Journal: bioRxiv

Article Title: Alternating polarity integrates chemical and mechanical cues to drive tissue morphogenesis

doi: 10.1101/2023.08.09.552692

Figure Lengend Snippet: Multiple regulators of the actin cytoskeleton co-localize to the same alternating cell edges. A-C) Immunostaining of candidate actin regulatory proteins α-actinin (A), Sallimus/Kettin/D-Titan (B), and SCAR (C). Dlg or β-catenin (magenta) marks cell outlines. α-actinin and Sallimus co-localize with F-actin (cyan) to alternating edges (yellow arrowheads), while Scar localizes to puncta bordering nascent alternating edges (yellow arrowheads). D) Schematic of two possible underlying causes for the localization of proteins to alternating edges. The localization pattern of SCAR above supports a model invoking alternating polarity (right) rather than alternating expression (left). E-F) Immunostaining of phosphoinositides PI4P (E) and PIP2 (F) reveals co-localization with F-actin (cyan) to alternating edges (yellow arrowheads). Dlg (magenta) marks cell outlines. PIP2 antibody staining may reflect either PI(3,4)P2 or PI(4,5)P2 (see main text). Anterior up. Scale bars = 20 μm.

Article Snippet: Primary antibodies used included the following: Baz/Par3 (kind gift of Jennifer Zallen , rabbit polyclonal ( ) or guinea pig polyclonal , 1:250-1:500); aPKC (Santa Cruz Biotechnology, PKCζ antibody (H1): sc-17781, mouse IgG2a, 1:50); Dlg (DSHB (Developmental Studies Hybridoma Bank) # 4F3 anti-discs large supernatant, mouse IgG1, 1:50); β-catenin: (DSHB # N2 7A1 Armadillo supernatant, mouse IgG2a supernatant, 1:20); α-actinin (DSHB # 2G3-3D7 supernatant, mouse IgG2a, 1:10); Kettin/Sallimus/Titin (DSHB # 1B8-3D9 supernatant, mouse IgG1, 1:10); SCAR (DSHB # P1C1-SCAR supernatant, mouse IgG1, 1:50); PI4P (Echelon Biosciences # Z-P004, clone PI4-2, mouse IgM, 8 μg/mL); PIP2 (Abcam # ab2335, clone KT10, mouse IgG2b, 1:100).

Techniques: Immunostaining, Expressing, Staining