Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet: Validation of candidate DGKθ-interacting proteins. (A) Streptavidin beads were used to isolate biotinylated proteins from DGKθ-GFP-APEX2 labeled cortical neuron lysates (DIV21), and biotinylation was confirmed biochemically for dynamin-1, synaptogyrin-1, Munc18-1, clathrin heavy chain 1, Syt1, CaMKIIα, Hsc70, and syntaxin-7. (B) DGKθ overexpressed in HEK cells is immunoprecipitated with a myc antibody. (C) DGKθ interacts with Hsc70, CaMKIIα, Syt1, synaptogyrin-1, dynamin-1, syntaxin-7, and Munc18-1 when both are overexpressed in HEK cells. Hsc70 interacts with DGKθ at its endogenous expression level. (D) Clathrin heavy chain 1 does not interact with DGKθ when overexpressed in HEK cells. All blots are representative of at least three separate experiments.

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques: Labeling, Immunoprecipitation, Expressing

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet: Syt1 increases DGKθ activity 10-fold over DGKθ alone. (A) List of candidate proteins for which an interaction with DGKθ was validated and their function. Syt1 has the highest percent basicity of the confirmed interactors. (B) DGKθ kinase activity was measured for DGKθ alone and for DGKθ (10 ng/μL) plus the addition of each confirmed interactor (500 ng/μL). Syt1 increased DGKθ activity 10-fold, while syntaxin-7 increased DGKθ activity four-fold. Error bars indicate SEM. Graph summarizes data from 2 experiments with three replicates each, n = 6, * p < 0.05, *** p < 0.0005, **** p < 0.0001 against DGKθ only, ratio paired t -test.

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques: Activity Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet: Model for DGKθ’s interaction with Syt1. Syt1 is a transmembrane protein on synaptic vesicles and the presynaptic membrane. Perhaps to aid in the membrane deformation required in the fusion and fission of vesicles, Syt1 binds to DGKθ and stimulates its kinase activity, producing local hotspots of PtdOH that generate negative membrane curvature. AZ = active zone, EZ = endocytic zone. Figure made with BioRender.com .

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques: Activity Assay

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet:

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques: Purification

Journal: Frontiers in Synaptic Neuroscience

Article Title: Identification of Synaptic DGKθ Interactors That Stimulate DGKθ Activity

doi: 10.3389/fnsyn.2022.855673

Figure Lengend Snippet:

Article Snippet: Rabbit anti-synaptotagmin1 , Cell Signaling Technology , 14558S , – , 1:1000.

Techniques:

Journal: The Journal of Neuroscience

Article Title: WT1 -Expressing Interneurons Regulate Left–Right Alternation during Mammalian Locomotor Activity

doi: 10.1523/JNEUROSCI.0328-18.2018

Figure Lengend Snippet: WT1-expressing neurons terminate in close proximity to populations of commissurally projecting interneurons. A, A 20-μm-thick section of a spinal cord 46 h after PRV-152 injection into the GS muscle on the left side and stained with antibodies to GFP (green) and WT1 (blue). At this time point, no WT1-expressing neurons on either side of the spinal cord have taken up the tracer. Contralateral region containing WT1-expressing cells (dashed box) is expanded to the right. B, To identify presumptive synapses on genetically defined interneuronal subtypes, we inspected the synaptotagmin+ “halo” (indicated by white arrows) surrounding each labeled nuclei for WT1+/synaptotagmin+ terminals. C–F, The 20-μm-thick sections cut from a P0 WT1CreERROSA26tdTomato spinal cord and stained with antibodies to tdTomato (red), the synaptic marker synatotagmin (blue), as well as (green) a nuclear marker of V2a cells (Chx10-, C), V1 cells (En1-, D), V0V cells (Evx1-, E), or DMRT3-expressing dI6 cells (F). WT1-expressing terminals (tdTomato+/synaptotagmin+ processes) were rare or absent nearby Chx10- or En1-expressing cells but were commonly seen in close proximity to Evx1+ and DMRT3+ neurons. C, D, Dashed boxes are expanded to the right. E, F, Arrow in the low-magnification image indicates the specific WT1 cell of interest in the panel to the right. In magnified images, arrows indicate presumptive WT1+ axon terminals. Double labeling of these processes is confirmed in orthogonal views to the right of E, F. Scale bars: Low-magnification images, 100 μm; High-magnification images: A, C, D, 20 μm; B, E, F, 5 μm.

Article Snippet: Primary antibodies used were as follows: WT1 (rabbit, 1:100, Santa Cruz Biotechnology, RRID: AB_632611 ), GFP (goat, 1:5000, gift from Eusera), glycine (rat, 1:1000, Immunosolutions, RRID: AB_10013222 ), En1 (gift from Jessel laboratory, Columbia University, guinea pig, 1:1000), Chx10 (mouse, 1:200, Santa Cruz Biotechnology, RRID: AB_10842442 ), DMRT3 (goat, 1:100, Santa Cruz Biotechnology, RRID: AB_2091664 ), synaptotagmin (rabbit, 1:200, Alomone Labs), Evx1 (mouse, 1:100, DSHB, RRID: AB_2246711 ), and NeuN (mouse, 1:500, Millipore, RRID: AB_177621 ).

Techniques: Expressing, Injection, Staining, Labeling, Marker