Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) PC12 cells co-transfected with GFP and empty vector (EV) or BASP1, either wild-type (WT) or phosphomutants at the phosphosites identified by MS (T31, S40, and S170) were analyzed for the number of branches and longest neurite length. N>300 cells per condition. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *q<0.05 comparison between phosphomimetic and phosphoablative mutants, ▴ q<0.05 comparison between phosphomutants and WT. (B) Representative western blot from primary embryonic rat cortical neurons transduced with either GFP alone or BASP1-GFP constructs: WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Blots were probed with anti-GFP to detect expression of the BASP1-GFP constructs and with anti-actin as a loading control. (C) Quantification of ATP levels from conditions in (B). N>30. One-way Anova with post hoc Tukey’s test. (D) Representative confocal images of MAP2-immunostained primary embryonic rat cortical neurons cultured under the conditions described in (B). Scale bar = 100 µm. (E) Quantification of dendrite complexity in the cultures shown in (D), including measurements of the maximal secondary neurite length, secondary and tertiary dendritic arborization. N≥30. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (F) Representative confocal images of F-actin stained with BODIPY™ 558/568 Phalloidin in primary embryonic rat cortical neurons cultured under the conditions described in (B). Neurons were treated with 2 µM latrunculin B (LatB) for 10 min, followed by washout and a 10 min recovery period to allow actin re-polymerization. Scale bar = 20 µm. (J) Quantification of the levels of F-actin stained with BODIPY™ 558/568 Phalloidin in primary embryonic rat cortical neurons cultured under the conditions described in (B). Mean fluorescent intensities were measured before latrunculin B treatment (Pre-LatB), 10 min after LatB exposure (LatB), and at 10 min and 20 min following LatB washout (10′ and 20′ Post-LatB). N>30. One-way Anova with Tukey’s or Games-Howell’s post-hoc tests (depending on the condition of homoscedasticity and heteroscedasticity), * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Transfection, Plasmid Preparation, Comparison, Western Blot, Transduction, Construct, Expressing, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Schematic representation of the immunoprecipitation (IP) procedure used to isolate BASP1-GFP WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutant interactors from transduced primary embryonic rat cortical neurons. GFP alone was used as a control. Biorender software was used to create the figure under an academic license. (B) Venn diagram showing all mass spectrometry hits from (A) that met the selection criteria detailed in the main text. (B) Venn diagram showing all mass spectrometry hits from (A) that met the selection criteria detailed in the main text. (C) Reactome pathway analysis for molecular processes for the common BASP1 interactors from (B). (D) Heat map representation of CaN-dependent phosphoproteomic hits associated with BASP1 after normalization to BASP1 WT. Each row corresponds to a protein for which a phosphorylated peptide was identified. One-way ANOVA followed by a multiple comparison test with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( E) Reactome pathway analysis for molecular processes from the CaN-dependent phosphoproteomic hits for BASP1.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Immunoprecipitation, Mutagenesis, Control, Software, Mass Spectrometry, Selection, Comparison

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Representative Western blot of GFP immunoprecipitates (IP) from HeLa cells transfected with GFP alone or BASP1-GFP constructs: wild-type (WT), phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Blots were probed with anti-GFP and anti-prohibitin 1 (Phb1). (B) MS quantification of prohibitin 1 (Phb1) co-immunoprecipitated using anti-GFP beads from primary embryonic rat cortical neurons transduced with GFP alone or BASP1-GFP constructs: WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (C) Representative Western blot of BASP1 from HeLa cells co-transfected with GFP alone or BASP1-GFP constructs: BASP1 WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants, along with either a 3’UTR-targeting siRNA against human BASP1 or non-targeting control siRNA. (D) Quantification of BASP1 signal normalized to GAPDH (loading control) from (C), with values further normalized to the non-targeting siRNA control within each experiment. N=3. One-way Anova with post hoc Dunnett’s test, * P <0.05. (E-G) Mitochondrial length (E) and number (G) analyzed in HeLa cells from (C) using MitoTracker Deep Red-assisted confocal microscopy (F). Scale bar = 10 µm. N>20. One-way Anova with post hoc Tukey’s test, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. (H) MS quantification of actin co-immunoprecipitated using anti-GFP beads from primary embryonic rat cortical neurons cultured under the conditions described in (B). One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (I) Representative Western blot of GFP co-immunoprecipitates from HeLa cells cultured under the conditions described in (A). Blots were probed with anti-GFP and anti-actin. (J) Representative confocal pictures of mitochodnira and actin from HeLa cells cultured under the conditions described in (C) and co-transfected with LifeAct-mScarlet to visualize actin. Time-lapse imaging was performed at 150-second intervals to monitor actin localization at mitochondria following 4 µM ionomycin stimulation. Scale bar = 10 µm. (K) Quantification of actin fluorescence intensity from (J) over time following 4 µM ionomycin treatment. (L) Representative confocal pictures of HeLa cells cultured in conditions described in (C) under 4 µM Ionomycin treatment. Actin was visualized by transient expression of LifeAct-mScarlet and mitochondria were labeled with MitoTracker Deep Red. Time-lapse imaging was performed at 15-second intervals. Arrows indicate actin recruitment to mitochondria with BASP1-GFP WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants followed by fission events only in BASP1 WT and D,D. Scale bar = 2 µm.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Transduction, Control, Confocal Microscopy, Cell Culture, Imaging, Fluorescence, Expressing, Labeling

