Journal: The Journal of Cell Biology

Article Title: δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions

doi: 10.1083/jcb.202303138

Figure Lengend Snippet: Astrocytic δ-catenin does not regulate neuron morphology or astrocyte–astrocyte interactions. (a) Two shRNAs targeting rat/mouse Ctnnd2 were generated: shCtnnd2-1 (sh1) and shCtnnd2-2 (sh2). Both shRNAs effectively knock down δ-catenin (δ-cat) expression in astrocytes compared to their respective controls in which the shRNA sequence was scrambled (shControl-1, crt-1, and shControl-2, crt-2). Loading control: β-tubulin (β-tub). n = 3 independent experiments. Unpaired t test with Welch’s correction. ** P < 0.01. (b) Schematic of neuron morphology assay. Astrocytes were isolated from P1 rat cortices to obtain purified astrocyte cultures. DIV 7 astrocytes are nucleoporated with pLKO.1 plasmid expressing hU6-shRNA and CAG-EGFP and seeded at a density of 80,000 astrocytes per coverslip. Neurons were isolated from another set of P1 rat cortices when astrocytes were DIV 7 and co-cultured on top of nucleoporated astrocytes for 5 d in NGM plus to allow for neurite growth. (c) Representative images of Tuj1-positive rat neurons after 5 d co-culture with shControl or shCtnnd2 nucleoporated astrocytes (not labeled). (d) Neuron morphology is unaffected by silencing of astrocytic Ctnnd2 in astrocyte–neuron co-culture paradigm (P = 0.58). Neuron morphology is quantified by Sholl analysis. n = 60 neurons per condition from three independent experiments. Linear mixed model with Tukey HSD. (e) Schematic of astrocyte mosaic culture. Astrocytes were isolated from P1 rat cortices to obtain purified astrocyte cultures. DIV 7 astrocytes are nucleoporated with pLKO.1 plasmid expressing hU6-shRNA and CAG-EGFP or a pPB-shRNA-mCherryCAAX plasmid. Nucleoporated astrocytes were seeded onto coverslips at a total density of 80,000 astrocytes per coverslip in a 9:1 ratio of pPB-shRNA-mCherryCAAX:pLOK.1-shRNA-EGFP for 48 h in NGM Plus. (f) Representative images of rat astrocytes nucleoporated with shControl-GFP or shCtnnd2-GFP after 48 h co-culture with astrocytes nucleoporated with shControl-mCherry or shCtnnd2-mCherry (not labeled). (g) Reduction in astrocyte morphology following δ-catenin knockdown in astrocyte–neuron co-culture assay is unrelated to defective astrocyte–astrocyte adhesion. Silencing Ctnnd2 did not influence wild-type astrocyte morphology (P = 0.18). Wild-type astrocyte morphology was unchanged when cultured with either shControl or shCtnnd2 astrocytes (P = 0.06). The same was also observed in shCtnnd2 astrocytes cultured with shControl or shCtnnd2 astrocytes (P = 0.91). Astrocyte morphology is quantified by Sholl analysis. n = 90 neurons per condition from three independent experiments. Linear mixed model with Tukey HSD. (h) Full-length human CTNND2 is insensitive to shCtnnd2. Western blot of HEK293 cell lysates transfected with shControl or shCtnnd2 in the presence or absence of HA-tagged, full-length human CTNND2 (hCTNND2). hCTNND2 expression was detected using an antibody against HA-tag. GFP expression denotes the presence of the shRNA construct. All protein molecular weights are expressed in kiloDaltons (kD). All data is presented as mean ± SEM. Scale bars: 20 μm.

Article Snippet: Astrocyte mosaic cultures were stained with chicken anti-GFP and rabbit anti-RFP (#600-401-379; Rockland) primary antibodies before being stained with Alexa Fluor 488 goat anti-chicken IgY(H+L) and Alexa Fluor 594 goat anti-rabbit IgG(H+L).

Techniques: Generated, Expressing, shRNA, Sequencing, Morphology Assay, Isolation, Purification, Plasmid Preparation, Cell Culture, Co-Culture Assay, Labeling, Co-culture Assay, Western Blot, Transfection, Construct

Journal: The Journal of Cell Biology

Article Title: δ-Catenin controls astrocyte morphogenesis via layer-specific astrocyte–neuron cadherin interactions

doi: 10.1083/jcb.202303138

Figure Lengend Snippet: δ-Catenin interacts with the juxtamembrane domain of N-cadherin to increase N-cadherin cell surface expression. (a) Schematic of domains within δ-catenin. (b) Schematic of the structure of classical cadherin. The JMD of cadherin is highlighted in green. (c) Model of predicted interaction between N-cadherin-JMD and δ-catenin ARM domain. δ-catenin ARM domain forms a solenoid structure (magenta). N-cadherin JMD (green) binds to a groove within this solenoid structure. (d) Model of electrostatic charges of wild-type human δ-catenin solenoid structure. N-cadherin JMD (green) is predicted to bind to a positively charged groove within the solenoid structure. (e) IP of N-cadherin by δ-catenin-HA pulldown. Deletion of JMD from N-cadherin abolishes N-Cadherin/δ-catenin interaction. Immunoblot of HEK293T cell lysates overexpressing δ-catenin-HA with GFP or full-length N-cadherin-GFP or N-cadherin without JMD-GFP. N-cadherin is detected by an anti-GFP antibody, while δ-catenin is detected by an anti-HA antibody. (f) Model of electrostatic charges of δ-catenin with an autism-linked point mutation R713C (left) and a polymorphism G810R (right). Both mutations are within the armadillo repeats, but only the R713C mutation (yellow dotted box) is within the groove predicted to be critical for N-cadherin JMD binding. (g) Immunoblot of WT, R713C, and G810R co-immunoprecipitated with full-length N-cadherin-GFP from HEK293T cell lysates. Point mutations to δ-catenin do not disrupt cadherin–catenin interaction. (h) Schematic of surface biotinylation experiment to determine if δ-catenin mutations alter N-cadherin cell surface expression. (i) Immunoblot of N-cadherin cell surface expression in HEK293T cell lysates transfected with WT, R713C, or G810R. (j) Expression of WT and G810 significantly increases N-cadherin expression at the cell surface (P = 0.006 and P = 0.014, respectively). Expression of R713C did not alter N-cadherin cell surface expression (P = 0.75). * P < 0.05; ** P < 0.01. n = 3 independent replicates. One-way ANOVA with Tukey HSD. All protein molecular weights are expressed in kiloDaltons (kD). All data are presented as mean ± SEM. Source data are available for this figure: .

