Journal: Life Science Alliance

Article Title: The CDKL5 kinase undergoes liquid–liquid phase separation driven by a serine-rich C-terminal region

doi: 10.26508/lsa.202503402

Figure Lengend Snippet: (A) Central upper panel . Super-resolution (Airyscan, Zeiss) confocal fluorescence micrograph of a differentiated NG108-15 neuronal cell expressing CDKL5-GFP. The left , lower middle , and right panels show, respectively, magnified details of the somatic compartment ( s ), the axon (a), and a varicosity ( v ). Note the intra- and extranuclear condensate-like foci ( arrowheads ). Scale bar: 10 μm. The image upper corners have been filled in dark gray for graphical purposes. (B) Neuropil of intermeshed neurites, with varicosities, of NG108-15 cells expressing CDKL5-GFP. Arrowheads indicate condensate-like CDKL5 foci in a varicosity ( left ) and along a neurite ( right ). Scale bar: 10 μm. (C) Two HEK293 cells expressing CDKL5-GFP. Arrowheads indicate condensates. Scale bar: 10 μm. (D) Correlative light and electron microscopy (CLEM) images of a HEK293 cell expressing CDKL5-GFP. The first panel from the left shows a confocal fluorescence image with intranuclear CDKL5-GFP condensates ( green ). The nucleus is stained with DAPI ( blue ), and mitochondria ( white arrow ) are marked by the MitoTracker dye ( red ). The third panel shows an EM image of a cell section at the same level of the confocal fluorescence image. The second panel is an overlay of the fluorescence and EM images. The two fluorescent condensate-like foci are highlighted by arrowheads and correspond to denser areas (boxed in yellow in the EM image) within the nuclear compartment, which are magnified in the two right panels . The MitoTracker signal overlaps with mitochondria, as expected. The two condensates are membraneless, and the larger one has an internal cavity also visible in the fluorescence image (see the Results section). Scale bars: 2.5 μm (larger images), 250 nm (smaller panels). (E) As in panel (D), for cytoplasmic condensate-like foci. The fourth image on the right illustrates a cluster of them (see yellow box in the EM image), two of which ( asterisks ) appear to be coalescing with each other. (D) Scale bars as in panel (D).

Article Snippet: After rinsing with PBS (3x), the cells were incubated with secondary fluorescent antibodies (Jackson ImmunoResearch, anti-rabbit [cat. #711-175-152] or anti-rat [cat. #712-165-153]; 1:1,000 dilution) in PBS containing 3% NDS and 0.5% Triton X-100 for 1 h at RT and rinsed with PBS (3x).

Techniques: Fluorescence, Expressing, Electron Microscopy, Staining

Journal: Life Science Alliance

Article Title: The CDKL5 kinase undergoes liquid–liquid phase separation driven by a serine-rich C-terminal region