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Representative confocal images of β-III-tubulin staining in DIV23 primary embryonic rat cortical neurons, transduced with either GFP alone or BASP1-GFP constructs: wild-type (WT), phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Neurons were subjected to mechanical injury in a scratch assay, and the staining was performed 24 hours post-injury. Scale bar = 50 µm. (B) Quantification of neurite outgrowth for the cultures in (A) using ImageJ/Fiji. N>950. One-Way Anova with Games-Howell’s post-hoc test, **** P <0.0001 (C) Representative immunofluorescence images of primary embryonic rat cortical neurons cultured under the conditions described in (A) showing immunostaining for mitochondrial markers TOMM20 and prohibitin 1 (Phb1). Scale bar = 5 µm. (D) Quantification of mitochondrial length in neurites for the cultures in (C) N>550. One-way Anova with Games-Howell’s post-hoc test, **** P <0.0001. (E) Representative live-cell images of primary embryonic rat cortical neurons cultured under the conditions described in (A) and stained with the fluorescent mitochondrial dye tetramethylrhodamine methyl ester (TMRM) acquired at 3-min intervals. Scale bar = 5 µm. (F) Quantification of TMRM intensities for the cultures in (E), normalized to GFP alone within each experiment. N=3. One-way Anova with post hoc Tukey’s test. (G) Quantification of α-syn in neurites from neurons cultured under the conditions described in (A). N>80. One-way Anova with Games-Howell’s post-hoc test, * P <0.05, ** P <0.01, *** P <0.001. (H) Representative immunofluorescence images of primary embryonic rat cortical neurons cultured under the conditions described in (A) showing staining for α-synuclein (α-syn). Scale bar = 5 µm.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Staining, Transduction, Construct, Wound Healing Assay, Immunofluorescence, Cell Culture, Immunostaining

Journal: Molecular and Cellular Biology

Article Title: Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

doi: 10.1128/MCB.00230-16

Figure Lengend Snippet: Generation of brain-specific PDK1 L155E mice. (A) Diagram depicting the 5′ end of the PDK1 gene from exons 2 to 7 (PDK1); the targeting construct containing the thymidine kinase (TK) negative selectable marker and the minigene cassette, which includes the PDK1 open reading frame from exons 3 to 14 plus the natural polyadenylation signals (A+) and is flanked by LoxP sites (CONSTRUCT); the targeted allele, which drives the expression of the PDK1 wild-type protein in control tissues (PDK1 WT); and the excised allele, in which the CRE recombinase-mediated deletion of the minigene cassette allows the expression of the PDK1 mutant protein (PDK1 L155E). The white boxes represent exons, the triangles represent LoxP sites, and the mutated exon 4 containing the Leu155Glu amino acid substitution is represented by black boxes labeled with asterisks. (B) Breeding strategy used for generation of mice expressing the PDK1 L155E mutant protein in brain. The number (n) and proportion (%) of mice of each genotype resulting from the depicted experimental breeding are indicated both at E15.5 and at birth (P0). **, the lower-than-expected frequency of PDK1fl/fl CRE+ pups was statistically significant (P < 0.005 by χ2 test). (C) PDK1 was affinity purified on PIF-Sepharose from liver or brain embryonic extracts of the indicated genotypes. The expression levels of the PDK1 wild-type protein were quantified on the indicated genotypes and tissues from the PDK1 immunoblot signals of the PIF-Sepharose pulldowns (middle blots), normalized by the total PDK1 protein levels derived from the whole tissue lysate immunoblot signals (upper blots), and represented as percentages of those of the PDK1+/fl CRE− controls (top). Each bar represents the means and standard errors of the mean for the immunoblot signals derived from two independent experiments. A representative Western blot is shown, where each lane corresponds to a sample derived from a different mouse. Immunoblots with the ERK1/2 antibody are also shown as controls for protein loading (bottom blots). **, P < 0.005 compared to controls.

