Journal: eLife

Article Title: An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging mouse neurons

doi: 10.7554/eLife.101533

Figure Lengend Snippet: ( A ) Schematic of cytoplasmic compartments and condensates and respective markers in parentheses that were assayed to colocalize with cytoplasmic POLK. ( B ) Cytoplasmic POLK (green) expression colocalizing with G3BP1 and LAMP1 (red) in fluorescent-nissl stained cells (blue) of mouse brain tissue in 18-month-old brain. Line scan in the boxed region shows POLK colocalizing with LAMP1 and G3BP1. ( C – F ) Cytoplasmic POLK (green), EEA1, CTSB, CTSD, and GBA1 (red) in fluorescent-nissl stained cells (blue) of mouse brain tissue in 18-month-old brain. Line scan in the boxed region shows level of POLK colocalization with the proteins. Highest colocalization with CTSD, partial with EEA1 and CTSB, and minimal GBA1.

Article Snippet: Antibody , Rat monoclonal anti-1D4B (LAMP1) , DSHB , RRID: AB_2134500 , IF (1:200).

Techniques: Expressing, Staining

Journal: eLife

Article Title: An altered cell-specific subcellular distribution of translesion synthesis DNA polymerase kappa (POLK) in aging mouse neurons

doi: 10.7554/eLife.101533

Figure Lengend Snippet: ( A ) Cytoplasmic POLK (green) expression colocalizing with G3BP1 (blue) in fluorescent-nissl stained cells (purple) of mouse brain tissue from M1 and S1 cortical regions in 18-month-old brain but not in young 1 month. ( B ) Cytoplasmic POLK (green) expression colocalizing with LAMP1 (blue) in fluorescent-nissl stained cells (purple) of mouse brain tissue from M1 and S1 cortical regions of 18-month brain. ( C ) Additional representative image with channel separation for immunofluorescence staining of wild-type mouse brain cortical areas S1, showing cytoplasmic POLK is colocalized with stress granule marker G3BP1 and endo/lysosomal marker LAMP1. Arrows indicate few representative sites of colocalization in both images.

Article Snippet: Antibody , Rat monoclonal anti-1D4B (LAMP1) , DSHB , RRID: AB_2134500 , IF (1:200).

Techniques: Expressing, Staining, Immunofluorescence, Marker

Journal: bioRxiv

Article Title: Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

doi: 10.64898/2026.04.20.719580

Figure Lengend Snippet: Representative images of the effects of mutations on the lysosomal localisation of sialin visualised by epifluorescence microscopy. Wild-type (WT) or different mutated human sialin (R39C, R39K, E194A, E262A, E264A and I266A) tagged with EGFP (green) constructs were transiently expressed in HeLa cells by electroporation. After two days, cells were fixed and analysed under fluorescence microscopy using LAMP1 immunostaining (red) to detect late endosomes and lysosomes. The scale bar represents 10 µm.

Article Snippet: Coverslips were then incubated for at least 1 h with mouse anti-LAMP1 antibodies (H4A3; Developmental Studies Hybridoma Bank) at 0.75 μg/mL in blocking buffer, washed, and incubated with Cy3-conjugated donkey antimouse antibodies (Jackson ImmunoResearch) at 1.4 μg/mL or Alexa Fluor 555 goat antimouse antibodies (Invitrogen) at 2μg/mL in the same buffer.

Techniques: Epifluorescence Microscopy, Construct, Electroporation, Fluorescence, Microscopy, Immunostaining

Journal: bioRxiv

Article Title: Molecular basis of Salla Disease: R39C Mutation Effects on the Lysosomal Transporter Sialin

doi: 10.64898/2026.04.20.719580

Figure Lengend Snippet: Mutagenesis experiments on the sialin R39 site. A and B) Wild-type (WT) or different mutated human sialin tagged with EGFP were transiently expressed in HeLa cells by electroporation. After two days, cells were fixed and analysed under fluorescence microscopy using LAMP1 immunostaining to detect late endosomes and lysosomes. In independent experiments, colocalisation was quantified using scatter plots of the sialin and LAMP1 pixel intensities. The graphs show the Pearson correlation coefficients normalised to that of WT (100%) in three different experiments for the mutants, except for R39C, 6 experiments, with 10 cells per experiment. The statistical analysis was performed by a one-sample t-test comparing the mean of the values for each mutant with a hypothetical mean value of 100: *= p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ****= p ≤ 0.0001). The red dotted line represents a Pearson coefficient of 0.5, above which we consider that sialin is targeted to lysosomes. It is calculated taking into consideration that the mean Pearson correlation coefficient for WT sialin is 0.73. C and D) Transport activity was measured in a whole-cell assay in which sialin was redirected to the plasma membrane by mutation of the lysosomal sorting motif to facilitate transport measurements (L22G L23G). Human L22G L23G sialin tagged with EGFP with/without mutations was transiently transfected by lipofection in HEK 293T cells. Human sialin (control) and its mutants were assayed for [ 3 H]Neu5Ac uptake at pH 5.0 in 3-6 independent experiments. The graphs show the transport activity relative to that of sialin L22G/L23G (100%) The statistical analysis was performed by a one-sample t-test comparing the mean of the values for each mutant with a hypothetical mean value of 100: * = p ≤ 0.05, ** = p ≤ 0.01, *** = p ≤ 0.001, ****= p ≤ 0.0001). E and F) Close-up views of ICH1 interactions in the completed and minimised models of the LO (E) and CO (F) conformations. Residues in ICH1 (green) are shown to interact with TM1 (orange), TM5 (purple), and other parts (grey) of sialin. Hydrogen bonding (green dashed lines), charge-charge (orange dashed lines), and apolar (purple dashed lines) interactions are shown for residues Y261 to L270 and R39. Hydrogen bonds between backbone atoms involved in the formation of alpha-helices are not shown and only polar hydrogens are displayed to improve visibility. Residues A26-C36 of the N-terminus are not shown to improve visibility.

Article Snippet: Coverslips were then incubated for at least 1 h with mouse anti-LAMP1 antibodies (H4A3; Developmental Studies Hybridoma Bank) at 0.75 μg/mL in blocking buffer, washed, and incubated with Cy3-conjugated donkey antimouse antibodies (Jackson ImmunoResearch) at 1.4 μg/mL or Alexa Fluor 555 goat antimouse antibodies (Invitrogen) at 2μg/mL in the same buffer.

Techniques: Mutagenesis, Electroporation, Fluorescence, Microscopy, Immunostaining, Activity Assay, Clinical Proteomics, Membrane, Transfection, Control