doi: 10.26508/lsa.202503402

Figure Lengend Snippet: (A) Representative confocal fluorescence images of HEK293 cells expressing either CDKL5-GFP ( upper row ) or GFP alone ( lower row ) and immunostained to reveal the presence of EB2 ( right column, purple ) or its S222 phosphorylated form (pEB2; left column, cyan ). Calibration bar: 10 μm. Note the strong increase of the pEB2 signal in cells expressing CDKL5-GFP. (B) Representative Western blot of lysates of HEK293 cells expressing either CDKL5-GFP ( left lane ) or GFP alone ( right lane ) to detect the levels of these two exogenous proteins (α-GFP immunoblotting, IB) and the endogenous CDKL5 target protein EB2, as such (α-EB2 IB) or phosphorylated at Ser222 (α-pEB2 IB). Vinculin, a housekeeping protein, was used as a lane loading control (α-vinculin IB). Note how EB2 is considerably more phosphorylated in CDKL5-GFP–expressing cells in comparison with what was observed in GFP-expressing cells. Together with the results shown in panel (A), these findings indicate that the exogenously expressed GFP-tagged kinase is catalytically active and dramatically enhances EB2 phosphorylation in HEK293 cells. (C) As in panel (A), for cells expressing ( green signal) either WT CDKL5, its two indicated disease-related mutants, or GFP alone (control). The cells were immunostained to reveal EB2 (in purple ) and pEB2 (in cyan ). Nuclei are stained using DAPI (in gray ). Images displaying the four merged channels for each construct are shown on the right ( merge ). Scale bar: 10 μm. (D) Bar graph displaying the quantification of the relative proportion of phosphorylated EB2 in the experiments shown in panel (C). Columns report for each group the mean ratio between the pEB2 and EB2 fluorescent intensities in each cell, expressed as variation (Δ) versus the GFP control group and normalized to the WT CDKL5-GFP group. Asterisks indicate statistically significant differences. Note how the relative phosphorylation level of EB2 is significantly lower in cells expressing the two truncated kinases than in cells expressing the full-length CDKL5-GFP kinase. (E) As in panel (B), for lysates of cells expressing either CDKL5-GFP, or one of the indicated GFP-tagged disease-related mutants, or GFP alone as a control. Immunoblotting was performed for each experimental group using α-pEB2 and α-EB2, α-GFP, and α-vinculin (vinc) antibodies. Vinculin was used as a lane loading control. (F) Bar graph displaying the optical density analysis of Western blotting experiments as shown in panel (E). The columns report the ratio of the vinculin-normalized band intensities for pEB2 and EB2, expressed as variation (Δ) versus the GFP control group, normalized to the WT CDKL5-GFP group in each experiment. Asterisks indicate statistically significant differences. Note how, after normalization to the expression level of the housekeeping protein, the relative phosphorylation level of EB2 is lower in cells expressing the two truncated kinases than in cells expressing the full-length CDKL5-GFP kinase. Source data are available for this figure.

Article Snippet: After rinsing with PBS (3x), the cells were incubated with secondary fluorescent antibodies (Jackson ImmunoResearch, anti-rabbit [cat. #711-175-152] or anti-rat [cat. #712-165-153]; 1:1,000 dilution) in PBS containing 3% NDS and 0.5% Triton X-100 for 1 h at RT and rinsed with PBS (3x).

Techniques: Fluorescence, Expressing, Western Blot, Control, Comparison, Phospho-proteomics, Staining, Construct

Journal: The Journal of Experimental Medicine

Article Title: Surface μ Heavy Chain Signals Down-Regulation of the V(D)J-Recombinase Machinery in the Absence of Surrogate Light Chain Components

doi: 10.1084/jem.20031523

Figure Lengend Snippet: μH chain is detectable on the surface of λ5 −/− , VpreB1 −/− VpreB2 −/− , and SLC −/− pre–B cells. (A) CD19 + BM cells were isolated by MACS from tet-μHC, tet-μHC λ5 −/− , and tet-μHC VpreB1 −/− VpreB2 −/− mice that had received Tet in drinking water for 7 d. Cells were expanded on stromal cells in medium containing IL-7 and Tet for 48 h, washed, and recultured for an additional 18 h in the presence of IL-7 with (shaded) or without Tet (unshaded). Cells were harvested and stained with biotinylated Abs against μH chain (goat polyclonal), pre-BCR (SL156), or SLC components (LM34, VP245, and R5). Surface-bound biotin was amplified and revealed by EAS and streptavidin-PE. Propidium iodide negative cells were analyzed by flow cytometry. (B) CD19 + BM cells were isolated by MACS from SLC −/− or Rag1 −/− (control) mice and expanded on stromal cells in medium containing IL-7 for 4 d. Cells were harvested and stained with FITC-labeled Abs against κ/λLC and biotinylated Abs against μHC (goat polyclonal) or λ5 (LM34). Surface-bound biotin was amplified and revealed aforementioned. Numbers represent the percentage of cells within the quadrants.

Article Snippet: For intracellular stainings, cells were fixed and permeabilized using the FIX & PERM ® kit (Caltag Laboratories) and the following antisera: FITC- or Cy™5-conjugated polyclonal goat anti–mouse IgM (μH chain specific) and FITC- or Cy™5-conjugated polyclonal donkey anti–rabbit IgG (Dianova); FITC-conjugated anti–κL-chain and anti–λ 1 +λ 2 L-chain mAbs (BD Biosciences); polyclonal rabbit anti-TdT (Supertechs); and polyclonal rabbit anti-Ku70 (DPC Biermann).

Techniques: Isolation, Staining, Amplification, Flow Cytometry, Labeling