Spectrin binding motifs regulate Scribble cortical dynamics and polarity function
- Institut Pasteur - CNRS UMR 3691, France
- INSERM, France
Figures
Figure 1 with 2 supplements
Dissection of the SCRIB β-spectrin interaction.
(A) Schematic representation of SCRIB and different C-terminal constructs used in this study. (B) GST-C, GST-C1 and GST-C2 pull-down on astrocytes cell extract. Samples were analyzed by ponceau …
Figure 1—figure supplement 1
SCRIB C-terminal sequence alignment.
Sequence alignment of SCRIB C-terminal domain of indicated different species. Boxes indicate the emplacement of the SADH motifs. Note that no sequences fitting the SADH consensus can be detected in …
Figure 1—figure supplement 2
Schematic representation of SCRIB, β2-spectrin and β3 spectrin proteins.
(A) The SCRIB C-terminal sequence encompassing the 1223–1630 region was used as a bait in the two hybrid screen. (B) Minimal β2-spectrin and β3 spectrin sequences recovered in the two hybrid screen …
Figure 2
SCRIB spectrin binding motifs bind to CH1 domains of the β spectrin family.
(A) Schematic representation of full length human β2 spectrin protein (left) and different β2 spectrin deletion constructs used in the study (right). (B) GST pull down using a GST-SCRIB C1 resin …
Figure 3 with 1 supplement
The SADH motif (SCRIB, ABLIM, dematin homology motif).
(A) Alignment of vertebrate SCRIB spectrin binding repeats 1 (pink), 2 (yellow) and 3 (green). Conserved residues are shown in blue. [+] indicates positively charged amino acids and Φ represents …
Figure 3—figure supplement 1
SADH domains in vertebrates.
(A) Schematic representation of SCRIB gene with spectrin binding motifs (up). Table displaying the distribution of SADH domains in SCRIB vertebrate protein sequences. SADH motifs could not be …
Figure 4 with 3 supplements
SADH motif influence SCRIB membrane stability and is required for SCRIB polarity function.
(A) 16HBE cells were transiently nucleofected with the indicated GFP constructs and analyzed by live confocal microscopy to calculate their cortical localization index. (n = 50 for each conditions). …
Figure 4—figure supplement 1
Cortical localization of SCRIB SADH mutant.
(A) Representative images of 16HBE cells nucleofected with the indicated GFP constructs acquired live and used for the CLI analysis. (B) Schematic representation of the process used for CLI …
Figure 4—figure supplement 2
SADH domains are implicated in SCRIB-mediated control of Cdc42 localization.
(A) Cherry-Cdc42 recruitment at the leading edge of migrating astrocytes cells expressing the indicated constructs. Results as shown are means ± SEM of three independent experiments, with a total of …
Figure 4—figure supplement 3
SCRIB SADH domain are required for tight junction assembly.
(A) WB analysis of SCRIB and actin expression in Caco-2 cells, 3 days after nucleofection with control siRNA (Ctrl) or siRNAs directed against SCRIB ORF (ORF1 and ORF2) or 3′ UTR (UTR1 and 2). (B) …
Figure 5 with 1 supplement
SADH motifs mutations in human pathology.
(A) Table of identified somatic SCRIB SADH motifs mutations in human cancers. Data sourced from COSMIC (http://cancer.sanger.ac.uk/cosmic). (B) GST-SCRIB SADH2 WT and SADH2 R1322W pull down assay on …
Figure 5—figure supplement 1
(A) 16HBE cells were transiently nucleofected with the indicated GFP constructs and analyzed by live confocal microscopy to calculate their cortical localization index.
(n = 50 for each conditions). (B) Quantitative analysis of FRAP experiment on 16HBE cells nucleofected with the indicated GFP constructs (n = 30 for each conditions).