Article Snippet: Astrocyte mosaic cultures were stained with chicken anti-GFP and rabbit anti-RFP (#600-401-379; Rockland) primary antibodies before being stained with Alexa Fluor 488 goat anti-chicken IgY(H+L) and Alexa Fluor 594 goat anti-rabbit IgG(H+L).

Techniques: Expressing, Western Blot, Mutagenesis, Binding Assay, Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: Capsaicin pretreatment alleviates postoperative pain and reduces primary sensory neuron Ca 2+ activity

doi: 10.1101/2021.05.21.445191

Figure Lengend Snippet: A . Spontaneous foot lifting as an index of spontaneous pain was counted at 3 h and 1 d after incision surgery. The number of spontaneous foot lifts by vehicle and capsaicin-treated (7 days prior to incision surgery) mice is plotted. B . Examples of traces of mouse movement in the open field test for assessment of spontaneous pain in naïve, vehicle-treated mice, and capsaicin-treated mice. C . Total walking distance in open field test of naïve, vehicle-treated, and capsaicin-treated mice. D . Average walking velocity in the open field test of naïve, vehicle-treated, and capsaicin-treated mice. E . Confocal microscopic immunohistochemistry image (40X) of β-III tubulin (sensory nerve fiber), TRPV1 channel, and GCaMP (GFP)-expressing primary sensory neuron fibers in skin from incision and capsaicin-treated Pirt-GCaMP3 mice. Area 1, skin surface; area 2, epidermis; area 3, dermis. Arrowheads indicate nerve fibers and nerve endings in epidermis. F . PLAP staining image (40X) in skin from vehicle-treated and capsaicin-treated Pirt-cre/PLAP flox mice. Black lines are sensory nerve fibers. G . Graph of PLAP-positive skin nerve fiber length and ratio of fluorescence intensities of β-III tubulin and TRPV1 channels from either incision only mice or capsaicin-treated incision mice. Cap-ICS, capsaicin-treated incision. Scale bars, 100 µm. Fig. A, incision, n = 6; Cap-ICS, n = 9. Fig. B-D, naïve, n = 5; incision, n = 5; Cap-ICS, n = 5. Fig. E and G, incision, n = 3; Cap-ICS, n = 3. Fig. F and G, incision, n = 3; Cap-ICS, n = 3. Data represent mean ± S.E.M.; T-test or Dunnett’s test; * p <0.05, ** p <0.01 incision vs. naïve or capsaicin-treated incision.

Article Snippet: Sections were transferred to slides and dried at 37°C for an hour, incubated with guinea pig anti-TRPV1 antibody (1:250, cat. no. ab10295, Abcam, Cambridge, MA, USA), chicken anti-GFP antibody (1:250, cat. no. CH23105, Neuromics, Edina, MN, USA), and rabbit anti-β-III tubulin antibody (1:250, cat. no. ab18207, Abcam) overnight at 4°C incubation of blocking solution.

Techniques: Immunohistochemistry, Expressing, Staining, Fluorescence

Journal: bioRxiv

Article Title: Spatial and temporal control of expression with light-gated LOV-LexA

doi: 10.1101/2021.10.19.465021

Figure Lengend Snippet: A . Schematics showing the timeline of the experiment, light regime as well as the construct selected for LOV-LexA. B-G . Fat bodies of second to third instar larvae with LOV-LexA expression controlled by Cg-GAL4 for larvae kept in the dark (B-D) and exposed to three 30s pulses of blue LED light at 1 Hz (E-G). Exposure to blue light appears to alter LOV-LexA cellular distribution (C, F) and leads to expression of LexAop-CsChrimson:Venus in fat body cells as detected with anti-GFP antibody (D, G). G . Exposure to blue light leads to an increase in the amount of green signal, derived from CsChrimson:Venus compared to the LOV-LexA signal, measured by the ratio of anti-GFP signal/anti-RFP signal.

Article Snippet: Cells were fixed with 4% paraformaldehyde overnight at 4°C, between 8 to 10 hours after addition of copper sulfate, and processed for immunostaining with standard protocols [for eg., 97], with antibodies anti-GFP chicken antibody dillution 1:2000 (Rockland, Catalogue # 600-901-215S; RRID: AB_1537403), anti-RFP rabbit antibody dilution 1:2000 (Rockland, Catalogue # 600-401-379, RRID: AB_11182807), anti-FLAG rat antibody dilution 1:300 (Novus Biologicals, Catalogue # NBP1-06712, RRID: AB_1625981).

Techniques: Construct, Expressing, Derivative Assay

Journal: Nature Communications

Article Title: A neural circuit for wind-guided olfactory navigation

doi: 10.1038/s41467-022-32247-7

Figure Lengend Snippet: Antibody sources and dilutions

Article Snippet: Chicken anti-GFP , Fisher Scientific , 1:50 , RRID:AB_1074893.

Techniques: