (Sigma, clone 9E10, M4439 1:500), mouse monoclonal anti-flag (Sigma, clone M2, 1:500), rabbit polyclonal anti-Rab8a (Millipore, ABC423, 1:1000), mouse anti-Golgin97 (Molecular Probes, A-21270, 1:100), rabbit polyclonal anti-β-COP (Invitrogen, PA1–061, 1:750), and mouse monoclonal p230/Golgin-245 (Becton Dickinson, 611,280, 1:400).

Techniques: Western Blot, Transfection, Mutagenesis, Staining, Stable Transfection, Expressing

Journal: The Journal of Biological Chemistry

Article Title: Neuronal activity promotes secretory autophagy for the extracellular release of α-synuclein

doi: 10.1016/j.jbc.2024.107419

Figure Lengend Snippet: Role of autophagosomes in neuronal activity-mediated α-syn secretion. Western blots of TCA-precipitated CM and cell lysates are shown. A , treatment with 1 nM TeNT for 24 h inhibits α-syn secretion in primary cortical neurons (n = 3). B , glutamate-induced α-syn and p62 secretion is blocked by adding 1 nM TeNT in primary cortical neurons (secreted α-syn: n = 4; F (3,12) = 39.812, p < 0.001, ANOVA); (secreted p62: n = 4; F (3,12) = 8.010, p = 0.003, ANOVA); (intracellular α-syn: n = 4; F (3,12) = 0.393, p = 0.76, ANOVA) (intracellular p62: n = 4; F (3,12) = 6.479, p = 0.007, ANOVA). C , overexpression of Rab7 increases α-syn secretion in wt-αS/SH cells (n = 4). D , glutamate-induced α-syn secretion is blocked by RAB8A knockdown in wt-αS/SH cells (n = 3; F (2,6) = 40.114, p < 0.001, ANOVA). A – D , graphs show secreted α-syn levels normalized to controls ( white columns ) and percentages of LDH release to positive controls ( orange columns ). Upper right graph of ( B ) shows secreted p62 levels normalized to controls ( blue columns ). Lower left and right graphs of ( B ) show ratios of relative intensities (intracellular α-syn to β-actin) normalized to controls ( gray columns ) and ratios of relative intensities (intracellular p62 to β-actin) normalized to controls ( cyan columns ), respectively. Intracellular α-syn levels and LDH release are unchanged in these experiments. Data represent mean ± SD. Data are analyzed by unpaired Student’s t test ( A and C ) and one-way ANOVA with Bonferroni’s post hoc tests ( B and D ). ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001. CM, conditioned media; Cont., control; Glu, glutamate; Sec. αS, secreted α-synuclein; Sec. p62, secreted p62; TeNT, tetanus toxin.

Article Snippet: The following primary antibodies were used: anti-α-syn (mouse monoclonal, 1:2500, 42/α-Synuclein, BD Transduction Laboratories), anti-β-actin (mouse monoclonal, 1:10,000, AC-15, Sigma-Aldrich), anti-LC3 (mouse monoclonal, 1:2000, 8E10, MBL), anti-p62 C-terminal (guinea pig polyclonal, 1:2000, PM066, MBL), anti-p62 (rabbit polyclonal, 1:200, #5114, Cell Signaling Technology), anti-Atg5 (mouse monoclonal, 1:1000, 4D3, MBL), anti-beclin 1 (rabbit monoclonal, 1:2500, EPR19662, Abcam), anti-phospho-AMPKα (Thr172) (rabbit monoclonal, 1:1000, 40H9, Cell Signaling Technology), anti-phospho-p70S6K (Thr389) (rabbit monoclonal, 1:1000, 108D2, Cell Signaling Technology), anti-Rab7 (rabbit monoclonal, 1:5000, EPR7589, Abcam), and anti-Rab8a (rabbit monoclonal, 1:5000, EPR 14873, Abcam), anti-FLAG M2 (mouse monoclonal, 1:1000, F1804, Sigma-Aldrich), anti-CD63 (rabbit monoclonal, 1:1000, EPR21151, Abcam), anti-amyloid-β precursor protein (APP) (mouse monoclonal, 1:1000, 22C11, Thermo Fisher), anti-flottilin-1 (mouse monoclonal, 1:1000, 18/Flotillin-1, BD Transduction Laboratories), anti-tau (rat monoclonal, 1:1000, RTM38, Wako), anti-SOD1 (rabbit polyclonal, 1:1000, GTX100554, GeneTex), anti-ubiquitin B (rabbit polyclonal, 1:1000, 10201-2-AP, Proteintech) antibodies.

Techniques: Activity Assay, Western Blot, Over Expression, Knockdown, Control