Article Snippet: For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro , washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone).

Techniques: Construct, Marker, Expressing, Mutagenesis, Labeling, Affinity Purification, Western Blot, Derivative Assay

Journal: Molecular and Cellular Biology

Article Title: Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

doi: 10.1128/MCB.00230-16

Figure Lengend Snippet: (A, C, and D) Microcephaly of PDK1fl/fl CRE+ mice. The organ volume was measured from physical histological sections of the E15.5 embryo head (A) and brain (C) or MRI images of one hemisphere of the adult brain (D) by using the Cavalieri method, as described in Materials and Methods. The data are represented as means and standard errors of the mean obtained for three different mice per genotype and are expressed as percentages of the controls. (C and D) Total-volume values and representative photographs of E15.5 embryonic brains (C) or adult brain left hemispheres (D) are shown at the bottom, where the scale bars correspond to 1 mm and 2 mm, respectively. (B) Mean body weights of mice of the indicated genotypes. The values represent the means and standard errors of the mean for the indicated numbers of mice (n). (E and F) The number of cells (E) and the cellular volume (F) were determined from E15.5 dissociated cortical neurons with a Scepter 2.0 handheld automated cell counter (Millipore). The data are represented as the means and standard errors of the mean for the indicated numbers of embryos obtained from 11 independent litters. *, P < 0.05; **, P < 0.005 compared to controls.

Article Snippet: For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro , washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone).

Techniques:

Journal: Molecular and Cellular Biology

Article Title: Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

doi: 10.1128/MCB.00230-16

Figure Lengend Snippet: Reduced neuronal-progenitor proliferation in PDK1fl/fl CRE− and PDK1fl/fl CRE+ embryos. (A) Representative micrographs of E15.5 embryonic cortical cultures of the indicated genotypes immunostained with the described antibodies. Insets show enlarged single-cell representative images of the nuclear staining of Ki67 in red and the cytoplasmic active caspase-3 staining in green. (B and C) The percentages of primary cortical neurons expressing the Ki67 proliferation marker (B) or the active caspase 3 apoptotic marker (C) were scored at the indicated time points. The bars correspond to the means and standard errors of the mean from 10 different fields per culture and three independent embryonic cultures per genotype. (D) Epifluorescence microscopy images of E15.5 embryonic brain coronal sections of the indicated genotypes immunostained with the described antibodies. DCX, doublecortin. (E and F) The intensity of doublecortin staining (INTDEN) (E) and the number of caspase 3-positive cells per field (F) were quantified and expressed as the means and standard errors of the mean from three independent sections per embryo obtained from three different embryos per genotype. A.U, arbitrary units. *, P < 0.05; **, P < 0.005 compared to controls.

Article Snippet: For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro , washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone).

Techniques: Staining, Expressing, Marker, Epifluorescence Microscopy

Journal: Molecular and Cellular Biology

Article Title: Mutation of the 3-Phosphoinositide-Dependent Protein Kinase 1 (PDK1) Substrate-Docking Site in the Developing Brain Causes Microcephaly with Abnormal Brain Morphogenesis Independently of Akt, Leading to Impaired Cognition and Disruptive Behaviors

doi: 10.1128/MCB.00230-16

Figure Lengend Snippet: Activation of S6K1, RSK, SGK1 and PKC but not Akt is inhibited in PDK1fl/fl CRE+ mice. (A) Cortical neurons from three independent embryos of the indicated genotypes were cultured for 6 DIV and then serum starved for 4 h and either left unstimulated (0) or stimulated with 50 ng/ml of BDNF for 15 min (15). (B) Whole-brain protein extracts were obtained at E15.5 from three independent embryos of each depicted genotype. Lysates were immunoblotted with the indicated antibodies to monitor the activation of Akt, S6K, RSK, SGK, and PKC. Each lane corresponds to a different embryo. Band densitometry quantification of the ratio between phosphorylated and total protein levels is shown at the bottom, where the bars represent the means and standard errors of the mean obtained for three different mice per genotype analyzed, and is expressed as a percentage of the BDNF-stimulated control samples (A) or control brain tissue samples (B). *, P < 0.05; **, P < 0.005 compared to controls.

Article Snippet: For the survival studies, cortical neurons obtained at E15.5 were cultured in complete neurobasal medium with the B27 supplement for 6 days in vitro , washed twice with Dulbecco's modified Eagle's medium (DMEM) without serum, and then either reincubated in conditioned medium or trophic factor deprived for 24 h in serum-free neurobasal medium in the absence or presence of 50 ng/ml of brain-derived neurotrophic factor (BDNF) (Alomone).

Techniques: Activation Assay, Cell Culture